A lab-on-phone instrument with varifocal microscope via a liquid-actuated aspheric lens (LAL)

Science.gov (United States)

Fuh, Yiin-Kuen; Lai, Zheng-Hong; Kau, Li-Han; Huang, Hung-Jui

2017-01-01

In this paper, we introduce a novel concept of liquid-actuated aspheric lens (LAL) with a built-in aspheric polydimethylsiloxane lens (APL) to enable the design of compact optical systems with varifocal microscopic imaging. The varifocal lens module consists of a sandwiched structures such as 3d printed syringe pump functionally serves as liquid controller. Other key components include two acrylic cylinders, a rigid separator, a APL/membrane composite (APLMC) embedded PDMS membrane. In functional operation, the fluidic controller was driven to control the pressure difference and ALPMC deformation. The focal length can be changed through the pressure difference. This is achieved by the adjustment of volume change of injected liquid such that a widely tunable focal length. The proposed LAL can transform to 3 modes: microscopic mode (APLMC only), convex-concave mode and biconcave mode. It is noticeable that LAL in the operation of microscopic mode is tunable in focus via the actuation of APLMC (focal length is from 4.3 to 2.3 mm and magnification 50X) and can rival the images quality of commercial microscopes. A new lab-on-phone device is economically feasible and functionally versatile to offer a great potential in the point of care applications. PMID:28650971

Linearization of the bradford protein assay.

Science.gov (United States)

Ernst, Orna; Zor, Tsaffrir

2010-04-12

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, the ratio of the absorbance measurements at 590 nm and 450 nm is strictly linear with protein concentration. This simple procedure increases the accuracy and improves the sensitivity of the assay about 10-fold, permitting quantification down to 50 ng of bovine serum albumin. Furthermore, the interference commonly introduced by detergents that are used to create the cell lysates is greatly reduced by the new protocol. A linear equation developed on the basis of mass action and Beer's law perfectly fits the experimental data.

A rapid and efficient branched DNA hybridization assay to titer lentiviral vectors.

Science.gov (United States)

Nair, Ayyappan; Xie, Jinger; Joshi, Sarasijam; Harden, Paul; Davies, Joan; Hermiston, Terry

2008-11-01

A robust assay to titer lentiviral vectors is imperative to qualifying their use in drug discovery, target validation and clinical applications. In this study, a novel branched DNA based hybridization assay was developed to titer lentiviral vectors by quantifying viral RNA genome copy numbers from viral lysates without having to purify viral RNA, and this approach was compared with other non-functional (p24 protein ELISA and viral RT-qPCR) and a functional method (reporter gene expression) used commonly. The RT-qPCR method requires purification of viral RNA and the accuracy of titration therefore depends on the efficiency of purification; this requirement is ameliorated in the hybridization assay as RNA is measured directly in viral lysates. The present study indicates that the hybridization based titration assay performed on viral lysates was more accurate and has additional advantages of being rapid, robust and not dependent on transduction efficiency in different cell types.

Endotoxin testing of proteins for parenteral administration using the Mono Mac 6 assay

DEFF Research Database (Denmark)

Moesby, Lise; Hansen, E W; Christensen, J D

2000-01-01

Pharmaceutical products containing proteins cause problems in testing for endotoxin and pyrogens. Many proteins interfere with the LAL test and the proteins are immunogenic in rabbits. The monocytic cell line Mono Mac 6 is an alternative assay for detection of endotoxin and other pyrogens....

Inhibition of lipopolysaccharide induced acute inflammation in lung by chlorination.

Science.gov (United States)

Zhang, Jinshan; Xue, Jinling; Xu, Bi; Xie, Jiani; Qiao, Juan; Lu, Yun

2016-02-13

Lipopolysaccharide (LPS, also called endotoxin) is a pro-inflammatory constituent of gram negative bacteria and cyanobacteria, which causes a potential health risk in the process of routine urban application of reclaimed water, such as car wash, irrigation, scenic water refilling, etc. Previous studies indicated that the common disinfection treatment, chlorination, has little effect on endotoxin activity removal measured by Limulus amebocyte lysate (LAL) assay. However, in this study, significant decrease of acute inflammatory effects was observed in mouse lung, while LAL assay still presented a moderate increase of endotoxin activity. To explore the possible mechanisms, the nuclear magnetic resonance (NMR) results showed the chlorination happened in alkyl chain of LPS molecules, which could affect the interaction between LPS and LPS-binding protein. Also the size of LPS aggregates was found to drop significantly after treatment, which could be another results of chlorination caused polarity change. In conclusion, our observation demonstrated that chlorination is effective to reduce the LPS induced inflammation in lung, and it is recommended to use health effect-based methods to assess risk removal of water treatment technologies. Copyright © 2015 Elsevier B.V. All rights reserved.

Measurement of endotoxin levels in blood of hemodialysis Patients by 'Lal' test and comparision of its efficacy with blood culture

Directory of Open Access Journals (Sweden)

Gh Vazirzadeh

2006-01-01

Full Text Available Introduction: Presently, bacteremia is the principal cause of morbidity in patients undergoing hemodialysis. Gram-negative bacteria account for approximately 50 percent of documented infections. Endotoxins released during lysis of gram negative bacteremia result in inflammatory and defense response by the body and if not treated promptly result in septic shock and ultimately death of the patient. This study describes the detection of endotoxins in blood of patients with bacteremia due to gram - negative bacteria by LAL test. Method: Blood samples of 278 hemodialysis patients were analyzed in this study and pathogens were isolated from blood culture samples. Then, their antibiotic sensitivity was determined. In patients with positive blood culture, endotoxin levels were measured by LAL-test. Results: Frequency of bacteremia in patients was 13.6% . The prevalence of gram – negative bacteremia was 44.7%. E coli were the major pathogens, while staphylococcus aureus was the most common gram positive bacterium. Endotoxin was detected in 15 patients (3.8 ± 1.08 EU/ml . The sensitivity and specificity of endotoxins for gram – negative bacteremia were 88% and 95%, respectively. Conclusion: The results indicate that the LAL method is a fast, sensitive and simple method. There was no significant difference between the results of blood culture and LAL – test ( P > 0.05 .

Linearization of the Bradford Protein Assay

OpenAIRE

Ernst, Orna; Zor, Tsaffrir

2010-01-01

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. This most common assay enables rapid and simple protein quantification in cell lysates, cellular fractions, or recombinant protein samples, for the purpose of normalization of biochemical measurements. However, an intrinsic nonlinearity compromises the sensitivity and accuracy of this method. It is shown that under standard assay conditions, t...

Molecular and Cellular Mechanisms of Septic Shock

Science.gov (United States)

1988-03-01

due to gastric dilation - volvulus (6Y). The LAL-chromogenic procedure iýs sensitive and reliable for detecting plasma LPS. LPS has a short initial T 1/2...portal injections of endotoxin. and intestinal ischemia. Endotoxin (LPS) was quantitated in canine plasma using the Limulus amebocyte lysate (LAL...CV=1O), and stability of diluted, heat-treated, frozen samples (at least 8 wk). Analysis of canine plasma samples following sublethal IV (jugular or

Porcine platelet lysate as a supplement for animal cell culture

Science.gov (United States)

Aldén, Anna; Gonzalez, Lorena; Persson, Anna; Christensson, Kerstin; Holmqvist, Olov

2007-01-01

A novel supplementation of cell growth media based on a porcine platelet lysate was developed for culture of animal-derived cells. The platelet lysate was produced from porcine blood and contained lysate of platelets and plasma components. It showed satisfactory microbiological integrity and it carried only low amount of endotoxins (platelet lysate supported well proliferation of Vero (African green monkey transformed kidney epithelial cells), Chinese hamster ovary (CHO) and hybridoma cells comparable to fetal bovine serum (FBS). Platelet lysate shows promise as a viable choice over FBS as it can be produced in large quantities, high lot-to-lot consistency and with an attractive price structure. Furthermore it is a strong alternative to FBS for ethical reasons. It is expected that it can be used as a general supplementation for most animal cells for research studies on the proliferation of cells and their expression of products. PMID:19002989

Platelet lysate: a replacement for fetal bovine serum in animal cell culture?

OpenAIRE

Johansson, Liselott; Klinth, Jeanna; Holmqvist, Olov; Ohlson, Sten

2003-01-01

A new cell culture supplement, platelet lysate, was evaluated with reference to fetal bovine serum (FBS), an established industrial medium for animal cell culture. Chemical and bacteriological profiles were conducted including the presence of platelet-derived growth factor (PDGF). PDGF was detected in the platelet lysate but it was not present in FBS. The platelet lysate medium demonstrated lack of microorganisms, mycoplasma and endotoxins. The platelet lysate was investigated in culture stud...

A dimensão musical de lalíngua e seus efeitos na prática com crianças autistas

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Beatriz Alves Viana

Full Text Available Resumo Este artigo resulta da experiência com autistas em uma extensão universitária articulada à rede de saúde mental e a um serviço de psicologia. Investiga-se a relação de lalíngua com a música e estabelece suas contribuições à clínica do autismo. Para tanto, foi feita a leitura de textos nos campos da psicanálise e da música. Como material explorado, utilizaram-se relatos de experiência de profissionais e trabalho no grupo de extensão. A relação de lalíngua com a música é apresentada a partir da consideração dos jogos rítmicos de presença e ausência do som no decorrer do tempo que trazem elementos marcantes à própria constituição do sujeito, situando-nos diante de uma diacronia, também presente em lalíngua. Com isso, pode-se pensar como o estudo desse conceito nos auxilia na compreensão da relação primordial do sujeito com Outro, especialmente no autismo.

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers.

Science.gov (United States)

Sovkova, Vera; Vocetkova, Karolina; Rampichova, Michala; Mickova, Andrea; Buzgo, Matej; Lukasova, Vera; Dankova, Jana; Filova, Eva; Necas, Alois; Amler, Evzen

2018-06-01

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

Detection of endotoxins in radiopharmaceutical preparations. III. Limulus test assessment using radiopharmaceutical preparations; correlation with the rabbit pyrogen test

Energy Technology Data Exchange (ETDEWEB)

Cohen, Y; Bahri, F; Bruneau, J; Dubuis, M; Dubuis, N; Merlin, L; Michaud, T; Peysson, S

1986-01-01

Experiments using 17 radiopharmaceuticals containing known amounts of added endotoxin show that none of them inhibits the pyrogenic reaction of the rabbit. Gelation of the Limulus amoebocyte lysate (LAL) is inhibited by 4 of them: colloidal erbium 169Er citrate, colloidal rhenium 186Re sulfide, colloidal technetium /sup 99m/Tc (Re) sulfide for liver scintigraphy and the colloidal technetium /sup 99m/Tc (Re) sulfide for lymphography. This inhibition is cancelled, either by dilution or after neutral pH adjustment. Both controls were performed on 313 batches of various radiopharmaceuticals, 95% of results were identical (93% negative, 2% positive). The remaining 5% correspond to positive LAL tests vs negative rabbit tests on the same batches. No negative LAL test vs positive rabbit test was observed.

A multiplex branched DNA assay for parallel quantitative gene expression profiling.

Science.gov (United States)

Flagella, Michael; Bui, Son; Zheng, Zhi; Nguyen, Cung Tuong; Zhang, Aiguo; Pastor, Larry; Ma, Yunqing; Yang, Wen; Crawford, Kimberly L; McMaster, Gary K; Witney, Frank; Luo, Yuling

2006-05-01

We describe a novel method to quantitatively measure messenger RNA (mRNA) expression of multiple genes directly from crude cell lysates and tissue homogenates without the need for RNA purification or target amplification. The multiplex branched DNA (bDNA) assay adapts the bDNA technology to the Luminex fluorescent bead-based platform through the use of cooperative hybridization, which ensures an exceptionally high degree of assay specificity. Using in vitro transcribed RNA as reference standards, we demonstrated that the assay is highly specific, with cross-reactivity less than 0.2%. We also determined that the assay detection sensitivity is 25,000 RNA transcripts with intra- and interplate coefficients of variance of less than 10% and less than 15%, respectively. Using three 10-gene panels designed to measure proinflammatory and apoptosis responses, we demonstrated sensitive and specific multiplex gene expression profiling directly from cell lysates. The gene expression change data demonstrate a high correlation coefficient (R(2)=0.94) compared with measurements obtained using the single-plex bDNA assay. Thus, the multiplex bDNA assay provides a powerful means to quantify the gene expression profile of a defined set of target genes in large sample populations.

Optimization of dendritic cell loading with tumor cell lysates for cancer immunotherapy.

Science.gov (United States)

Hatfield, Paul; Merrick, Alison E; West, Emma; O'Donnell, Dearbhaile; Selby, Peter; Vile, Richard; Melcher, Alan A

2008-09-01

The immune response to cancer is critically determined by the way in which tumor cells die. As necrotic, stress-associated death can be associated with activation of antitumor immunity, whole tumor cell antigen loading strategies for dendritic cell (DC)-based vaccination have commonly used freeze-thaw "necrotic" lysates as an immunogenic source of tumor-associated antigens. In this study, the effect of such lysates on the ability of DCs to mature in response to well-established maturation stimuli was examined, and methods to enhance lysate-induced DC activation explored. Freeze-thaw lysates were prepared from murine tumor cell lines and their effects on bone marrow-derived DC maturation and function examined. Unmodified freeze-thaw tumor cell lysates inhibited the toll-like receptor-induced maturation and function of bone marrow-derived DCs, preventing up-regulation of CD40, CD86, and major histocompatibility complex class II, and reducing secretion of inflammatory cytokines [interleukin (IL)-12 p70, tumor necrosis factor-alpha, and IL-6]. Although IL-10 secretion was increased by lysate-pulsed DCs, this was not responsible for the observed suppression of IL-12. Although activation of the nuclear factor-kappaB pathway remained intact, the kinase activity of phosphorylated p38 mitogen-activated protein kinase was inhibited in lysate-pulsed DCs. Lysate-induced DC suppression was partially reversed in vitro by induction of tumor cell stress before lysis, and only DCs loaded with stressed lysates afforded protection against tumor challenge in vivo. These data suggest that ex vivo freeze-thaw of tumor cells does not effectively mimic in vivo immunogenic necrosis, and advocates careful characterization and optimization of tumor cell-derived vaccine sources for cancer immunotherapy.

Highly active antiretroviral therapy and outcome of AIDS-related Burkitt's lymphoma or leukemia. Results of the PETHEMA-LAL3/97 study.

Science.gov (United States)

Oriol, Albert; Ribera, Josep-Maria; Brunet, Salut; del Potro, Eloy; Abella, Eugènia; Esteve, Jordi

2005-07-01

Short, intensive cycles of chemotherapy have resulted in improved survival in BurkittOs lymphoma/leukemia (BL) in adults. The prognosis of patients with immunodeficiency virus (HIV)-associated BL is considered to be poor, but these patients have seldom been treated with BL-specific protocols. However, a study (PETHEMA-LAL3/97) in which patients with BL were treated regardless of their HIV status failed to find differences between HIV-infected and immunocompetent individuals. Furthermore, patients who received highly active antiretroviral therapy (HAART) seemed to have a slightly better disease-free survival than those who did not (p=0.051). We extended the follow-up analysis to elucidate the role of HAART in the survival of HIV-infected patients included in the PETHEMA-LAL3/97 protocol.

Effect of platelet lysate on human cells involved in different phases of wound healing.

Science.gov (United States)

Barsotti, Maria Chiara; Chiara Barsotti, Maria; Losi, Paola; Briganti, Enrica; Sanguinetti, Elena; Magera, Angela; Al Kayal, Tamer; Feriani, Roberto; Di Stefano, Rossella; Soldani, Giorgio

2013-01-01

Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization). Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2) and inflammatory response evaluation (NFκB). Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v). Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (pplatelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

High sensitivity pyrogen testing in water and dialysis solutions.

Science.gov (United States)

Daneshian, Mardas; Wendel, Albrecht; Hartung, Thomas; von Aulock, Sonja

2008-07-20

The dialysis patient is confronted with hundreds of litres of dialysis solution per week, which pass the natural protective barriers of the body and are brought into contact with the tissue directly in the case of peritoneal dialysis or indirectly in the case of renal dialysis (hemodialysis). The components can be tested for living specimens or dead pyrogenic (fever-inducing) contaminations. The former is usually detected by cultivation and the latter by the endotoxin-specific Limulus Amoebocyte Lysate Assay (LAL). However, the LAL assay does not reflect the response of the human immune system to the wide variety of possible pyrogenic contaminations in dialysis fluids. Furthermore, the test is limited in its sensitivity to detect extremely low concentrations of pyrogens, which in their sum result in chronic pathologies in dialysis patients. The In vitro Pyrogen Test (IPT) employs human whole blood to detect the spectrum of pyrogens to which humans respond by measuring the release of the endogenous fever mediator interleukin-1beta. Spike recovery checks exclude interference. The test has been validated in an international study for pyrogen detection in injectable solutions. In this study we adapted the IPT to the testing of dialysis solutions. Preincubation of 50 ml spiked samples with albumin-coated microspheres enhanced the sensitivity of the assay to detect contaminations down to 0.1 pg/ml LPS or 0.001 EU/ml in water or saline and allowed pyrogen detection in dialysis concentrates or final working solutions. This method offers high sensitivity detection of human-relevant pyrogens in dialysis solutions and components.

Determination of bacterial endotoxin (pyrogen) in radiopharmaceuticals by the gel clot method. Validation

International Nuclear Information System (INIS)

Fukumori, Neuza Taeko Okasaki

2008-01-01

Before the Limulus amebocyte lysate (LAL) test, the only available means of pirogenicity testing for parenteral drugs and medical devices was the United States Pharmacopoeia (USP) rabbit pyrogen test. Especially for radiopharmaceuticals, the LAL assay is the elective way to determine bacterial endotoxin. The aim of this work was to validate the gel clot method for some radiopharmaceuticals without measurable interference. The FDA's LALTest guideline defines interference as a condition that causes a significant difference between the endpoints of a positive water control and positive product control series using a standard endotoxin. Experiments were performed in accordance to the USP bacterial endotoxins test in the 131 I- m-iodobenzylguanidine; the radioisotopes Gallium-67 and Thallium-201; the lyophilized reagents DTPA, Phytate, GHA, HSA and Colloidal Tin. The Maximum Valid Dilution (MVD) was calculated for each product based upon the clinical dose of the material and a twofold serial dilution below the MVD was performed in duplicate to detect interferences. The labeled sensitivity of the used LAL reagent was 0.125 EU mL -1 (Endotoxin Units per milliliter). For validation, a dilution series was performed, a twofold dilution of control standard endotoxin (CSE) from 0.5 to 0.03 EU mL -1 , to confirm the labeled sensitivity of the LAL reagent being tested in sterile and non pyrogenic water, in quadruplicate. The same dilution series was performed with the CSE and the product in the 1:100 dilution factor, in three consecutive batches of each radiopharmaceutical. The products 131 I-m-iodobenzylguanidine, Gallium-67, Thallium-201, DTPA, HSA and Colloidal Tin were found compatible with the LAL test at a 1:100 dilution factor. Phytate and GHA showed some interference in the gel clot test. Other techniques to determine endotoxins as the chromogenic (color development) and the turbidimetric test (turbidity development), were also assessed to get valuable quantitative and

Determination of bacterial endotoxin (pyrogen) in radiopharmaceuticals by the gel clot method. Validation; Determinacao de endotoxina bacteriana (pirogenio) em radiofarmacos pelo metodo de formacao de gel. Validacao

Energy Technology Data Exchange (ETDEWEB)

Fukumori, Neuza Taeko Okasaki

2008-07-01

Before the Limulus amebocyte lysate (LAL) test, the only available means of pirogenicity testing for parenteral drugs and medical devices was the United States Pharmacopoeia (USP) rabbit pyrogen test. Especially for radiopharmaceuticals, the LAL assay is the elective way to determine bacterial endotoxin. The aim of this work was to validate the gel clot method for some radiopharmaceuticals without measurable interference. The FDA's LALTest guideline defines interference as a condition that causes a significant difference between the endpoints of a positive water control and positive product control series using a standard endotoxin. Experiments were performed in accordance to the USP bacterial endotoxins test in the {sup 131}I- m-iodobenzylguanidine; the radioisotopes Gallium-67 and Thallium-201; the lyophilized reagents DTPA, Phytate, GHA, HSA and Colloidal Tin. The Maximum Valid Dilution (MVD) was calculated for each product based upon the clinical dose of the material and a twofold serial dilution below the MVD was performed in duplicate to detect interferences. The labeled sensitivity of the used LAL reagent was 0.125 EU mL{sup -1} (Endotoxin Units per milliliter). For validation, a dilution series was performed, a twofold dilution of control standard endotoxin (CSE) from 0.5 to 0.03 EU mL{sup -1}, to confirm the labeled sensitivity of the LAL reagent being tested in sterile and non pyrogenic water, in quadruplicate. The same dilution series was performed with the CSE and the product in the 1:100 dilution factor, in three consecutive batches of each radiopharmaceutical. The products {sup 131}I-m-iodobenzylguanidine, Gallium-67, Thallium-201, DTPA, HSA and Colloidal Tin were found compatible with the LAL test at a 1:100 dilution factor. Phytate and GHA showed some interference in the gel clot test. Other techniques to determine endotoxins as the chromogenic (color development) and the turbidimetric test (turbidity development), were also assessed to get valuable

Determination of bacterial endotoxin (pyrogen) in radiopharmaceuticals by the gel clot method. Validation; Determinacao de endotoxina bacteriana (pirogenio) em radiofarmacos pelo metodo de formacao de gel. Validacao

Energy Technology Data Exchange (ETDEWEB)

Fukumori, Neuza Taeko Okasaki

2008-07-01

Before the Limulus amebocyte lysate (LAL) test, the only available means of pirogenicity testing for parenteral drugs and medical devices was the United States Pharmacopoeia (USP) rabbit pyrogen test. Especially for radiopharmaceuticals, the LAL assay is the elective way to determine bacterial endotoxin. The aim of this work was to validate the gel clot method for some radiopharmaceuticals without measurable interference. The FDA's LALTest guideline defines interference as a condition that causes a significant difference between the endpoints of a positive water control and positive product control series using a standard endotoxin. Experiments were performed in accordance to the USP bacterial endotoxins test in the {sup 131}I- m-iodobenzylguanidine; the radioisotopes Gallium-67 and Thallium-201; the lyophilized reagents DTPA, Phytate, GHA, HSA and Colloidal Tin. The Maximum Valid Dilution (MVD) was calculated for each product based upon the clinical dose of the material and a twofold serial dilution below the MVD was performed in duplicate to detect interferences. The labeled sensitivity of the used LAL reagent was 0.125 EU mL{sup -1} (Endotoxin Units per milliliter). For validation, a dilution series was performed, a twofold dilution of control standard endotoxin (CSE) from 0.5 to 0.03 EU mL{sup -1}, to confirm the labeled sensitivity of the LAL reagent being tested in sterile and non pyrogenic water, in quadruplicate. The same dilution series was performed with the CSE and the product in the 1:100 dilution factor, in three consecutive batches of each radiopharmaceutical. The products {sup 131}I-m-iodobenzylguanidine, Gallium-67, Thallium-201, DTPA, HSA and Colloidal Tin were found compatible with the LAL test at a 1:100 dilution factor. Phytate and GHA showed some interference in the gel clot test. Other techniques to determine endotoxins as the chromogenic (color development) and the turbidimetric test (turbidity development), were also assessed to get

Effect of platelet lysate on human cells involved in different phases of wound healing.

Directory of Open Access Journals (Sweden)

Maria Chiara Barsotti

Full Text Available BACKGROUND: Platelets are rich in mediators able to positively affect cell activity in wound healing. Aim of this study was to characterize the effect of different concentrations of human pooled allogeneic platelet lysate on human cells involved in the different phases of wound healing (inflammatory phase, angiogenesis, extracellular matrix secretion and epithelialization. METHODOLOGY/PRINCIPAL FINDINGS: Platelet lysate effect was studied on endothelial cells, monocytes, fibroblasts and keratinocytes, in terms of viability and proliferation, migration, angiogenesis, tissue repair pathway activation (ERK1/2 and inflammatory response evaluation (NFκB. Results were compared both with basal medium and with a positive control containing serum and growth factors. Platelet lysate induced viability and proliferation at the highest concentrations tested (10% and 20% v/v. Whereas both platelet lysate concentrations increased cell migration, only 20% platelet lysate was able to significantly promote angiogenic activity (p<0.05 vs. control, comparably to the positive control. Both platelet lysate concentrations activated important inflammatory pathways such as ERK1/2 and NFκB with the same early kinetics, whereas the effect was different for later time-points. CONCLUSION/SIGNIFICANCE: These data suggest the possibility of using allogeneic platelet lysate as both an alternative to growth factors commonly used for cell culture and as a tool for clinical regenerative application for wound healing.

Detection of microbial translocation in HIV and SIV infection using the Limulus amebocyte lysate assay is masked by serum and plasma.

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Ashwin Balagopal

Full Text Available Microbial translocation (MT is thought to be a major contributor to the pathogenesis of HIV-related immune activation, and circulating lipopolysaccharide (LPS from gram-negative bacteria is the principle measurement of this process. However, related research has been impeded by inconsistent LPS test results.Specimens were obtained from HIV-infected adults enrolled in the PEARLS study (ACTG A5175 and HIV-HCV co-infected participants enrolled in a study of liver disease staging using MRI elastography. Pig-tailed macaque specimens were obtained from SIV-infected and -uninfected animals. Samples were tested for LPS using the LAL assay with diazo-coupling modifications to improve sensitive detection.When exogenous LPS was added to macaque plasma, >25% inhibition of LPS detection was found in 10/10 (100% samples at 20% plasma concentration compared to control; in contrast 5/10 (50% samples at 2% plasma concentration (p = 0.07 and 0/10 (0% at 0.1% plasma concentration (p = 0.004 showed >25% inhibition of LPS detection. Similarly, when LPS was added to human serum, >25% inhibition of LPS detection was found in 5/12 (42% of samples at 2% serum concentration compared to control, while 0/12 (0% of samples in 0.1% serum showed >25% inhibition of LPS detection (p = 0.07. Likewise, LPS detection in human sera without exogenous LPS was improved by dilution: LPS was detected in 2/12 (17% human samples in 2% serum, ranging from 3,436-4,736 pg/mL, compared to 9/12 (75% samples in 0.1% serum, ranging from 123 pg/mL -60,131 pg/mL (p = 0.016. In a separate validation cohort of HIV-HCV co-infected participants sampled at two different times on the same day, LPS measured in 0.2% plasma and with diazo-coupling was closely correlated between the first and second samples (R = 0.66, p<0.05.Undiluted serum and plasma mask LPS detection. The extent of MT may be substantially underestimated.

A novel protease activity assay using a protease-responsive chaperone protein

International Nuclear Information System (INIS)

Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

2009-01-01

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

A novel protease activity assay using a protease-responsive chaperone protein

Energy Technology Data Exchange (ETDEWEB)

Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

2009-06-05

Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation.

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Moreira Teixeira, Liliana S; Leijten, Jeroen C H; Wennink, Jos W H; Chatterjea, Anindita G; Feijen, Jan; van Blitterswijk, Clemens A; Dijkstra, Pieter J; Karperien, Marcel

2012-05-01

In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and anti-inflammatory cytokines, but mechanically unstable. We hypothesized that the advantages of these systems may be combined in one hydrogel, which can be easily translated into clinical settings. Platelet lysate was successfully incorporated into Dex-TA polymer solution prior to gelation. After enzymatic crosslinking, rheological and morphological evaluations were performed. Subsequently, the effect of platelet lysate on cell migration, adhesion, proliferation and multi-lineage differentiation was determined. Finally, we evaluated the integration potential of this gel onto osteoarthritis-affected cartilage. The mechanical properties and covalent attachment of Dex-TA to cartilage tissue during in situ gel formation were successfully combined with the advantages of platelet lysate, revealing the potential of this enhanced hydrogel as a cell-free approach. The addition of platelet lysate did not affect the mechanical properties and porosity of Dex-TA hydrogels. Furthermore, platelet lysate derived anabolic growth factors promoted proliferation and triggered chondrogenic differentiation of mesenchymal stromal cells. Copyright © 2012 Elsevier Ltd. All rights reserved.

Comparison between mixed lysate antigen and α-actinin antigen in ELISA for serodiagnosis of trichomoniasis.

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Kim, Seung-Ryong; Kim, Jung-Hyun; Park, Soon-Jung; Lee, Hye-Yeon; Kim, Yong-Suk; Kim, Yu-Mi; Hong, Yeon-Chul; Ryu, Jae-Sook

2015-10-01

The aim of this study was to identify an antigen suitable for ELISA for serodiagnosis of Trichomonas vaginalis (T. vaginalis) infection. Mixed lysate antigen (Ag) from eight strains of T. vaginalis and recombinant α-actinin protein was compared. The sera of three groups were examined by ELISA: 73 women infected with trichomoniasis served as a positive control, 31 male volunteers as a negative control, and 424 women attending an outpatient health screening at Hanyang University Guri Hospital. Based on the cutoff optical density for each Ag obtained with a negative control, the serosensitivity of the mixed lysate Ag (79.5%) was significantly higher than that of the α-actinin (52.1%) in the 73 patients with trichomoniasis. The specificity using lysate Ag and α-actinin was 100% and 96.8%, respectively. On the other hand, the positivity rate in 424 outpatients was 39.2% and 11.8% with mixed lysate and α-actinin Ag, respectively. Taken together, mixed lysate Ag showed higher sensitivity and specificity than α-actinin. Therefore, mixed lysate may be a better Ag than α-actinin for ELISA for the diagnosis of trichomoniasis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Virally inactivated human platelet concentrate lysate induces regulatory T cells and immunosuppressive effect in a murine asthma model.

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Lee, Yueh-Lun; Lee, Lin-Wen; Su, Chen-Yao; Hsiao, George; Yang, Yi-Yuan; Leu, Sy-Jye; Shieh, Ying-Hua; Burnouf, Thierry

2013-09-01

Platelet concentrate lysates (PCLs) are increasingly used in regenerative medicine. We have developed a solvent/detergent (S/D)-treated PCL. The functional properties of this preparation should be unveiled. We hypothesized that, due to transforming growth factor-β1 (TGF-β1) content, PCLs may exert immunosuppressive and anti-inflammatory functions. PCL was prepared by S/D treatment, oil extraction, and hydrophobic interaction chromatography. The content of TGF-β in PCL was determined by enzyme-linked immunosorbent assay. Cultured CD4+ T cells were used to investigate the effects of PCL on expression of transcription factor forkhead box P3 (Foxp3), the inhibition of T-cell proliferation, and cytokine production. The regulatory function of PCL-converted CD4+ T cells was analyzed by suppressive assay. The BALB/c mice were given PCL-converted CD4+ T cells before ovalbumin (OVA) sensitization and challenge using an asthma model. Inflammatory parameters, such as the level of immunoglobulin E (IgE), airway hyperresponsiveness (AHR), bronchial lavage fluid eosinophils, and cytokines were assayed. Recombinant human (rHu) TGF-β1 was used as control. PCL significantly enhanced the development of CD4+Foxp3+-induced regulatory T cells (iTregs). Converted iTregs produced neither Th1 nor Th2 cytokines and inhibited normal T-cell proliferation. PCL- and rHuTGF-β-converted CD4+ T cells prevented OVA-induced asthma. PCL- and rHuTGF-β-modified T cells both significantly reduced expression levels of OVA-specific IgE and significantly inhibited the development of AHR, airway eosinophilia, and Th2 responses in mice. S/D-treated PCL promotes Foxp3+ iTregs and exerts immunosuppressive and anti-inflammatory properties. This finding may help to understand the clinical properties of platelet lysates. © 2013 American Association of Blood Banks.

Platelet lysates produced from expired platelet concentrates support growth and osteogenic differentiation of mesenchymal stem cells.

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Sandra Mjoll Jonsdottir-Buch

Full Text Available BACKGROUND: Mesenchymal stem cells are promising candidates in regenerative cell therapy. Conventional culture methods involve the use of animal substances, specifically fetal bovine serum as growth supplement. Since the use of animal-derived products is undesirable for human applications, platelet lysates produced from human platelets are an attractive alternative. This is especially true if platelet lysates from already approved transfusion units at blood banks can be utilized. The purpose of this study was to produce human platelet lysates from expired, blood bank-approved platelet concentrates and evaluate their use as growth supplement in the culture of mesenchymal stem cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, bone marrow-derived mesenchymal stem cells were cultured with one of three culture supplements; fetal bovine serum, lysates from freshly prepared human platelet concentrates, or lysates from expired human platelet concentrates. The effects of these platelet-derived culture supplements on basic mesenchymal stem cell characteristics were evaluated. All cultures maintained the typical mesenchymal stem cell surface marker expression, trilineage differentiation potential, and the ability to suppress in vitro immune responses. However, mesenchymal stem cells supplemented with platelet lysates proliferated faster than traditionally cultured cells and increased the expression of the osteogenic marker gene RUNX-2; yet no difference between the use of fresh and expired platelet concentrates was observed. CONCLUSION/SIGNIFICANCE: Our findings suggest that human platelet lysates produced from expired platelet concentrates can be used as an alternative to fetal bovine serum for mesenchymal stem cell culture to the same extent as lysates from fresh platelets.

The potential of lipopolysaccharide as a real-time biomarker of bacterial contamination in marine bathing water.

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Sattar, Anas A; Jackson, Simon K; Bradley, Graham

2014-03-01

The use of total lipopolysaccharide (LPS) as a rapid biomarker for bacterial pollution was investigated at a bathing and surfing beach during the UK bathing season. The levels of faecal indicator bacteria Escherichia coli (E. coli), the Gram-positive enterococci, and organisms commonly associated with faecal material, such as total coliforms and Bacteroides, were culturally monitored over four months to include a period of heavy rainfall and concomitant pollution. Endotoxin measurement was performed using a kinetic Limulus Amebocyte Lysate (LAL) assay and found to correlate well with all indicators. Levels of LPS in excess of 50 Endotoxin Units (EU) mL(-1) were found to correlate with water that was unsuitable for bathing under the current European regulations. Increases in total LPS, mainly from Gram-negative indicator bacteria, are thus a potential real-time, qualitative method for testing bacterial quality of bathing waters.

Platelet lysate and chondroitin sulfate loaded contact lenses to heal corneal lesions.

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Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Delfino, Alessio; Riva, Federica; Icaro Cornaglia, Antonia; Marrubini, Giorgio; Musitelli, Giorgio; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

2016-07-25

Hemoderivative tear substitutes contain various ephiteliotrophic factors, such as growth factors (GF), involved in ocular surface homeostasis without immunogenic properties. The aim of the present work was the loading of platelet lysate into contact lenses to improve the precorneal permanence of platelet lysate growth factors on the ocular surface to enhance the treatment of corneal lesions. To this purpose, chondroitin sulfate, a sulfated glycosaminoglycan, which is normally present in the extracellular matrix, was associated with platelet lysate. In fact, chondroitin sulfate is capable of electrostatic interaction with positively charged growth factors, in particular, with bFGF, IGF, VEGF, PDGF and TGF-β, resulting in their stabilization and reduced degradation in solution. In the present work, various types of commercially available contact lenses have been loaded with chondroitin sulfate or chondroitin sulfate in association with platelet lysate to achieve a release of growth factors directly onto the corneal surface lesions. One type of contact lenses (PureVision(®)) showed in vitro good proliferation properties towards corneal cells and were able to enhance cut closure in cornea constructs. Copyright © 2016 Elsevier B.V. All rights reserved.

Dendritic Cells Loaded with Pancreatic Cancer Stem Cells (CSCs) Lysates Induce Antitumor Immune Killing Effect In Vitro

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Yin, Tao; Shi, Pengfei; Gou, Shanmiao; Shen, Qiang; Wang, Chunyou

2014-01-01

According to the cancer stem cells (CSCs) theory, malignant tumors may be heterogeneous in which a small population of CSCs drive the progression of cancer. Because of their intrinsic abilities, CSCs may survive a variety of treatments and then lead to therapeutic resistance and cancer recurrence. Pancreatic CSCs have been reported to be responsible for the malignant behaviors of pancreatic cancer, including suppression of immune protection. Thus, development of immune strategies to eradicate pancreatic CSCs may be of great value for the treatment of pancreatic cancer. In this study, we enriched pancreatic CSCs by culturing Panc-1 cells under sphere-forming conditions. Panc-1 CSCs expressed low levels of HLA-ABC and CD86, as measured by flow cytometry analysis. We further found that the Panc-1 CSCs modulate immunity by inhibiting lymphocyte proliferation which is promoted by phytohemagglutinin (PHA) and anti-CD3 monoclonal antibodies. The monocyte derived dendritic cells (DCs) were charged with total lysates generated from Panc-1 CSCs obtained from tumor sphere culturing. After co-culturing with lymphocytes at different ratios, the Panc-1 CSCs lysates modified DC effectively promoted lymphocyte proliferation. The activating efficiency reached 72.4% and 74.7% at the ratios of 1∶10 and 1∶20 with lymphocytes. The activated lymphocytes secreted high levels of INF-γ and IL-2, which are strong antitumor cytokines. Moreover, Panc-1 CSCs lysates modified DC induced significant cytotoxic effects of lymphocytes on Panc-1 CSCs and parental Panc-1 cells, respectively, as shown by lactate dehydrogenase (LDH) assay. Our study demonstrates that the development of CSCs-based vaccine is a promising strategy for treating pancreatic cancer. PMID:25521461

Dendritic cells loaded with pancreatic Cancer Stem Cells (CSCs lysates induce antitumor immune killing effect in vitro.

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Tao Yin

Full Text Available According to the cancer stem cells (CSCs theory, malignant tumors may be heterogeneous in which a small population of CSCs drive the progression of cancer. Because of their intrinsic abilities, CSCs may survive a variety of treatments and then lead to therapeutic resistance and cancer recurrence. Pancreatic CSCs have been reported to be responsible for the malignant behaviors of pancreatic cancer, including suppression of immune protection. Thus, development of immune strategies to eradicate pancreatic CSCs may be of great value for the treatment of pancreatic cancer. In this study, we enriched pancreatic CSCs by culturing Panc-1 cells under sphere-forming conditions. Panc-1 CSCs expressed low levels of HLA-ABC and CD86, as measured by flow cytometry analysis. We further found that the Panc-1 CSCs modulate immunity by inhibiting lymphocyte proliferation which is promoted by phytohemagglutinin (PHA and anti-CD3 monoclonal antibodies. The monocyte derived dendritic cells (DCs were charged with total lysates generated from Panc-1 CSCs obtained from tumor sphere culturing. After co-culturing with lymphocytes at different ratios, the Panc-1 CSCs lysates modified DC effectively promoted lymphocyte proliferation. The activating efficiency reached 72.4% and 74.7% at the ratios of 1∶10 and 1∶20 with lymphocytes. The activated lymphocytes secreted high levels of INF-γ and IL-2, which are strong antitumor cytokines. Moreover, Panc-1 CSCs lysates modified DC induced significant cytotoxic effects of lymphocytes on Panc-1 CSCs and parental Panc-1 cells, respectively, as shown by lactate dehydrogenase (LDH assay. Our study demonstrates that the development of CSCs-based vaccine is a promising strategy for treating pancreatic cancer.

Performance of a Multiplex Serological Helicobacter pylori Assay on a Novel Microfluidic Assay Platform

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Angela Filomena

2017-10-01

Full Text Available Infection with Helicobacter pylori (H. pylori occurs in 50% of the world population, and is associated with the development of ulcer and gastric cancer. Serological diagnostic tests indicate an H. pylori infection by detecting antibodies directed against H. pylori proteins. In addition to line blots, multiplex assay platforms provide smart solutions for the simultaneous analysis of antibody responses towards several H. pylori proteins. We used seven H. pylori proteins (FliD, gGT, GroEL, HpaA, CagA, VacA, and HP0231 and an H. pylori lysate for the development of a multiplex serological assay on a novel microfluidic platform. The reaction limited binding regime in the microfluidic channels allows for a short incubation time of 35 min. The developed assay showed very high sensitivity (99% and specificity (100%. Besides sensitivity and specificity, the technical validation (intra-assay CV = 3.7 ± 1.2% and inter-assay CV = 5.5 ± 1.2% demonstrates that our assay is also a robust tool for the analysis of the H. pylori-specific antibody response. The integration of the virulence factors CagA and VacA allow for the assessment of the risk for gastric cancer development. The short assay time and the performance of the platform shows the potential for implementation of such assays in a clinical setting.

Estimativa da incerteza em ensaio de detecção de endotoxina bacteriana pelo método de gelificação Estimation of uncertainty in the detection of bacterial endotoxin by gel-clot method

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Felipe Rebello Lourenço

2005-12-01

Full Text Available Desde a publicação da ISO 17025:1999, o interesse em métodos para estimativa da incerteza em ensaios qualitativos, do tipo "passa/não passa", têm ganho grande importância. Uma forma de estimar e informar a incerteza deste tipo de ensaio é o uso das probabilidades de respostas-falsas, particularmente falsos-positivos e falsos-negativos, determinados a partir do teorema de Bayes. O objetivo deste artigo é estabelecer um método para a estimativa de incerteza em ensaios de detecção de endotoxina bacteriana pelo método in vitro Limulus Amebocyte Lysate (LAL. Considerando a confirmação da sensibilidade do LAL e a validação do teste, a probabilidade de uma resposta falsa corresponde à soma da probabilidade dos resultados falso-negativos e falso-positivos. A partir dos resultados obtidos foi verificado que a etapa da confirmação da sensibilidade do LAL contribui para a incerteza de forma mais significativa (67,6% que a etapa de validação do teste (32,4%. Através de um procedimento simples, descrito neste artigo, e de dados obtidos a partir da confirmação da sensibilidade do LAL e validação do teste para um produto em questão é possível obter uma estimativa de incerteza razoável para o ensaio de detecção de endotoxinas bacterianas pelo método de gelificação.Since the publication of ISO 17025:1999, the interest in methods for estimation of the uncertainty in qualitative analysis, such as 'pass/fail', have became more important. The usual form of estimating and informing the uncertainty in this kind of analysis is the use of false-response rates, particularly false-positive and false-negative, determinated from Bayes theorem. The aim of this paper is establish a method for estimation of the uncertainty in the detection of bacterial endotoxins by in vitro Limulus Amebocyte Lysate (LAL test. Considering the confirmation of LAL sensitivity and the validation of the test, the probability of a false-response corresponds to the

Platelet lysate formulations based on mucoadhesive polymers for the treatment of corneal lesions.

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Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Del Fante, Claudia; Perotti, Cesare; Scudeller, Luigia; Caramella, Carla

2011-02-01

Growth factors contained in platelet α-granules initiate and modulate tissue repair and are proposed for the treatment of soft and hard-tissue surgical conditions and in the management of non-healing wounds. Platelet lysate is a hemoderivative obtained from platelet-rich plasma and is capable of releasing a pool of growth factors. Many medical and surgical techniques have been proposed for the treatment of corneal lesions; management of these conditions remains problematic and healing with standard protocols is unattainable. The aim of this study was to develop formulations suitable for prolonging the contact of platelet lysate with the damaged cornea for the time necessary to exert a therapeutic effect. Two vehicles, one based on polyacrylic acid and one based on chitosan, were autoclaved and loaded with platelet lysate and the resultant formulations were characterized for rheology, mucoadhesion, vehicle compatibility and stability. The proliferation effect was tested on two cell culture types (rabbit corneal epithelial cells and fibroblasts). An in-vitro wound-healing test was performed on fibroblasts. In both cases the formulations were compared with platelet lysate diluted with saline at the same concentration. Both formulations maintained the rheological and mucoadhesive properties of the vehicles and the proliferative activity of platelet lysate. The chitosan formulation was able to significantly enhance epithelial cell growth even after storage of up to 2 weeks (in-use conditions), while the polyacrylic acid formulation was less efficient, probably due to the characteristics of the cell model used. The in-vitro wound-healing test performed on fibroblasts confirmed the differences between the two vehicles. The effect induced by the platelet lysate and chitosan formulation was faster than that of the polyacrylic acid formulation and complete in-vitro wound repair was achieved within 48 h. © 2010 The Authors. JPP © 2010 Royal Pharmaceutical Society.

Thermal precipitation fluorescence assay for protein stability screening.

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Fan, Junping; Huang, Bo; Wang, Xianping; Zhang, Xuejun C

2011-09-01

A simple and reliable method of protein stability assessment is desirable for high throughput expression screening of recombinant proteins. Here we described an assay termed thermal precipitation fluorescence (TPF) which can be used to compare thermal stabilities of recombinant protein samples directly from cell lysate supernatants. In this assay, target membrane proteins are expressed as recombinant fusions with a green fluorescence protein tag and solubilized with detergent, and the fluorescence signals are used to report the quantity of the fusion proteins in the soluble fraction of the cell lysate. After applying a heat shock, insoluble protein aggregates are removed by centrifugation. Subsequently, the amount of remaining protein in the supernatant is quantified by in-gel fluorescence analysis and compared to samples without a heat shock treatment. Over 60 recombinant membrane proteins from Escherichia coli were subject to this screening in the presence and absence of a few commonly used detergents, and the results were analyzed. Because no sophisticated protein purification is required, this TPF technique is suitable to high throughput expression screening of recombinant membrane proteins as well as soluble ones and can be used to prioritize target proteins based on their thermal stabilities for subsequent large scale expression and structural studies. Copyright © 2011 Elsevier Inc. All rights reserved.

Comparative Effects of Platelet-Rich Plasma, Platelet Lysate, and Fetal Calf Serum on Mesenchymal Stem Cells.

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Lykov, A P; Bondarenko, N A; Surovtseva, M A; Kim, I I; Poveshchenko, O V; Pokushalov, E A; Konenkov, V I

2017-10-01

We studied the effects of human platelet-rich plasma and platelet lysate on proliferation, migration, and colony-forming properties of rat mesenchymal stem cells. Platelet-rich plasma and platelet lysate stimulated the proliferation, migration, and colony formation of mesenchymal stem cells. A real-time study showed that platelet-rich plasma produces the most potent stimulatory effect, while both platelet-rich plasma and platelet lysate stimulated migration of cells.

Detection of endotoxins and other pyrogens using human whole blood.

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Fennrich, S; Fischer, M; Hartung, T; Lexa, P; Montag-Lessing, T; Sonntag, H G; Weigandt, M; Wendel, A

1999-01-01

When cells of the immune system, i.e. primarily blood monocytes and macrophages, come into contact with pyrogens (fever-inducing contaminations) they release mediators transmitting the fever reaction through the organism to the thermoregulatory centres of the brain. The new test discussed here exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample. If there is pyrogen contamination, the endogenous pyrogen interleukin-1 is released, which is then determined by ELISA. According to the pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample. In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species. Compared to the well established in vitro alternative, i.e. the limulus amebocyte lysate assay (LAL), the new blood assay is not restricted to endotoxins of gram-negative bacteria, it is not affected by endotoxin-binding blood proteins and it reflects the potency of different endotoxin preparations in mammals. Here, interim results of the ongoing optimization and pre-validation are reported and the present state of the evaluation for biological and pharmaceutical drugs are presented.

Measurement of operative plasma endotoxin levels in jaundiced and non-jaundiced patients.

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Pain, J A; Bailey, M E

1987-01-01

A study of portal plasma endotoxin levels was performed using a chromogenic limulus amoebocyte lysate (LAL) assay. The assay proved sensitive and reproducible. In only 1 of 25 healthy subjects was the systemic plasma endotoxin level above 100 pg/ml (equivalent Escherichia coli 0111B4). In 30 non-jaundiced patients undergoing surgery the mean (+SEM) portal plasma endotoxin level (60 + 9 pg/ml) was significantly higher (p less than 0.05) than the mean level in the systemic blood (46 + 6 pg/ml), supporting the concept of endotoxin absorption from the intestine into the portal blood. In 20 patients with obstructive jaundice undergoing surgery 42% of portal, 45% of inferior mesenteric and 35% of systemic venous plasma endotoxin levels were above 100 pg/ml. There were significantly higher levels in the portal (p less than 0.05) and inferior mesenteric (p less than 0.05) compared with the systemic blood. Neither the presence of malignancy nor the duration of surgery appeared to influence endotoxin absorption. The significance of raised plasma endotoxin levels in obstructive jaundice is discussed.

Serum-converted platelet lysate can substitute for fetal bovine serum in human mesenchymal stromal cell cultures.

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Mojica-Henshaw, Mariluz P; Jacobson, Pam; Morris, Julie; Kelley, Linda; Pierce, Jan; Boyer, Michael; Reems, Jo-Anna

2013-12-01

Fetal bovine serum (FBS) is commonly used as a serum supplement for culturing human mesenchymal stromal cells (hMSCs). However, human cells grown in FBS, especially for extended periods, risk potential exposure to bovine immunogenic proteins and infectious agents. To address this issue, we investigated the ability of a novel human platelet serum supplement to substitute for FBS in hMSC cultures. Platelet lysate-serum (PL-serum) was converted from platelet lysate-plasma (PL-plasma) that was manufactured from pooled platelet-rich plasma (PRP) apheresis units. Growth factor levels and the number of residual intact platelets in PL-serum and PL-plasma were compared with enzyme-linked immunosorbent assays and flow cytometry, respectively. Proliferation responses of hMSCs cultured in PL-serum, PL-plasma, or FBS were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the immunophenotype of harvested hMSCs was evaluated by flow cytometry and tri-lineage differentiation potential was evaluated by assessing adipogenic, osteogenic and chondrogenic development. Selected growth factor levels in PL-serum were not significantly different from PL-plasma (P > 0.05). hMSC cultures supplemented with PL-serum had comparable growth kinetics to PL-plasma, and hMSC yields were consistently greater than with FBS. hMSCs harvested from cultures supplemented with PL-serum, PL-plasma or FBS had similar cell surface phenotypes and maintained tri-lineage differentiation potential. PL-serum, similar to PL-plasma, can substitute for FBS in hMSC cultures. Use of PL-serum, in contrast to PL-plasma, has an added advantage of not requiring addition of a xenogeneic source of heparin, providing a completely xeno-free culture medium. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Autologous Dendritic Cells Pulsed with Allogeneic Tumor Cell Lysate in Mesothelioma: From Mouse to Human.

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Aerts, Joachim G J V; de Goeje, Pauline L; Cornelissen, Robin; Kaijen-Lambers, Margaretha E H; Bezemer, Koen; van der Leest, Cor H; Mahaweni, Niken M; Kunert, André; Eskens, Ferry A L M; Waasdorp, Cynthia; Braakman, Eric; van der Holt, Bronno; Vulto, Arnold G; Hendriks, Rudi W; Hegmans, Joost P J J; Hoogsteden, Henk C

2018-02-15

Purpose: Mesothelioma has been regarded as a nonimmunogenic tumor, which is also shown by the low response rates to treatments targeting the PD-1/PD-L1 axis. Previously, we demonstrated that autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy increased T-cell response toward malignant mesothelioma. However, the use of autologous tumor material hampers implementation in large clinical trials, which might be overcome by using allogeneic tumor cell lines as tumor antigen source. The purpose of this study was to investigate whether allogeneic lysate-pulsed DC immunotherapy is effective in mice and safe in humans. Experimental Design: First, in two murine mesothelioma models, mice were treated with autologous DCs pulsed with either autologous or allogeneic tumor lysate or injected with PBS (negative control). Survival and tumor-directed T-cell responses of these mice were monitored. Results were taken forward in a first-in-human clinical trial, in which 9 patients were treated with 10, 25, or 50 million DCs per vaccination. DC vaccination consisted of autologous monocyte-derived DCs pulsed with tumor lysate from five mesothelioma cell lines. Results: In mice, allogeneic lysate-pulsed DC immunotherapy induced tumor-specific T cells and led to an increased survival, to a similar extent as DC immunotherapy with autologous tumor lysate. In the first-in-human clinical trial, no dose-limiting toxicities were established and radiographic responses were observed. Median PFS was 8.8 months [95% confidence interval (CI), 4.1-20.3] and median OS not reached (median follow-up = 22.8 months). Conclusions: DC immunotherapy with allogeneic tumor lysate is effective in mice and safe and feasible in humans. Clin Cancer Res; 24(4); 766-76. ©2017 AACR . ©2017 American Association for Cancer Research.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

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Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Achado dunhas lousas con arte megalítico en Fonte Tourón Lalín, Pontevedra

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Cabrejas Domínguez, Elena

2009-12-01

Full Text Available In an archaeological context before a works execution took place (an electric half tension line, some engraved gravestones were found around the area of Fonte Touron, Council of Lalín. These gravestones have engravings representative of megalithism and nowadays they became boundary markers of the yards in which they are. The features of the engravings, besides the very shape of the stones and the context where other mounds are located, and above everything, the information provided by local neighbors that relate the stones of this mound to one set apart some years ago, points that they were part of a megalithic monument, and it is highly probable that they were the orthostats of a chamber.

No contexto dos traballos arqueolóxicos previos á execución dunha obra (unha liña eléctrica de media tensión, localizáronse unhas lousas con gravados na zona de Fonte Tourón, en Lalín. Trátase dunhas lousas que presentan uns gravados de estilo propio do megalitismo e que agora forman parte dos lindes das fincas nas que se atopan. As características destes gravados, sumado á propia forma das pedras, a un contexto onde se localizan outras mámoas e sobre todo, á información dos veciños que vinculan estas pedras cunha mámoa desfeita fai uns anos, apuntan a que formaron parte dun monumento megalítico, e que moi probablemente se trata dos esteos dunha cámara.

Evaluation of the limulus amoebocyte lysate test in conjunction with a gram negative bacterial plate count for detecting irradiation of chicken

Science.gov (United States)

Scotter, Susan L.; Wood, Roger; McWeeny, David J.

A study to evaluate the potential of the Limulus amoebocyte lysate (LAL) test in conjuction with a Gram negative bacteria (GNB) plate count for detecting the irradiation of chicken is described. Preliminary studies demonstrated that chickens irradiated at an absorbed dose of 2.5 kGy could be differentiated from unirradiated birds by measuring levels of endotoxin and of numbers of GNB on chicken skin. Irradiated birds were found to have endotoxin levels similar to those found in unirradiated birds but significantly lower numbers of GNB. In a limited study the test was found to be applicable to birds from different processors. The effect of temperature abuse on the microbiological profile, and thus the efficacy of the test, was also investigated. After temperature abuse, the irradiated birds were identifiable at worst up to 3 days after irradiation treatment at the 2.5 kGy level and at best some 13 days after irradiation. Temperature abuse at 15°C resulted in rapid recovery of surviving micro-organisms which made differentiation of irradiated and unirradiated birds using this test unreliable. The microbiological quality of the bird prior to irradiation treatment also affected the test as large numbers of GNB present on the bird prior to irradiation treatment resulted in larger numbers of survivors. In addition, monitoring the developing flora after irradiation treatment and during subsequent chilled storage also aided differentiation of irradiated and unirradiated birds. Large numbers of yeasts and Gram positive cocci were isolated from irradiated carcasses whereas Gram negative oxidative rods were the predominant spoilage flora on unirradiated birds.

Evaluation of the Limulus amoebocyte lysate test in conjunction with a gram negative bacterial plate count for detecting irradiation of chicken

International Nuclear Information System (INIS)

Scotter, S.L.; Wood, R.; McWeeny, D.J.

1990-01-01

A study to evaluate the potential of the Limulus amoebocyte lysate (LAL) test in conjunction with a Gram negative bacterial (GNB) plate count for detecting the irradiation of chicken is described. Preliminary studies demonstrated that chickens irradiated at an absorbed dose of 2.5 kGy could be differentiated from unirradiated birds by measuring levels of endotoxin and of numbers of GNB on chicken skin. Irradiated birds were found to have endotoxin levels similar to those found in unirradiated birds but significantly lower numbers of GNB. In a limited study the test was found to be applicable to birds from different processors. The effect of temperature abuse on the microbiological profile, and thus the efficacy of the test, was also investigated. After temperature abuse, the irradiated birds were identifiable at worst up to 3 days after irradiation treatment at the 2.5 kGy level and at best some 13 days after irradiation. Temperature abuse at 15 0 C resulted in rapid recovery of surviving micro-organisms which made differentiation of irradiated and unirradiated birds using this test unreliable. The microbiological quality of the bird prior to irradiation treatment also affected the test as large numbers of GNB present on the bird prior to irradiation treatment resulted in larger numbers of survivors. In addition, monitoring the developing flora after irradiation treatment amd during subsequent chilled storage also aided differentiation of irradiated and unirradiated birds. Large numbers of yeast and Gram positive cocci were isolated from irradiated carcasses whereas Gram negative oxidative rods were the predominant spoilage flora on unirradiated birds. (author)

Evaluation of the Limulus amoebocyte lysate test in conjunction with a gram negative bacterial plate count for detecting irradiation of chicken

Energy Technology Data Exchange (ETDEWEB)

Scotter, S L; Wood, R; McWeeny, D J [Ministry of Agriculture, Fisheries and Food, Norwich (UK). Food Science Lab.

1990-01-01

A study to evaluate the potential of the Limulus amoebocyte lysate (LAL) test in conjunction with a Gram negative bacterial (GNB) plate count for detecting the irradiation of chicken is described. Preliminary studies demonstrated that chickens irradiated at an absorbed dose of 2.5 kGy could be differentiated from unirradiated birds by measuring levels of endotoxin and of numbers of GNB on chicken skin. Irradiated birds were found to have endotoxin levels similar to those found in unirradiated birds but significantly lower numbers of GNB. In a limited study the test was found to be applicable to birds from different processors. The effect of temperature abuse on the microbiological profile, and thus the efficacy of the test, was also investigated. After temperature abuse, the irradiated birds were identifiable at worst up to 3 days after irradiation treatment at the 2.5 kGy level and at best some 13 days after irradiation. Temperature abuse at 15{sup 0}C resulted in rapid recovery of surviving micro-organisms which made differentiation of irradiated and unirradiated birds using this test unreliable. The microbiological quality of the bird prior to irradiation treatment also affected the test as large numbers of GNB present on the bird prior to irradiation treatment resulted in larger numbers of survivors. In addition, monitoring the developing flora after irradiation treatment amd during subsequent chilled storage also aided differentiation of irradiated and unirradiated birds. Large numbers of yeast and Gram positive cocci were isolated from irradiated carcasses whereas Gram negative oxidative rods were the predominant spoilage flora on unirradiated birds. (author).

Platelet Lysate-Modified Porous Silicon Microparticles for Enhanced Cell Proliferation in Wound Healing Applications.

Science.gov (United States)

Fontana, Flavia; Mori, Michela; Riva, Federica; Mäkilä, Ermei; Liu, Dongfei; Salonen, Jarno; Nicoletti, Giovanni; Hirvonen, Jouni; Caramella, Carla; Santos, Hélder A

2016-01-13

The new frontier in the treatment of chronic nonhealing wounds is the use of micro- and nanoparticles to deliver drugs or growth factors into the wound. Here, we used platelet lysate (PL), a hemoderivative of platelets, consisting of a multifactorial cocktail of growth factors, to modify porous silicon (PSi) microparticles and assessed both in vitro and ex vivo the properties of the developed microsystem. PL-modified PSi was assessed for its potential to induce proliferation of fibroblasts. The wound closure-promoting properties of the microsystem were then assessed in an in vitro wound healing assay. Finally, the PL-modified PSi microparticles were evaluated in an ex vivo experiment over human skin. It was shown that PL-modified PSi microparticles were cytocompatible and enhanced the cell proliferation in different experimental settings. In addition, this microsystem promoted the closure of the gap between the fibroblast cells in the wound healing assay, in periods of time comparable with the positive control, and induced a proliferation and regeneration process onto the human skin in an ex vivo experiment. Overall, our results show that PL-modified PSi microparticles are suitable microsystems for further development toward applications in the treatment of chronic nonhealing wounds.

Human platelet lysate as a promising growth-stimulating additive for culturing of stem cells and other cell types.

Science.gov (United States)

Shanskii, Ya D; Sergeeva, N S; Sviridova, I K; Kirakozov, M S; Kirsanova, V A; Akhmedova, S A; Antokhin, A I; Chissov, V I

2013-11-01

We compared the composition and biological activity of fetal calf serum and platelet lysate from donor platelet concentrate. In platelet lysate, the concentrations of alkaline phosphatase, lactate dehydrogenase, creatinine, and mineral metabolism parameters were lower, while parameters of lipid and protein metabolism were higher than in fetal calf serum. The concentrations of growth factors (platelet-derived (AA, AB, BB), vascular endothelial, insulin-like, and transforming growth factor β) in platelet lysate 1.7-148.7-fold surpassed the corresponding parameters in fetal calf serum. After replacement of fetal calf serum with platelet lysate in the culture medium (0, 25, 50, 75, and 100%), the count of multipotent mesenchymal stromal cells on day 7 (in comparison with day 1) increased by 154.8, 206.6, 228.2, 367.7, and 396.5%, respectively. Thus, platelet lysate can be an adequate non-xenogenic alternative for fetal calf serum.

In-Solution SH2 Domain Binding Assay Based on Proximity Ligation.

Science.gov (United States)

Machida, Kazuya

2017-01-01

Protein-protein interactions mediated by SH2 domains confer specificity in tyrosine kinase pathways. Traditional assays for assessing interactions between an SH2 domain and its interacting protein such as far-Western and pull-down are inherently low throughput. We developed SH2-PLA, an in-solution SH2 domain binding assay, that takes advantage of the speed and sensitivity of proximity ligation and real-time PCR. SH2-PLA allows for rapid assessment of SH2 domain binding to a target protein using only a few microliters of cell lysate, thereby making it an attractive new tool to study tyrosine kinase signaling.

The effect of lysate of spleen cells after low dose radiation (LDR) on NK activity

International Nuclear Information System (INIS)

Lu Duicai; Su Liaoyuan

2003-01-01

To find effect of lysate of spleen cells after LDR on NK activity of CD 57 cells or non-CD 57 cells, lysate of spleen cells after LDR were extracted. McAb (anti CD 57 cells) was used to separate CD 57 cells from human peripheral blood by Panning direct method. The CD 57 cells and non-CD 57 cells were used as effective cells. K 562 cells labelled by 3 H-TdR were used as target cells. The ratio of effect cells to target cells was 10:1. NK activity of CD 57 cells or non-CD 5 -7 cells with the lysate of spleen cells after LDR was reflected by the efficiency of anti tumor cells. The 3 H-TdR incorporation in K 562 cells cultured with non-CD 57 cells was significantly lower than that with CD 57 cells. After use of the lysate of spleen cells after LDR, NK activities of CD 57 cells and non-CD 57 cells were 1.24 and 1.58 respectively. They were both increased obviously compared with control groups. The effect of anti K 562 cells of non-CD 57 cells is even greater than that of CD 57 cells. The lysate of spleen cells after LDR can enhance the effect of both non-CD 57 cells and CD 57 cells

Effect of Different Adjuvants on Protection and Side-Effects Induced by Helicobacter suis Whole-Cell Lysate Vaccination.

Directory of Open Access Journals (Sweden)

Iris Bosschem

Full Text Available Helicobacter suis (H. suis is a widespread porcine gastric pathogen, which is also of zoonotic importance. The first goal of this study was to investigate the efficacy of several vaccine adjuvants (CpG-DNA, Curdlan, Freund's Complete and Incomplete, Cholera toxin, administered either subcutaneously or intranasally along with H. suis whole-cell lysate, to protect against subsequent H. suis challenge in a BALB/c infection model. Subcutaneous immunization with Freund's complete (FC/lysate and intranasal immunization with Cholera toxin (CT/lysate were shown to be the best options for vaccination against H. suis, as determined by the amount of colonizing H. suis bacteria in the stomach, although adverse effects such as post-immunization gastritis/pseudo-pyloric metaplasia and increased mortality were observed, respectively. Therefore, we decided to test alternative strategies, including sublingual vaccine administration, to reduce the unwanted side-effects. A CCR4 antagonist that transiently inhibits the migration of regulatory T cells was also included as a new adjuvant in this second study. Results confirmed that immunization with CT (intranasally or sublingually is among the most effective vaccination protocols, but increased mortality was still observed. In the groups immunized subcutaneously with FC/lysate and CCR4 antagonist/lysate, a significant protection was observed. Compared to the FC/lysate immunized group, gastric pseudo-pyloric metaplasia was less severe or even absent in the CCR4 antagonist/lysate immunized group. In general, an inverse correlation was observed between IFN-γ, IL-4, IL-17, KC, MIP-2 and LIX mRNA expression and H. suis colonization density, whereas lower IL-10 expression levels were observed in partially protected animals.

Culture of human cell lines by a pathogen-inactivated human platelet lysate.

Science.gov (United States)

Fazzina, R; Iudicone, P; Mariotti, A; Fioravanti, D; Procoli, A; Cicchetti, E; Scambia, G; Bonanno, G; Pierelli, L

2016-08-01

Alternatives to the use of fetal bovine serum (FBS) have been investigated to ensure xeno-free growth condition. In this study we evaluated the efficacy of human platelet lysate (PL) as a substitute of FBS for the in vitro culture of some human cell lines. PL was obtained by pools of pathogen inactivated human donor platelet (PLT) concentrates. Human leukemia cell lines (KG-1, K562, JURKAT, HL-60) and epithelial tumor cell lines (HeLa and MCF-7) were cultured with either FBS or PL. Changes in cell proliferation, viability, morphology, surface markers and cell cycle were evaluated for each cell line. Functional characteristics were analysed by drug sensitivity test and cytotoxicity assay. Our results demonstrated that PL can support growth and expansion of all cell lines, although the cells cultured in presence of PL experienced a less massive proliferation compared to those grown with FBS. We found a comparable percentage of viable specific marker-expressing cells in both conditions, confirming lineage fidelity in all cultures. Functionality assays showed that cells in both FBS- and PL-supported cultures maintained their normal responsiveness to adriamycin and NK cell-mediated lysis. Our findings indicate that PL is a feasible serum substitute for supporting growth and propagation of haematopoietic and epithelial cell lines with many advantages from a perspective of process standardization, ethicality and product safety.

Validation of the L.A.L. technique (gel cloth) in radiodiagnosis and radioisotopes

International Nuclear Information System (INIS)

Morote, M.; Otero, M.; Chavez, G.

1999-01-01

Based on the validation of the LAL technique (gel cloth), real endotoxin limits (EL), expressed in endotoxin units per milliliter (EU/mL), as well as maximum dilution volume (MDV) were determined in seven radiodiagnosis agents (RDA): medronate, pentetate, sulfur colloid, albumin macroaggregated, succimer, disofenin, and pyrophosphates, which showed M.D.V. values of: 1:80; 1:16; 1:128; 1:64; 1:4; 1:128; 1:64 and EL of 20 EU/mL; 4 EU/mL; 32 EU/mL; 16 EU/mL; 1 EU/mL and 16 EU/mL, respectively. Radioisotope Tc-99m presents a MDV of 1:1 and an EL below 0,25 EU/mL. Clear interference in the acidity of sulfur colloid (pH below 2) was also found against pyrotell reaction, even in the sixth dilution of the product. M.D.V. results also showed that dilutions are even 100 times smaller than the theoretical values

Platelet lysate obtained via plateletpheresis performed in standing and awake equine donors.

Science.gov (United States)

Sumner, Scarlett M; Naskou, Maria C; Thoresen, Merrilee; Copland, Ian; Peroni, John F

2017-07-01

Platelet preparations containing growth factors, attachment factors, and enzymes are appealing to enhance healing of injured tissues and as an alternative to xenogenic serum in cell culture media. Plateletpheresis is commonly used to collect platelets in human medicine but has not been validated in horses. Plateletpheresis to collect platelet concentrate was performed on six female, mixed breed, chemically restrained horses using commercially available apheresis equipment. Before and immediately after plateletpheresis, we performed physical examinations and collected blood for chemistry and coagulation panels and then again at 8, 16, 24, and 48 hours after the procedure. To produce platelet lysate, the platelet concentrate underwent two freeze-thaw cycles followed by centrifugation and filtration processing. The platelet lysate was then analyzed for cellular debris, fibrinogen, and growth factors. The collected platelet concentration contained a mean platelet yield of 390 × 10 3 /μL. Donor platelet count decreased from a mean of 193 × 10 3 /μL to 138 × 10 3 /μL after plateletpheresis, but no individual was at risk for hemorrhage. Pooled platelet lysate had minimal cellular residue and contained growth factor concentrations at 6.1 ng/mL for transforming growth factor-β1, at 3.5 ng/mL for platelet-derived growth factor-BB, and at 13.8 ng/mL for vascular endothelial growth factor-A. Plateletpheresis using commercially available apheresis equipment is a feasible option for collecting platelet concentrate from equine donors. The lysate generated from the apheresis product contains growth factors and has potential to be used as a fetal bovine serum substitute for cell culture. © 2017 AABB.

An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

DEFF Research Database (Denmark)

Rasmussen, Randi Engelberth; Erstad, Simon Matthé; Ramos Martinez, Erick Miguel

2016-01-01

microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls...... used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at -20 °C without losing the enzyme activities. The permeabilization process...... for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism...

Assay to detect lipid peroxidation upon exposure to nanoparticles.

Science.gov (United States)

Potter, Timothy M; Neun, Barry W; Stern, Stephan T

2011-01-01

This chapter describes a method for the analysis of human hepatocarcinoma cells (HEP G2) for lipid peroxidation products, such as malondialdehyde (MDA), following treatment with nanoparticle formulations. Oxidative stress has been identified as a likely mechanism of nanoparticle toxicity, and cell-based in vitro systems for evaluation of nanoparticle-induced oxidative stress are widely considered to be an important component of biocompatibility screens. The products of lipid peroxidation, lipid hydroperoxides, and aldehydes, such as MDA, can be measured via a thiobarbituric acid reactive substances (TBARS) assay. In this assay, which can be performed in cell culture or in cell lysate, MDA combines with thiobarbituric acid (TBA) to form a fluorescent adduct that can be detected at an excitation wavelength of 530 nm and an emission wavelength of 550 nm. The results are then expressed as MDA equivalents, normalized to total cellular protein (determined by Bradford assay).

flyLAL покоряет Эстонию / Екатерина Таклая

Index Scriptorium Estoniae

Таклая, Екатерина

2009-01-01

Leedule kuuluv lennufirma Avia Solutions Group lõi Eestisse lennufirma flyLAL Charters Eesti, millega sõlmisid lepingud TEZ Tour, Novatours ja Horizon Travel. Estonian Air ei pea uut firmat konkurendiks

Exosomes: novel effectors of human platelet lysate activity

Directory of Open Access Journals (Sweden)

E Torreggiani

2014-09-01

Full Text Available Despite the popularity of platelet-rich plasma (PRP and platelet lysate (PL in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF, vascular endothelial growth factor (VEGF, platelet-derived growth factor (PDGF-BB and transforming growth factor beta 1 (TGF-β1 as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

Promoter Engineering Reveals the Importance of Heptameric Direct Repeats for DNA Binding by Streptomyces Antibiotic Regulatory Protein-Large ATP-Binding Regulator of the LuxR Family (SARP-LAL) Regulators in Streptomyces natalensis.

Science.gov (United States)

Barreales, Eva G; Vicente, Cláudia M; de Pedro, Antonio; Santos-Aberturas, Javier; Aparicio, Jesús F

2018-05-15

The biosynthesis of small-size polyene macrolides is ultimately controlled by a couple of transcriptional regulators that act in a hierarchical way. A Streptomyces antibiotic regulatory protein-large ATP-binding regulator of the LuxR family (SARP-LAL) regulator binds the promoter of a PAS-LuxR regulator-encoding gene and activates its transcription, and in turn, the gene product of the latter activates transcription from various promoters of the polyene gene cluster directly. The primary operator of PimR, the archetype of SARP-LAL regulators, contains three heptameric direct repeats separated by four-nucleotide spacers, but the regulator can also bind a secondary operator with only two direct repeats separated by a 3-nucleotide spacer, both located in the promoter region of its unique target gene, pimM A similar arrangement of operators has been identified for PimR counterparts encoded by gene clusters for different antifungal secondary metabolites, including not only polyene macrolides but peptidyl nucleosides, phoslactomycins, or cycloheximide. Here, we used promoter engineering and quantitative transcriptional analyses to determine the contributions of the different heptameric repeats to transcriptional activation and final polyene production. Optimized promoters have thus been developed. Deletion studies and electrophoretic mobility assays were used for the definition of DNA-binding boxes formed by 22-nucleotide sequences comprising two conserved heptameric direct repeats separated by four-nucleotide less conserved spacers. The cooperative binding of PimR SARP appears to be the mechanism involved in the binding of regulator monomers to operators, and at least two protein monomers are required for efficient binding. IMPORTANCE Here, we have shown that a modulation of the production of the antifungal pimaricin in Streptomyces natalensis can be accomplished via promoter engineering of the PAS-LuxR transcriptional activator pimM The expression of this gene is

Human Platelet Lysate as a Replacement for Fetal Bovine Serum in Limbal Stem Cell Therapy.

Science.gov (United States)

Suri, Kunal; Gong, Hwee K; Yuan, Ching; Kaufman, Stephen C

2016-10-01

To evaluate the use of human platelet lysate (HPL) as an alternative supplement for limbal explant culture. Culture media were prepared using either 10% pooled HPL (PHPL), single donor HPL, or fetal bovine serum (FBS). Limbal tissues, obtained from the Minnesota Lions Eye Bank, were cultured in each medium on plastic plates or on denuded amniotic membrane (AM). Immunofluorescence staining was performed for ABCG2, tumor protein p63α, and cytokeratin 3 (K3). Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of ABCG2 and p63. Limbal explants grown in each medium were labeled with bromodeoxyuridine (BrdU) to assess the proliferative capacity in each medium. Concentration of growth factors including epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and platelet derived growth factor (PDGF) in HPL and PHPL was compared to that in human serum (HS). Immunofluorescence staining on AM showed prominent expression of ABCG2, p63α but sparse expression of K3 in HPL and PHPL supplemented medium. Real time-PCR showed 1.7 fold higher expression of ABCG2 in PHPL supplemented medium (p = 0.03), and similar expression of p63 in HPL and PHPL supplemented medium compared to FBS medium. The proliferation assay showed that LSCs retained their proliferative potential in HPL supplemented medium. Higher concentration of growth factors were found in HPL, compared to HS. Human platelet lysate has higher concentration of grown factors and is effective in maintaining growth and stem cell phenotype of corneal limbal explant cultures.

FRET microscopy autologous tumor lysate processing in mature dendritic cell vaccine therapy

Directory of Open Access Journals (Sweden)

Ridolfi Ruggero

2010-06-01

Full Text Available Abstract Background Antigen processing by dendritic cells (DC exposed to specific stimuli has been well characterized in biological studies. Nonetheless, the question of whether autologous whole tumor lysates (as used in clinical trials are similarly processed by these cells has not yet been resolved. Methods In this study, we examined the transfer of peptides from whole tumor lysates to major histocompatibility complex class II molecules (MHC II in mature dendritic cells (mDC from a patient with advanced melanoma. Tumor antigenic peptides-MHC II proximity was revealed by Förster Resonance Energy Transfer (FRET measurements, which effectively extends the application of fluorescence microscopy to the molecular level ( Results We detected significant energy transfer between donor and acceptor-labelled antibodies against HLA-DR at the membrane surface of mDC. FRET data indicated that fluorescent peptide-loaded MHC II molecules start to accumulate on mDC membranes at 16 hr from the maturation stimulus, steeply increasing at 22 hr with sustained higher FRET detected up to 46 hr. Conclusions The results obtained imply that the patient mDC correctly processed the tumor specific antigens and their display on the mDC surface may be effective for several days. These observations support the rationale for immunogenic efficacy of autologous tumor lysates.

Platelet lysate-based pro-angiogenic nanocoatings.

Science.gov (United States)

Oliveira, Sara M; Pirraco, Rogério P; Marques, Alexandra P; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

2016-03-01

Human platelet lysate (PL) is a cost-effective and human source of autologous multiple and potent pro-angiogenic factors, such as vascular endothelial growth factor A (VEGF A), fibroblast growth factor b (FGF b) and angiopoietin-1. Nanocoatings previously characterized were prepared by layer-by-layer assembling incorporating PL with marine-origin polysaccharides and were shown to activate human umbilical vein endothelial cells (HUVECs). Within 20 h of incubation, the more sulfated coatings induced the HUVECS to the form tube-like structures accompanied by an increased expression of angiogenic-associated genes, such as angiopoietin-1 and VEGF A. This may be a cost-effective approach to modify 2D/3D constructs to instruct angiogenic cells towards the formation of neo-vascularization, driven by multiple and synergistic stimulations from the PL combined with sulfated polysaccharides. The presence, or fast induction, of a stable and mature vasculature inside 3D constructs is crucial for new tissue formation and its viability. This has been one of the major tissue engineering challenges, limiting the dimensions of efficient tissue constructs. Many approaches based on cells, growth factors, 3D bioprinting and channel incorporation have been proposed. Herein, we explored a versatile technique, layer-by-layer assembling in combination with platelet lysate (PL), that is a cost-effective source of many potent pro-angiogenic proteins and growth factors. Results suggest that the combination of PL with sulfated polyelectrolytes might be used to introduce interfaces onto 2D/3D constructs with potential to induce the formation of cell-based tubular structures. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

USE OF BACTERIAL LYSATES IN COMPLEX TREATMENT OF RHINOSINUSITIDIS

Directory of Open Access Journals (Sweden)

E.A. Vishneva

2011-01-01

Full Text Available Rhinosinusitis is a common term that means inflammation of the mucosa of paranasal and nasal cavities. In the last 10 years there was a three-fold increase in rhinosinusitis’ morbidity rates and annual increase of hospital admissions on the average of 1,5–2%. The prevalence of various forms of rhinosinusitidis among adult population is 5 to 15% and 5% among children. Such ubiquitous spreading is one of the leading factors that stipulate socio-economic burden of the disease. According to various studies, rhinosinusitidis dramatically decrease quality of life: these patients show more inferior results in tests upon pain sensitivity and social activity in comparison with patients with coronary heart disease and COPD; in approximately 26% of patients rhinosinusitidis are followed by development or progression of depression. Rhinosinusitis treatment is one of the hottest issues in modern otorhinolaryngology, that demand more financial support with every year. Achievments in immunology have become grounds for immunocorrection as part of complex treatment of such patients. Bacterial lysates are used as preventive medications.Key words: rhinosinusitidis, immunomodulators, bacterial lysates. (Voprosy sovremennoi pediatrii — Current Pediatrics. — 2011; 10 (6: 157–161

Performances of the Alpha-X RF gun on the PHIL accelerator at LAL

Science.gov (United States)

Vinatier, T.; Bruni, C.; Roux, R.; Brossard, J.; Chancé, S.; Cayla, J. N.; Chaumat, V.; Xu, G.; Monard, H.

2015-10-01

The Alpha-X RF-gun was designed to produce an ultra-short (laser-driven plasma accelerator with a short wavelength accelerating medium. It has been demonstrated on PHIL (Photo-Injector at LAL) that the coaxial RF coupling, chosen to preserve the gun field cylindrical symmetry, is perfectly understood and allows reaching the required peak accelerating field of 100 MV/m giving beam energy of 6.3 MeV. Moreover, a quite low beam rms relative energy spread of 0.15% at 3.8 MeV has been measured, completely agreeing with simulations. Dark current, quantum efficiencies and dephasing curves measurements have also been performed. They all show high values of the field enhancement factor β, which can be explained by the preparation of the photocathodes. Finally, measurements on the transverse phase-space have been carried out, with some limitations given by the difficult modelization of one of the PHIL solenoid magnets and by the enlargement of the beam transverse dimensions due to the use of YAG screens. These measurements give a normalized rms transverse emittance around 5π mm mrad, which does not fulfill the requirement for the Alpha-X project.

Analysis of Reparative Activity of Platelet Lysate: Effect on Cell Monolayer Recovery In Vitro and Skin Wound Healing In Vivo.

Science.gov (United States)

Sergeeva, N S; Shanskii, Ya D; Sviridova, I K; Karalkin, P A; Kirsanova, V A; Akhmedova, S A; Kaprin, A D

2016-11-01

Platelet lysate prepared from donor platelet concentrate and pooled according to a developed technique stimulates migration of multipotent mesenchymal stromal cells of the human adipose tissue and promotes healing of the monolayer defect in cultures of human fibroblasts and multipotent mesenchymal stromal cells in vitro in concentrations close those of fetal calf serum (5-10%). Lysate of platelets from platelet-rich rat blood plasma stimulated healing of the skin defect by promoting epithelialization and granulation tissue formation. The regenerative properties of platelet lysate in vivo increased with increasing its concentration.

A new scintillation proximity assay-based approach for the detection of KRAS mutations

Energy Technology Data Exchange (ETDEWEB)

Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee [Korea Atomic Energy Research Institute (KAERI), Daejeon (Korea, Republic of). Radioisotope Research Div.

2016-04-01

KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with {sup 125}I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

A new scintillation proximity assay-based approach for the detection of KRAS mutations

International Nuclear Information System (INIS)

Lee, So-Young; Lim, Jae-Cheong; Cho, Eun-Ha; Jung, Sung-Hee

2016-01-01

KRAS is very commonly mutated resulting in a constitutively activated protein, which is independent of epidermal growth factor receptor (EGFR) ligand binding and resistant to anti-EGFR therapy. Although KRAS is frequently studied, there is still no uniform standard for detecting of KRAS mutations. In this report, a new scintillation proximity assay-based approach is described that determines the relative affinities of wild-type and mutated KRAS to the anti-KRAS antibody. We performed in vitro experiments using normal human colonic cells (CCD18Co), KRAS wild type (Caco-2) and KRAS mutant (HCT 116) cell lines to determine the relative affinities of wild type or mutated KRAS toward an anti-KRAS monoclonal antibody. The process consists of two primary steps: immunoprecipitation from cell lysate to enrich the KRAS protein and the scintillation proximity assay of the immunoprecipitant to determine the relative affinity against the antibody. A fixed concentration of cell lysates was purified by the immunoprecipitation method. The expressions of the KRAS protein in all cell lines was quantitatively confirmed by western blot analysis. For the scintillation proximity assay, the KRAS standard protein was radiolabeled with 125 I by a simple mixing process in the iodogen tube immediately at room temperature immediately before use. The obtained CPM (count per minute) values of were used to calculate the KRAS concentration using purified KRAS as the standard. The calculated relative affinities of 7 μg of Caco-2 and HCT 116 immunoprecipitants for the anti-KRAS antibody were 77 and 0%, respectively. The newly developed scintillation proximity assay-based strategy determines the relative affinities of wild-type or mutated KRAS towards the anti-KRAS monoclonal antibody. This determination can help distinguish mutated KRAS from the wild type protein. The new SPA based approach for detecting KRAS mutations is applicable to many other cancer-related mutations.

Tumour cell lysate-loaded dendritic cell vaccine induces biochemical and memory immune response in castration-resistant prostate cancer patients.

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Reyes, D; Salazar, L; Espinoza, E; Pereda, C; Castellón, E; Valdevenito, R; Huidobro, C; Inés Becker, M; Lladser, A; López, M N; Salazar-Onfray, F

2013-09-17

Recently, we produced a tumour antigen-presenting cells (TAPCells) vaccine using a melanoma cell lysate, called TRIMEL, as an antigen source and an activation factor. Tumour antigen-presenting cells induced immunological responses and increased melanoma patient survival. Herein, we investigated the effect of TAPCells loaded with prostate cancer cell lysates (PCCL) as an antigen source, and TRIMEL as a dendritic cell (DC) activation factor; which were co-injected with the Concholepas concholepas haemocyanin (CCH) as an adjuvant on castration-resistant prostate cancer (CRPC) patients. The lysate mix capacity, for inducing T-cell activation, was analysed by flow cytometry and Elispot. Delayed-type hypersensitivity (DTH) reaction against PCCL, frequency of CD8(+) memory T cells (Tm) in blood and prostate-specific antigen (PSA) levels in serum were measured in treated patients. The lysate mix induced functional mature DCs that were capable of activating PCCL-specific T cells. No relevant adverse reactions were observed. Six out of 14 patients showed a significant decrease in levels of PSA. DTH(+) patients showed a prolonged PSA doubling-time after treatment. Expansion of functional central and effector CD8(+) Tm were detected. Treatment of CRPC patients with lysate-loaded TAPCells and CCH as an adjuvant is safe: generating biochemical and memory immune responses. However, the limited number of cases requires confirmation in a phase II clinical trial.

Assessment of bone healing ability of calcium phosphate cements loaded with platelet lysate in rat calvarial defects.

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Babo, Pedro S; Carvalho, Pedro P; Santo, Vítor E; Faria, Susana; Gomes, Manuela E; Reis, Rui L

2016-11-01

Injectable calcium phosphate cements have been used as a valid alternative to autologous bone grafts for bone augmentation with the additional advantage of enabling minimally invasive implantation procedures and for perfectly fitting the tissue defect. Nevertheless, they have low biodegradability and lack adequate biochemical signaling to promote bone healing and remodeling. In previous in vitro studies, we observed that the incorporation of platelet lysate directly into the cement paste or loaded in hyaluronic acid microspheres allowed to modulate the cement degradation and the in vitro expression of osteogenic markers in seeded human adipose derived stem cells. The present study aimed at investigating the possible effect of this system in new bone formation when implanted in calvarial bilateral defects in rats. Different formulations were assessed, namely plain calcium phosphate cements, calcium phosphate cements loaded with human platelet lysate, hybrid injectable formulations composed of the calcium phosphate cement incorporating hyaluronin acid non-loaded microparticles (20% hyaluronin acid) or with particles loaded with platelet lysate. The degradability and new bone regrowth were evaluated in terms of mineral volume in the defect, measured by micro-computed tomography and histomorphometric analysis upon 4, 8 and 12 weeks of implantation. We observed that the incorporation of hyaluronin acid microspheres induced an overly rapid cement degradation, impairing the osteoconductive properties of the cement composites. Moreover, the incorporation of platelet lysate induced higher bone healing than the materials without platelet lysate, up to four weeks after surgery. Nevertheless, this effect was not found to be significant when compared to the one observed in the sham-treated group. © The Author(s) 2016.

Topical gel formulation and stability assessment of platelet lysate based on turbidimetric method

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Soliman Mohammadi Samani

2015-06-01

Full Text Available Platelet-rich growth factors have attracted attentions of scientists and clinical practitioners who are involved in wound healing and regenerative medicine extensively, according to their unprecedented potential of promoting and catalyzing healing process. Platelet-rich growth factors are cost-benefit, available and more stable than recombinant human growth factors. These appealing characteristics have converted PRGF to one of the popular candidates for treatment of variety of wounds. According to these valuable properties, we decided to formulate and assess the effect of different excipients on the stability of such valuable protein based formulations. Different excipients have been chosen according to their effective ness on the stability of proteins and their application in other similar formulations. The stabilizing effect of excipients was evaluated by measuring heat-induced aggregation of growth factors by turbidimetric assay. Glycerol, glycine and dextrose were chosen as stabilizing excipients for these formulations. The results show that dextrose has more stabilizing effect on prevention of heat induced aggregation of the platelet lysate growth factors than glycerol and glycine. All of the formulations also contained antioxidant, chelating agents, preservative and carbopol934 in order to form appropriate gel.

Intranasal Coadministration of the Cry1Ac Protoxin with Amoebal Lysates Increases Protection against Naegleria fowleri Meningoencephalitis

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Rojas-Hernández, Saúl; Rodríguez-Monroy, Marco A.; López-Revilla, Rubén; Reséndiz-Albor, Aldo A.; Moreno-Fierros, Leticia

2004-01-01

Cry1Ac protoxin has potent mucosal and systemic adjuvant effects on antibody responses to proteins or polysaccharides. In this work, we examined whether Cry1Ac increased protective immunity against fatal Naegleria fowleri infection in mice, which resembles human primary amoebic meningoencephalitis. Higher immunoglobulin G (IgG) than IgA anti-N. fowleri responses were elicited in the serum and tracheopulmonary fluids of mice immunized by the intranasal or intraperitoneal route with N. fowleri lysates either alone or with Cry1Ac or cholera toxin. Superior protection against a lethal challenge with 5 × 104 live N. fowleri trophozoites was achieved for immunization by the intranasal route. Intranasal immunization of N. fowleri lysates coadministered with Cry1Ac increased survival to 100%; interestingly, immunization with Cry1Ac alone conferred similar protection to that achieved with amoebal lysates alone (60%). When mice intranasally immunized with Cry1Ac plus lysates were challenged with amoebae, both IgG and IgA mucosal responses were rapidly increased, but only the increased IgG response persisted until day 60 in surviving mice. The brief rise in the level of specific mucosal IgA does not exclude the role that this isotype may play in the early defense against this parasite, since higher IgA responses were detected in nasal fluids of mice intranasally immunized with lysates plus either Cry1Ac or cholera toxin, which, indeed, were the treatments that provided the major protection levels. In contrast, serum antibody responses do not seem to be related to the protection level achieved. Both acquired and innate immune systems seem to play a role in host defense against N. fowleri infection, but further studies are required to elucidate the mechanisms involved in protective effects conferred by Cry1Ac, which may be a valuable tool to improve mucosal vaccines. PMID:15271892

Implementation of a Multiplex and Quantitative Proteomics Platform for Assessing Protein Lysates Using DNA-Barcoded Antibodies.

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Lee, Jinho; Geiss, Gary K; Demirkan, Gokhan; Vellano, Christopher P; Filanoski, Brian; Lu, Yiling; Ju, Zhenlin; Yu, Shuangxing; Guo, Huifang; Bogatzki, Lisa Y; Carter, Warren; Meredith, Rhonda K; Krishnamurthy, Savitri; Ding, Zhiyong; Beechem, Joseph M; Mills, Gordon B

2018-06-01

Molecular analysis of tumors forms the basis for personalized cancer medicine and increasingly guides patient selection for targeted therapy. Future opportunities for personalized medicine are highlighted by the measurement of protein expression levels via immunohistochemistry, protein arrays, and other approaches; however, sample type, sample quantity, batch effects, and "time to result" are limiting factors for clinical application. Here, we present a development pipeline for a novel multiplexed DNA-labeled antibody platform which digitally quantifies protein expression from lysate samples. We implemented a rigorous validation process for each antibody and show that the platform is amenable to multiple protocols covering nitrocellulose and plate-based methods. Results are highly reproducible across technical and biological replicates, and there are no observed "batch effects" which are common for most multiplex molecular assays. Tests from basal and perturbed cancer cell lines indicate that this platform is comparable to orthogonal proteomic assays such as Reverse-Phase Protein Array, and applicable to measuring the pharmacodynamic effects of clinically-relevant cancer therapeutics. Furthermore, we demonstrate the potential clinical utility of the platform with protein profiling from breast cancer patient samples to identify molecular subtypes. Together, these findings highlight the potential of this platform for enhancing our understanding of cancer biology in a clinical translation setting. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Absence of micronucleus formation in CHO-K1 cells cultivated in platelet lysate enriched medium.

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Bernardi, Martina; Adami, Valentina; Albiero, Elena; Madeo, Domenico; Rodeghiero, Francesco; Astori, Giuseppe

2014-03-01

Human platelet lysate (PL) represents an effective substitute of fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) cultivation. Compared to FBS, PL favors MSC proliferation significantly shortening the population doubling time and avoiding the risks related to the use of animal derivatives. Growth factors contained in the platelets are released upon platelet disruption following freezing/thawing cycles or as we have recently described by using ultrasound. We have investigated whether the increased cell proliferation achieved by using PL could induce mitotic stress and whether the potential formation of free radicals during PL production by ultrasound could cause chromosomal instability in mammalian cells. We have applied an image analysis assisted high content screening (HCS) in vitro micronucleus assay in the Chinese Hamster Ovarian K1 (CHO-K1) rodent mammalian cell line. PL was produced by sonication; for the micronucleus assay, CHO-K1 cells were exposed to increasing concentrations of PL. Cytokinesis was blocked by cytochalasin B, nuclei were stained with bisbenzimide and images were acquired and analyzed automatically using an HCS system, both with a 20× and a 10× objective. Our results suggest that growth stimulus induced by the use of PL did not significantly increase micronucleus formation in CHO-K1 cells compared to negative control. Micronucleus testing in conjunction with HCS could represent a valid tool to evaluate the safety of ancillary materials used in the production of cell-based medicinal products. Copyright © 2013 Elsevier GmbH. All rights reserved.

Electrospun silk fibroin fibers for storage and controlled release of human platelet lysate.

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Pignatelli, Cataldo; Perotto, Giovanni; Nardini, Marta; Cancedda, Ranieri; Mastrogiacomo, Maddalena; Athanassiou, Athanassia

2018-04-17

Human platelet lysate (hPL) is a pool of growth factors and cytokines able to induce regeneration of different tissues. Despite its good potentiality as therapeutic tool for regenerative medicine applications, hPL has been only moderately exploited in this field. A more widespread adoption has been limited because of its rapid degradation at room temperature that decreases its functionality. Another limiting factor for its extensive use is the difficulty of handling the hPL gels. In this work, silk fibroin-based patches were developed to address several points: improving the handling of hPL, enabling their delivery in a controlled manner and facilitating their storage by creating a device ready to use with expanded shelf life. Patches of fibroin loaded with hPL were synthesized by electrospinning to take advantage of the fibrous morphology. The release kinetics of the material was characterized and tuned through the control of fibroin crystallinity. Cell viability assays, performed with primary human dermal fibroblasts, demonstrated that fibroin is able to preserve the hPL biological activity and prolong its shelf-life. The strategy of storing and preserving small active molecules within a naturally-derived, protein-based fibrous scaffold was successfully implemented, leading to the design of a biocompatible device, which can potentially simplify the storage and the application of the hPL on a human patient, undergoing medical procedures such as surgery and wound care. Human platelets lysate (hPL) is a mixture of growth factors and cytokines able to induce the regeneration of damaged tissues. This study aims at enclosing hPL in a silk fibroin electrospun matrix to expand its utilization. Silk fibroin showed the ability to preserve the hPL activity at temperature up to 60 °C and the manipulation of fibroin's crystallinity provided a tool to modulate the hPL release kinetic. This entails the possibility to fabricate the hPL silk fibroin patches in advance and

SWATH Mass Spectrometry Performance Using Extended Peptide MS/MS Assay Libraries*

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Wu, Jemma X.; Song, Xiaomin; Pascovici, Dana; Zaw, Thiri; Care, Natasha; Krisp, Christoph; Molloy, Mark P.

2016-01-01

The use of data-independent acquisition methods such as SWATH for mass spectrometry based proteomics is usually performed with peptide MS/MS assay libraries which enable identification and quantitation of peptide peak areas. Reference assay libraries can be generated locally through information dependent acquisition, or obtained from community data repositories for commonly studied organisms. However, there have been no studies performed to systematically evaluate how locally generated or repository-based assay libraries affect SWATH performance for proteomic studies. To undertake this analysis, we developed a software workflow, SwathXtend, which generates extended peptide assay libraries by integration with a local seed library and delivers statistical analysis of SWATH-quantitative comparisons. We designed test samples using peptides from a yeast extract spiked into peptides from human K562 cell lysates at three different ratios to simulate protein abundance change comparisons. SWATH-MS performance was assessed using local and external assay libraries of varying complexities and proteome compositions. These experiments demonstrated that local seed libraries integrated with external assay libraries achieve better performance than local assay libraries alone, in terms of the number of identified peptides and proteins and the specificity to detect differentially abundant proteins. Our findings show that the performance of extended assay libraries is influenced by the MS/MS feature similarity of the seed and external libraries, while statistical analysis using multiple testing corrections increases the statistical rigor needed when searching against large extended assay libraries. PMID:27161445

Lysate of engineered Escherichia coli supports high-level conversion of glucose to 2,3-butanediol.

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Kay, Jennifer E; Jewett, Michael C

2015-11-01

Cell-free metabolic engineering (CFME) is emerging as a powerful approach for the production of target molecules and pathway debugging. Unfortunately, high cofactor costs, limited cofactor and energy regeneration, and low volumetric productivities hamper the widespread use and practical implementation of CFME technology. To address these challenges, we have developed a cell-free system that harnesses ensembles of catalytic proteins prepared from crude lysates, or extracts, of cells to fuel highly active heterologous metabolic conversions. As a model pathway, we selected conversion of glucose to 2,3-butanediol (2,3-BD), a medium level commodity chemical with many industrial applications. Specifically, we engineered a single strain of Escherichia coli to express three pathway enzymes necessary to make meso-2,3-BD (m2,3-BD). We then demonstrated that lysates from this strain, with addition of glucose and catalytic amounts of cofactors NAD+ and ATP, can produce m2,3-BD. Endogenous glycolytic enzymes convert glucose to pyruvate, the starting intermediate for m2,3-BD synthesis. Strikingly, with no strain optimization, we observed a maximal synthesis rate of m2,3-BD of 11.3 ± 0.1 g/L/h with a theoretical yield of 71% (0.36 g m2,3-BD/g glucose) in batch reactions. Titers reached 82 ± 8 g/L m2,3-BD in a 30 h fed-batch reaction. Our results highlight the ability for high-level co-factor regeneration in cell-free lysates. Further, they suggest exciting opportunities to use lysate-based systems to rapidly prototype metabolic pathways and carry out molecular transformations when bioconversion yields (g product/L), productivities (g product/L/h), or cellular toxicity limit commercial feasibility of whole-cell fermentation. Copyright © 2015 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Platelet lysate mucohadesive formulation to treat oral mucositis in graft versus host disease patients: a new therapeutic approach.

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Del Fante, Claudia; Perotti, Cesare; Bonferoni, Maria Cristina; Rossi, Silvia; Sandri, Giuseppina; Ferrari, Franca; Scudeller, Luigia; Caramella, Carla Marcella

2011-09-01

Optimal treatment of oral mucositis (OM) due to graft versus host disease (GvHD) is currently not available. Platelet-derived growth factors (PDGFs) have high capability for tissue healing and may play a role in repairing the mucosal barrier. The aim of the present work was to develop a mucoadhesive formulation to administer platelet lysate to oral cavity prolonging contact time of platelet lysate with oral mucosa. The mucoadhesive formulation was characterized for in vitro properties (PDGF-AB concentration, mucoadhesive properties, cytotoxicity, fibroblast proliferation, wound healing). Moreover, a preliminary clinical study on seven GvHD patients with OM refractory to other therapies was conducted, to evaluate feasibility, safety, and efficacy. GVPL (mucoadhesive gel vehicle mixed with platelet lysate)showed good mucoadhesive properties; additionally, it was characterized by good biocompatibility in vitro on fibroblasts and it was able to enhance fibroblast proliferation and wound healing, maintaining the efficacy for up to 14 days following storage at 2-8°C. In vivo, clinical response was good-to-complete in five, fair in one, none in the remaining one. The in vitro results indicate that GVPL has optimal mucoadhesive and healing enhancer properties, maintained over time (up to 14 days); preliminary clinical results suggest that oral application of platelet lysate-loaded mucoadhesive formulation is feasible, safe, well tolerated, and effective. A larger controlled randomized study is needed.

Rosette Assay: Highly Customizable Dot-Blot for SH2 Domain Screening.

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Ng, Khong Y; Machida, Kazuya

2017-01-01

With a growing number of high-throughput studies, structural analyses, and availability of protein-protein interaction databases, it is now possible to apply web-based prediction tools to SH2 domain-interactions. However, in silico prediction is not always reliable and requires experimental validation. Rosette assay is a dot blot-based reverse-phase assay developed for the assessment of binding between SH2 domains and their ligands. It is conveniently customizable, allowing for low- to high-throughput analysis of interactions between various numbers of SH2 domains and their ligands, e.g., short peptides, purified proteins, and cell lysates. The binding assay is performed in a 96-well plate (MBA or MWA apparatus) in which a sample spotted membrane is incubated with up to 96 labeled SH2 domains. Bound domains are detected and quantified using a chemiluminescence or near-infrared fluorescence (IR) imaging system. In this chapter, we describe a practical protocol for rosette assay to assess interactions between synthesized tyrosine phosphorylated peptides and a library of GST-tagged SH2 domains. Since the methodology is not confined to assessment of SH2-pTyr interactions, rosette assay can be broadly utilized for ligand and drug screening using different protein interaction domains or antibodies.

A novel whole-cell lysate kinase assay identifies substrates of the p38 MAPK in differentiating myoblasts

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Knight James DR

2012-03-01

Full Text Available Abstract Background The p38α mitogen-activated protein kinase (MAPK is a critical mediator of myoblast differentiation, and does so in part through the phosphorylation and regulation of several transcription factors and chromatin remodelling proteins. However, whether p38α is involved in processes other than gene regulation during myogenesis is currently unknown, and why other p38 isoforms cannot compensate for its loss is unclear. Methods To further characterise the involvement of p38α during myoblast differentiation, we developed and applied a simple technique for identifying relevant in vivo kinase substrates and their phosphorylation sites. In addition to identifying substrates for one kinase, the technique can be used in vitro to compare multiple kinases in the same experiment, and we made use of this to study the substrate specificities of the p38α and β isoforms. Results Applying the technique to p38α resulted in the identification of seven in vivo phosphorylation sites on six proteins, four of which are cytoplasmic, in lysate derived from differentiating myoblasts. An in vitro comparison with p38β revealed that substrate specificity does not discriminate these two isoforms, but rather that their distinguishing characteristic appears to be cellular localisation. Conclusion Our results suggest p38α has a novel cytoplasmic role during myogenesis and that its unique cellular localisation may be why p38β and other isoforms cannot compensate for its absence. The substrate-finding approach presented here also provides a necessary tool for studying the hundreds of protein kinases that exist and for uncovering the deeper mechanisms of phosphorylation-dependent cell signalling.

Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture

International Nuclear Information System (INIS)

Yoshito, Daniele

2011-01-01

For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

A midgut lysate of the Riptortus pedestris has antibacterial activity against LPS O-antigen-deficient Burkholderia mutants.

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Jang, Ho Am; Seo, Eun Sil; Seong, Min Young; Lee, Bok Luel

2017-02-01

Riptortus pedestris, a common pest in soybean fields, harbors a symbiont Burkholderia in a specialized posterior midgut region of insects. Every generation of second nymphs acquires new Burkholderia cells from the environment. We compared in vitro cultured Burkholderia with newly in vivo colonized Burkholderia in the host midgut using biochemical approaches. The bacterial cell envelope of in vitro cultured and in vivo Burkholderia differed in structure, as in vivo bacteria lacked lipopolysaccharide (LPS) O-antigen. The LPS O-antigen deficient bacteria had a reduced colonization rate in the host midgut compared with that of the wild-type Burkholderia. To determine why LPS O-antigen-deficient bacteria are less able to colonize the host midgut, we examined in vitro survival rates of three LPS O-antigen-deficient Burkholderia mutants and lysates of five different midgut regions. The LPS O-antigen-deficient mutants were highly susceptible when cultured with the lysate of a specific first midgut region (M1), indicating that the M1 lysate contains unidentified substance(s) capable of killing LPS O-antigen-deficient mutants. We identified a 17 kDa protein from the M1 lysate, which was enriched in the active fractions. The N-terminal sequence of the protein was determined to be a soybean Kunitz-type trypsin inhibitor. These data suggest that the 17 kDa protein, which was originated from a main soybean source of the R. pedestris host, has antibacterial activity against the LPS O-antigen deficient (rough-type) Burkholderia. Copyright © 2016 Elsevier Ltd. All rights reserved.

Identification of heme oxygenase-1 stimulators by a convenient ELISA-based bilirubin quantification assay.

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Rücker, Hannelore; Amslinger, Sabine

2015-01-01

The upregulation of heme oxygenase-1 (HO-1) has proven to be a useful tool for fighting inflammation. In order to identify new HO-1 inducers, an efficient screening method was developed which can provide new lead structures for drug research. We designed a simple ELISA-based HO-1 enzyme activity assay, which allows for the screening of 12 compounds in parallel in the setting of a 96-well plate. The well-established murine macrophage cell line RAW264.7 is used and only about 26µg of protein from whole cell lysates is needed for the analysis of HO-1 activity. The quantification of HO-1 activity is based on an indirect ELISA using the specific anti-bilirubin antibody 24G7 to quantify directly bilirubin in the whole cell lysate, applying a horseradish peroxidase-tagged antibody together with ortho-phenylenediamine and H2O2 for detection. The bilirubin is produced on the action of HO enzymes by converting their substrate heme to biliverdin and additional recombinant biliverdin reductase together with NADPH at pH 7.4 in buffer. This sensitive assay allows for the detection of 0.57-82pmol bilirubin per sample in whole cell lysates. Twenty-three small molecules, mainly natural products with an α,β-unsaturated carbonyl unit such as polyphenols, including flavonoids and chalcones, terpenes, an isothiocyanate, and the drug oltipraz were tested at typically 6 or 24h incubation with RAW264.7 cells. The activity of known HO-1 inducers was confirmed, while the chalcones cardamonin, flavokawain A, calythropsin, 2',3,4'-trihydroxy-4-methoxychalcone (THMC), and 2',4'-dihydroxy-3,4-dimethoxychalcone (DHDMC) were identified as new potent HO-1 inducers. The highest inductive power after 6h incubation was found at 10µM for DHDMC (6.1-fold), carnosol (3.9-fold), butein (3.1-fold), THMC (2.9-fold), and zerumbone (2.5-fold). Moreover, the time dependence of HO-1 protein production for DHDMC was compared to its enzyme activity, which was further evaluated in the presence of

Platelet lysate enhances synovial fluid multipotential stromal cells functions: Implications for therapeutic use.

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Altaie, Ala; Baboolal, Thomas G; Wall, Owen; Jones, Elena; McGonagle, Dennis

2018-03-01

Although intra-articular injection of platelet products is increasingly used for joint regenerative approaches, there are few data on their biological effects on joint-resident multipotential stromal cells (MSCs), which are directly exposed to the effects of these therapeutic strategies. Therefore, this study investigated the effect of platelet lysate (PL) on synovial fluid-derived MSCs (SF-MSCs), which in vivo have direct access to sites of cartilage injury. SF-MSCs were obtained during knee arthroscopic procedures (N = 7). Colony forming unit-fibroblast (CFU-F), flow-cytometric phenotyping, carboxyfluorescein succinimidyl ester-based immunomodulation for T-cell and trilineage differentiation assays were performed using PL and compared with standard conditions. PL-enhanced SF-MSC (PL-MSC) proliferation as CFU-F colonies was 1.4-fold larger, and growing cultures had shorter population-doubling times. PL-MSCs and fetal calf serum (FCS)-MSCs had the same immunophenotype and similar immunomodulation activities. In chondrogenic and osteogenic differentiation assays, PL-MSCs produced 10% more sulfated-glycosaminoglycan (sGAG) and 45% less Ca ++ compared with FCS-MSCs, respectively. Replacing chondrogenic medium transforming growth factor-β3 with 20% or 50% PL further increased sGAG production of PL-MSCs by 69% and 95%, respectively, compared with complete chondrogenic medium. Also, Dulbecco's Modified Eagle's Medium high glucose (HG-DMEM) plus 50% PL induced more chondrogenesis compared with HG-DMEM plus 10% FCS and was comparable to complete chondrogenic medium. This is the first study to assess SF-MSC responses to PL and provides biological support to the hypothesis that PL may be capable of modulating multiple functional aspects of joint resident MSCs with direct access to injured cartilage. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Vaccination with OK-432 followed by TC-1 tumor lysate leads to significant antitumor effects.

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Chen, I-Ju; Yen, Chih-Feng; Lin, Kun-Ju; Lee, Chyi-Long; Soong, Yung-Kuei; Lai, Chyong-Huey; Lin, Cheng-Tao

2011-07-01

Human papillomavirus (HPV) infects large numbers of women worldwide and is present in more than 99% of all cervical cancer. TC-1 cell is a cell line with high expression of E7 antigen of HPV type 16 and its cell lysate has been demonstrated as an ideal inducer of E7-specific, antitumor immunity. OK-432 (Picibanil), a penicillin-killed Streptococcus pyogenes, has been reported with potent immunomodulation properties in cancer treatment by stimulating the maturation of dendritic cells (DCs) and secretion of Th-1 type cytokines. The current study demonstrated that a protocol to immunize the C57BL/6 mice with OK-432 followed by treatment with TC-1 lysate can generate markedly increased immune responses of E7-specific CD4(+) T cells and a moderate increase of natural killer (NK) cell, as well as a satisfactorily protective and therapeutic antitumor effect by triggering the DCs to prime T cells. Depletion of lymphocyte subset in vivo suggested that the antitumor effects could be dominantly executed by CD8+ T cells and followed by NK cells, and both of these reactions were induced by the generation of robust E7-specific CD4(+) T helper cell response. These findings warrant OK-432 combination with tumor-lysate as an effective and safe vaccine in future clinical application of cervical cancer.

Human platelet lysate: Replacing fetal bovine serum as a gold standard for human cell propagation?

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Burnouf, Thierry; Strunk, Dirk; Koh, Mickey B C; Schallmoser, Katharina

2016-01-01

The essential physiological role of platelets in wound healing and tissue repair builds the rationale for the use of human platelet derivatives in regenerative medicine. Abundant growth factors and cytokines stored in platelet granules can be naturally released by thrombin activation and clotting or artificially by freeze/thaw-mediated platelet lysis, sonication or chemical treatment. Human platelet lysate prepared by the various release strategies has been established as a suitable alternative to fetal bovine serum as culture medium supplement, enabling efficient propagation of human cells under animal serum-free conditions for a multiplicity of applications in advanced somatic cell therapy and tissue engineering. The rapidly increasing number of studies using platelet derived products for inducing human cell proliferation and differentiation has also uncovered a considerable variability of human platelet lysate preparations which limits comparability of results. The main variations discussed herein encompass aspects of donor selection, preparation of the starting material, the possibility for pooling in plasma or additive solution, the implementation of pathogen inactivation and consideration of ABO blood groups, all of which can influence applicability. This review outlines the current knowledge about human platelet lysate as a powerful additive for human cell propagation and highlights its role as a prevailing supplement for human cell culture capable to replace animal serum in a growing spectrum of applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

The effect of platelet lysate supplementation of a dextran-based hydrogel on cartilage formation

NARCIS (Netherlands)

Moreira Teixeira, Liliana; Leijten, Jeroen Christianus Hermanus; Wennink, J.W.H.; Ganguly, Anindita; Feijen, Jan; van Blitterswijk, Clemens; Dijkstra, Pieter J.; Karperien, Hermanus Bernardus Johannes

2012-01-01

In situ gelating dextran-tyramine (Dex-TA) injectable hydrogels have previously shown promising features for cartilage repair. Yet, despite suitable mechanical properties, this system lacks intrinsic biological signals. In contrast, platelet lysate-derived hydrogels are rich in growth factors and

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization

Directory of Open Access Journals (Sweden)

Yvonne Wanner

2017-01-01

Full Text Available Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL- cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

Platelet Lysate: The Better Choice for Jaw Periosteal Cell Mineralization.

Science.gov (United States)

Wanner, Yvonne; Umrath, Felix; Waidmann, Marc; Reinert, Siegmar; Alexander, Dorothea

2017-01-01

Previously, we demonstrated a high quality of minerals formed by serum-free cultured jaw periosteal cells (JPCs) by Raman spectroscopy but the mineralization extent was not satisfactory. In the present study, we analyzed the proliferation and mineralization potential of human platelet lysate- (hPL-) cultured JPCs in comparison to that of FCS-cultured JPCs. By cell impedance measurements, we detected significantly higher population doubling times of PL-cultured JPCs in comparison to FCS-cultured JPCs. However, this result was not based on lower proliferation activities but on diminished cell sizes which JPCs develop under PL cultivation. The measurements of the metabolic activities clearly showed significantly higher cell proliferation rates under PL culturing. Equivalent levels of the mesenchymal cell markers CD29, CD45, CD73, CD90, and CD105 were detected, but there were significantly increased MSCA-1 levels under PL cultivation. While JPCs only occasionally mineralize under FCS culture conditions, the mineralization potential was significantly stronger under PL cultivation. Moreover, in 4 of 5 analyzed patient cells, the addition of dexamethasone was proved no longer necessary for strong mineralization of PL-cultured JPCs. We conclude that in vitro cultivation of JPCs with platelet lysate is a suitable alternative to FCS culture conditions and a powerful tool for the development of high-quality TE constructs using jaw periosteal cells.

Composition of growth factors and cytokines in lysates obtained from fresh versus stored pathogen-inactivated platelet units.

Science.gov (United States)

Sellberg, Felix; Berglund, Erik; Ronaghi, Martin; Strandberg, Gabriel; Löf, Helena; Sommar, Pehr; Lubenow, Norbert; Knutson, Folke; Berglund, David

2016-12-01

Platelet lysate is a readily available source of growth factors, and other mediators, which has been used in a variety of clinical applications. However, the product remains poorly standardized and the present investigation evaluates the composition of platelet lysate obtained from either fresh or stored pathogen-inactivated platelet units. Platelet pooled units (n = 10) were obtained from healthy blood donors and tested according to standard procedures. All units were pathogen inactivated using amotosalen hydrochloride and UVA exposure. Platelet lysate was subsequently produced at two separate time-points, either from fresh platelet units or after 5 days of storage, by repeated freeze-thaw cycles. The following mediators were determined at each time-point: EGF, FGF-2, VEGF, IGF-1, PDGF-AB/BB, BMP-2, PF4, TGF-β isoform 1, IL-1β, IL-2, IL-6, IL-10, IL-12p70, 1L-17A, TNF-α, and IFN-γ. The concentration of growth factors and cytokines was affected by time in storage. Notably, TGF-β, PDGF-AB/BB, and PF4 showed an increase of 27.2% (p product, which potentially may influence the clinical effects. Copyright © 2016. Published by Elsevier Ltd.

Development of a dual luciferase activity and fluorescamine protein assay adapted to a 384 micro-well plate format: Reducing variability in human luciferase transactivation cell lines aimed at endocrine active substances

Science.gov (United States)

Brennan, Jennifer; Tillitt, Donald E.

2018-01-01

There is a need to adapt cell bioassays to 384-well and 1536-well formats instead of the traditional 96-well format as high-throughput screening (HTS) demands increase. However, the sensitivity and performance of the bioassay must be re-verified in these higher micro-well plates, and verification of cell health must also be HT (high-throughput). We have adapted two commonly used human breast luciferase transactivation cell bioassays, the recently re-named estrogen agonist/antagonist screening VM7Luc4E2 cell bioassay (previously designated BG1Luc4E2) and the androgen/glucocorticoid screening MDA-kb2 cell bioassay, to 384-well formats for HTS of endocrine-active substances (EASs). This cost-saving adaptation includes a fast, accurate, and easy measurement of protein amount in each well via the fluorescamine assay with which to normalize luciferase activity of cell lysates without requiring any transfer of the cell lysates. Here we demonstrate that by accounting for protein amount in the cell lysates, antagonistic agents can easily be distinguished from cytotoxic agents in the MDA-kb2 and VM7Luc4E2 cell bioassays. Additionally, we demonstrate via the fluorescamine assay improved interpretation of luciferase activity in wells along the edge of the plate (the so-called “edge effect”), thereby increasing usable wells to the entire plate, not just interior wells.

Performances of the Alpha-X RF gun on the PHIL accelerator at LAL

Energy Technology Data Exchange (ETDEWEB)

Vinatier, T., E-mail: vinatier@lal.in2p3.fr [Laboratoire de l' Accélérateur Linéaire (LAL), Université Paris Sud, UMR 8607, bâtiment 200, 91898 Orsay Cedex (France); Bruni, C. [Laboratoire de l' Accélérateur Linéaire (LAL), Université Paris Sud, UMR 8607, bâtiment 200, 91898 Orsay Cedex (France); Roux, R. [Laboratoire de l' Accélérateur Linéaire (LAL), Université Paris Sud, UMR 8607, bâtiment 200, 91898 Orsay Cedex (France); Laboratoire d' Etude des Eléments Légers, CEA IRAMIS, bâtiment 524, 91191 Gif sur Yvette Cedex (France); Brossard, J. [Laboratoire de l' Accélérateur Linéaire (LAL), Université Paris Sud, UMR 8607, bâtiment 200, 91898 Orsay Cedex (France); Laboratoire Astroparticule et Cosmologie, Université Paris 7, UMR 7164, bâtiment Condorcet, 75205 Paris Cedex (France); Chancé, S.; Cayla, J.N.; Chaumat, V. [Laboratoire de l' Accélérateur Linéaire (LAL), Université Paris Sud, UMR 8607, bâtiment 200, 91898 Orsay Cedex (France); and others

2015-10-11

The Alpha-X RF-gun was designed to produce an ultra-short (<100 fs rms), 100 pC and 6.3 MeV electron beam with a normalized rms transverse emittance of 1π mm mrad for a gun peak accelerating field of 100 MV/m. Such beams will be required by the Alpha-X project, which aims to study a laser-driven plasma accelerator with a short wavelength accelerating medium. It has been demonstrated on PHIL (Photo-Injector at LAL) that the coaxial RF coupling, chosen to preserve the gun field cylindrical symmetry, is perfectly understood and allows reaching the required peak accelerating field of 100 MV/m giving beam energy of 6.3 MeV. Moreover, a quite low beam rms relative energy spread of 0.15% at 3.8 MeV has been measured, completely agreeing with simulations. Dark current, quantum efficiencies and dephasing curves measurements have also been performed. They all show high values of the field enhancement factor β, which can be explained by the preparation of the photocathodes. Finally, measurements on the transverse phase-space have been carried out, with some limitations given by the difficult modelization of one of the PHIL solenoid magnets and by the enlargement of the beam transverse dimensions due to the use of YAG screens. These measurements give a normalized rms transverse emittance around 5π mm mrad, which does not fulfill the requirement for the Alpha-X project.

A Novel Detection Platform for Shrimp White Spot Syndrome Virus Using an ICP11-Dependent Immunomagnetic Reduction (IMR) Assay.

Science.gov (United States)

Liu, Bing-Hsien; Lin, Yu-Chen; Ho, Chia-Shin; Yang, Che-Chuan; Chang, Yun-Tsui; Chang, Jui-Feng; Li, Chun-Yuan; Cheng, Cheng-Shun; Huang, Jiun-Yan; Lee, Yen-Fu; Hsu, Ming-Hung; Lin, Feng-Chun; Wang, Hao-Ching; Lo, Chu-Fang; Yang, Shieh-Yueh; Wang, Han-Ching

2015-01-01

Shrimp white spot disease (WSD), which is caused by white spot syndrome virus (WSSV), is one of the world's most serious shrimp diseases. Our objective in this study was to use an immunomagnetic reduction (IMR) assay to develop a highly sensitive, automatic WSSV detection platform targeted against ICP11 (the most highly expressed WSSV protein). After characterizing the magnetic reagents (Fe3O4 magnetic nanoparticles coated with anti ICP11), the detection limit for ICP11 protein using IMR was approximately 2 x 10(-3) ng/ml, and the linear dynamic range of the assay was 0.1~1 x 10(6) ng/ml. In assays of ICP11 protein in pleopod protein lysates from healthy and WSSV-infected shrimp, IMR signals were successfully detected from shrimp with low WSSV genome copy numbers. We concluded that this IMR assay targeting ICP11 has potential for detecting the WSSV.

Real-Time Detection and Identification of Chlamydophila Species in Veterinary Specimens by Using SYBR Green-Based PCR Assays

DEFF Research Database (Denmark)

Nordentoft, Steen; Kabell, Susanne; Pedersen, Karl

2011-01-01

of Chlamydiaceae and differentiate the most prevalent veterinary Chlamydophila species: Cp. psittaci, Cp. abortus, Cp. felis, and Cp. caviae. By adding bovine serum albumin to the master mixes, target DNA could be detected directly in crude lysates of enzymatically digested conjunctival or pharyngeal swabs...... or tissue specimens from heart, liver, and spleen without further purification. The assays were evaluated on veterinary specimens where all samples were screened using a family-specific PCR, and positive samples were further tested using species-specific PCRs. Cp. psittaci was detected in 47 birds, Cp...... with a highly sensitive family-specific PCR, we were able to screen for Chlamydiaceae in veterinary specimens and confirm the species in positive samples with additional PCR assays....

Combinations of SPR and MS for Characterizations of Native and Recombinant Proteins in Cell Lysates

DEFF Research Database (Denmark)

Borch, Jonas; Roepstorff, Peter

2006-01-01

Surface plasmon resonance and mass spectrometry (SPR-MS) has been combined for quality check of recombinant 6xHis-tagged 14-3-3 proteins expressed in Escherichia coli. Lysates were injected over an SPR sensorchip with immobilized Ni2+ for SPR analysis of the specific Ni2+ binding response...... and stability. To validate the identity, intactness and homogeneity of the captured proteins were eluted for mass spectrometric analysis of intact molecular weight and peptide mass mapping. Additionally, the captured recombinant proteins were investigated for specific binding to known phosphorylated ligands...... of 14-3-3 proteins in order to test their activity. Specific binding of recombinant and native 14-3-3 proteins in complex mixtures to immobilized phosphopeptides and subsequent elution was also tested by SPR-MS. Ammonium sulfate precipitate fractions from lysates of E. coli expressing 14-3-3 protein...

Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds.

Science.gov (United States)

Bonferoni, Maria Cristina; Sandri, Giuseppina; Rossi, Silvia; Dellera, Eleonora; Invernizzi, Alessandro; Boselli, Cinzia; Cornaglia, Antonia Icaro; Del Fante, Claudia; Perotti, Cesare; Vigani, Barbara; Riva, Federica; Caramella, Carla; Ferrari, Franca

2018-02-09

Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

Association of Alpha Tocopherol and Ag Sulfadiazine Chitosan Oleate Nanocarriers in Bioactive Dressings Supporting Platelet Lysate Application to Skin Wounds

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Maria Cristina Bonferoni

2018-02-01

Full Text Available Chitosan oleate was previously proposed to encapsulate in nanocarriers some poorly soluble molecules aimed to wound therapy, such as the anti-infective silver sulfadiazine, and the antioxidant α tocopherol. Because nanocarriers need a suitable formulation to be administered to wounds, in the present paper, these previously developed nanocarriers were loaded into freeze dried dressings based on chitosan glutamate. These were proposed as bioactive dressings aimed to support the application to wounds of platelet lysate, a hemoderivative rich in growth factors. The dressings were characterized for hydration capacity, morphological aspect, and rheological and mechanical behavior. Although chitosan oleate nanocarriers clearly decreased the mechanical properties of dressings, these remained compatible with handling and application to wounds. Preliminary studies in vitro on fibroblast cell cultures demonstrated good compatibility of platelet lysate with nanocarriers and bioactive dressings. An in vivo study on a murine wound model showed an accelerating wound healing effect for the bioactive dressing and its suitability as support of the platelet lysate application to wounds.

Platelet lysate as an autologous alternative for fetal bovine serum in cardiovascular tissue engineering

NARCIS (Netherlands)

Riem Vis, P.W.; Bouten, C.V.C.; Sluijter, J.P.G.; Herwerden, van L.A.; Kluin, J.

2010-01-01

There is an ongoing search for alternative tissue culture sera to engineer autologous tissues, since use of fetal bovine serum (FBS) is limited under Good Tissue Practice (GTP) guidelines. We compared FBS with human Platelet-lysate (PL) in media for in vitro cell culture. A threefold increase in

Residents’ Awareness of Urban and Social Living Quality: The Example of Prof. Dr. Ahmet Taner Kışlalı Neighborhood in Çayyolu/Ankara

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Ahmet Tanju Gültekin

2015-12-01

Full Text Available Since 1980, the dynamics of globalization have directed the processes of social, cultural, and political adjustment as well as those of economic integration and especially capital towards urban development, forming urban space. The wave of globalization in Turkey, within a process of economic change in the context of neo liberal policies, urban space has been turned into the most profitable area of investment in the unearned income (economic rent sector. The residential sector has been included in this process quickly and easily through effective and widespread marketing strategies. Over the last two decades of this development, the Ankara-Çayyolu District has become segregated economically and socially between its upper and upper-middle class residents in the urban context. This segregation is evident in residential areas with high-level physical and spatial quality. On the other hand, in addition to the measurable physical and spatial standards of housing quality, it is also important to find out if the quality of living meets the social and cultural expectations of its residents. Starting from this point of view, we examine residents’ perceptions of their physical and social environment in our chosen sample area in the Prof. Dr. Ahmet Taner Kışlalı Neighborhood, and their demands and awareness of housing quality. The story of this neighborhood, which begins in 1980, at a time when neo liberal policies and the power to form urban space was all-important, is defined here by the residents’ quality of living and awareness of it. Our results obtained are expected to help guide the creation of liveable housing areas in the future.Keywords: Housing Users, Urban Living Quality, Social Living, Awareness, Ankara- Çayyolu-Prof. Dr. Ahmet Taner Kışlalı Neighborhood.

Overcoming the bottleneck of platelet lysate supply in large-scale clinical expansion of adipose-derived stem cells: A comparison of fresh versus three types of platelet lysates from outdated buffy coat-derived platelet concentrates.

Science.gov (United States)

Glovinski, Peter V; Herly, Mikkel; Mathiasen, Anders B; Svalgaard, Jesper D; Borup, Rehannah; Talman, Maj-Lis M; Elberg, Jens J; Kølle, Stig-Frederik T; Drzewiecki, Krzysztof T; Fischer-Nielsen, Anne

2017-02-01

Platelet lysates (PL) represent a promising replacement for xenogenic growth supplement for adipose-derived stem cell (ASC) expansions. However, fresh platelets from human blood donors are not clinically feasible for large-scale cell expansion based on their limited supply. Therefore, we tested PLs prepared via three methods from outdated buffy coat-derived platelet concentrates (PCs) to establish an efficient and feasible expansion of ASCs for clinical use. PLs were prepared by the freeze-thaw method from freshly drawn platelets or from outdated buffy coat-derived PCs stored in the platelet additive solution, InterSol. Three types of PLs were prepared from outdated PCs with platelets suspended in either (1) InterSol (not manipulated), (2) InterSol + supplemented with plasma or (3) plasma alone (InterSol removed). Using these PLs, we compared ASC population doubling time, cell yield, differentiation potential and cell surface markers. Gene expression profiles were analyzed using microarray assays, and growth factor concentrations in the cell culture medium were measured using enzyme-linked immunosorbent assay (ELISA). Of the three PL compositions produced from outdated PCs, removal of Intersol and resuspension in plasma prior to the first freezing process was overall the best. This specific outdated PL induced ASC growth kinetics, surface markers, plastic adherence and differentiation potentials comparable with PL from fresh platelets. ASCs expanded in PL from fresh versus outdated PCs exhibited different expressions of 17 overlapping genes, of which 10 were involved in cellular proliferation, although not significantly reflected by cell growth. Only minor differences in growth factor turnover were observed. PLs from outdated platelets may be an efficient and reliable source of human growth supplement allowing for large-scale ASC expansion for clinical use. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights

A protein chip membrane-capture assay for botulinum neurotoxin activity

International Nuclear Information System (INIS)

Marconi, Severine; Ferracci, Geraldine; Berthomieu, Maelys; Kozaki, Shunji; Miquelis, Raymond; Boucraut, Jose; Seagar, Michael

2008-01-01

Botulinum neurotoxins A and B (BoNT/A and B) are neuromuscular blocking agents which inhibit neurotransmission by cleaving the intra-cellular presynaptic SNARE proteins SNAP-25 and VAMP2, localized respectively in plasma membrane and synaptic vesicles. These neurotoxins are both dangerous pathogens and powerful therapeutic agents with numerous clinical and cosmetic applications. Consequently there is a need for in vitro assays of their biological activity to screen for potential inhibitors and to replace the widely used in vivo mouse assay. Surface plasmon resonance (SPR) was used to measure membrane vesicle capture by antibodies against SNAP-25 and VAMP2. Substrate cleavage by BoNTs modified capture providing a method to assay toxin activity. Firstly using synaptic vesicles as a substrate, a comparison of the EC 50 s for BoNT/B obtained by SPR, ELISA or flow cytometry indicated similar sensitivity although SPR assays were more rapid. Sonication of brain or neuronal cultures generated plasma membrane fragments with accessible intra-cellular epitopes adapted to measurement of BoNT/A activity. SPR responses were proportional to antigen concentration permitting detection of as little as 4 pM SNAP-25 in crude lysates. BoNT/A activity was assayed using monoclonal antibodies that specifically recognize a SNAP-25 epitope generated by the proteolytic action of the toxin. Incubation of intact primary cultured neurons with BoNT/A yielded an EC 50 of 0.5 pM. The SPR biosensor method was sensitive enough to monitor BoNT/A and B activity in cells cultured in a 96-well format providing an alternative to experimental animals for toxicological assays

A contribution to the study of pyrogenic substances in radiopharmaceutical preparations. Comparison between methods using Rabbit and those using Limulus amebocytes lysate

International Nuclear Information System (INIS)

Bruneau, Jacky.

1982-10-01

We have studied two methods for pyrogenic substances detection. We used: the hyperthermic action of these substances after injection in Rabbit, and the gelation reaction of a Limulus amebocytes lysate. To apply these two methods of pyrogenic substances detection to the radiopharmaceutical preparations, we have conceived and designed a material allowing their handling in compliance with the radioactive safety norms. We have compared the sensitivity, reliability and reproducibility of these methods, one based on gelation of Limulus amebocytes lysate in presence of endotoxins, the other on the hyperthermic action of these same endotoxins in the rabbit when injected intravenously or through the suboccipital route. The discussion of the results obtained, shows that the method using the Limulus amebocytes lysate is more sensitive, less expansive and less dangerous. This method particulary well adapted to the control of radiopharmaceutical preparations, brings an additional security to the patients for whom these products are destined [fr

[Development and evaluation of a pyrogen test based on human whole blood

Science.gov (United States)

Hartung, Thomas; Fennrich, Stefan; Fischer, Matthias; Montag-Lessing, Thomas; Wendel, Albrecht

1998-01-01

When cells of the immune system, especially blood monocytes and macrophages, come into contact with pyrogenic (fever-inducing) contaminations, they secrete messenger molecules which initiate an hyperthermic reaction in the organism. Of this group of endogenous pyrogens, most is known about interleukin-1 (IL-1). A new pyrogen test makes use of this reaction as a system for detection: The substances which are to be screened are incubated with a small volume of blood from a healthy donor. Any pyrogens present induce the production of IL-1 which can be detected by ELISA. This test has a higher sensitivity and is more economical than the conventional pyrogen test in rabbits and furthermore reflects the reaction of the relevant species. In contrast to the customary alternative method, the Limulus amoebocyte lysate test (LAL), this test is not restricted to endotoxins from Gram-negative bacteria and is also not hindered by substances which bind endotoxins, such as blood proteins, to the same extent. Consequently, more than 50 non-endotoxin pyrogens have already been traced by this test. The whole blood test is even superior to the LAL in regard to the detection of endotoxins: in a comparison of about 60 endotoxins, there was a correlation of the potency of the individual endotoxins between the whole blood test and the pyrogen test in rabbits, but neither test correlated with the LAL test. In some cases, endotoxins with equal effects in the LAL test differed in potency in the human blood model by a factor of 10 000. A method has been developed by which cryopreserved blood can be put to use in the test. In this way, blood donations from a donor can be pre-tested so that uniform material may be employed in the test. This test opens up entirely new perspectives on pyrogen testing for Gram-positive or fungal pyrogens as well as in medicinal products. In addition, it could fill the dangerous security gap which might result from the limitations of testing medications and blood

Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide microarray.

Science.gov (United States)

Sharif, Suhela; Shi, Jie; Bourakba, Mostafa; Ruijtenbeek, Rob; Pieters, Roland J

2017-09-01

O-GlcNAcylation is a post-translational modification resulting from the addition of an N-acetylglucosamine moiety to the hydroxyl groups of serine and threonine residues of nuclear and cytoplasmic proteins. In addition, O-GlcNAcylated proteins can be phosphorylated, which suggests the possibility for crosstalk between O-GlcNAcylation and phosphorylation. Dysregulation of O-GlcNAcylation affects cell signaling, transcriptional regulation, cell cycle control and can e.g. lead to tumorigenesis and tumor metastasis. There is a strong demand for efficient analytical techniques to better detect and investigate this abundant modification and its role in cancer. Herein we demonstrated the utility of an O-GlcNAcylated peptide array to examine O-GlcNAcase (OGA) activity and substrate specificity of both purified protein as well cell lysates of different cancer cell lines. Using this microarray, we clearly observed OGA activity and also inhibition thereof by OGA inhibitor thiamet G. Interestingly, different levels of OGA activity were observed of lysates derived from different cancer cell lines. This suggests that the tool may be useful in cancer research and biomarker development. Copyright © 2017 Elsevier Inc. All rights reserved.

Platelet lysate 3D scaffold supports mesenchymal stem cell chondrogenesis: an improved approach in cartilage tissue engineering.

Science.gov (United States)

Moroz, Andrei; Bittencourt, Renata Aparecida Camargo; Almeida, Renan Padron; Felisbino, Sérgio Luis; Deffune, Elenice

2013-01-01

Articular lesions are still a major challenge in orthopedics because of cartilage's poor healing properties. A major improvement in therapeutics was the development of autologous chondrocytes implantation (ACI), a biotechnology-derived technique that delivers healthy autologous chondrocytes after in vitro expansion. To obtain cartilage-like tissue, 3D scaffolds are essential to maintain chondrocyte differentiated status. Currently, bioactive 3D scaffolds are promising as they can deliver growth factors, cytokines, and hormones to the cells, giving them a boost to attach, proliferate, induce protein synthesis, and differentiate. Using mesenchymal stem cells (MSCs) differentiated into chondrocytes, one can avoid cartilage harvesting. Thus, we investigated the potential use of a platelet-lysate-based 3D bioactive scaffold to support chondrogenic differentiation and maintenance of MSCs. The MSCs from adult rabbit bone marrow (n = 5) were cultivated and characterized using three antibodies by flow cytometry. MSCs (1 × 10(5)) were than encapsulated inside 60 µl of a rabbit platelet-lysate clot scaffold and maintained in Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 supplemented with chondrogenic inductors. After 21 days, the MSCs-seeded scaffolds were processed for histological analysis and stained with toluidine blue. This scaffold was able to maintain round-shaped cells, typical chondrocyte metachromatic extracellular matrix deposition, and isogenous group formation. Cells accumulated inside lacunae and cytoplasm lipid droplets were other observed typical chondrocyte features. In conclusion, the usage of a platelet-lysate bioactive scaffold, associated with a suitable chondrogenic culture medium, supports MSCs chondrogenesis. As such, it offers an alternative tool for cartilage engineering research and ACI.

Comparison of allogeneic platelet lysate and fetal bovine serum for in vitro expansion of equine bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Seo, Jong-pil; Tsuzuki, Nao; Haneda, Shingo; Yamada, Kazutaka; Furuoka, Hidefumi; Tabata, Yasuhiko; Sasaki, Naoki

2013-10-01

Mesenchymal stem cells (MSCs) are promising candidates for cell-based therapy and tissue engineering approaches. Fetal bovine serum (FBS) is commonly used for in vitro MSC expansion; however, the use of FBS may be associated with ethical, scientific, and safety issues. This study aimed to compare the ability of allogeneic platelet lysate (PL) and FBS to cause equine bone marrow-derived MSC expansion. MSCs were isolated from bone marrow aspirate in media supplemented with either PL or FBS, and cell proliferation properties and characteristics were examined. There were no significant differences in MSC yield, colony-forming unit-fibroblast (CFU-F) assay, and population doubling time between PL and FBS cultures. In addition, both PL-MSCs and FBS-MSCs showed similar results in term of ALP staining, osteogenic differentiation, and RT-PCR, although there were subtle differences in morphology, growth pattern, and adhesive properties. These results suggest that PL is a suitable alternative to FBS for use in equine MSC expansion, without the problems related to FBS use. Published by Elsevier India Pvt Ltd.

Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate.

Science.gov (United States)

Wagner, Eric R; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M; Chase, Steven; Westendorf, Jennifer J; Dietz, Allan B; van Wijnen, Andre J; Yaszemski, Michael J; Kakar, Sanjeev

2015-11-01

Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer "neoligament" seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell-cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p engineering and ligament regeneration.

Clinical Benefit of Allogeneic Melanoma Cell Lysate-Pulsed Autologous Dendritic Cell Vaccine in MAGE-Positive Colorectal Cancer Patients

DEFF Research Database (Denmark)

Toh, Han Chong; Wang, Who-Whong; Chia, Whay Kuang

2009-01-01

PURPOSE: We evaluated the clinical benefit of an allogeneic melanoma cell lysate (MCL)-pulsed autologous dendritic cell (DC) vaccine in advanced colorectal cancer patients expressing at least one of six MAGE-A antigens overexpressed by the cell line source of the lysate. EXPERIMENTAL DESIGN: DCs...... were cultured from peripheral blood mononuclear cells (PBMC), pulsed with the allogeneic MCL, and matured using cytokines that achieved high CD83- and CCR7-expressing DCs. Each patient received up to 10 intradermal vaccinations (3-5 x 10(6) cells per dose) at biweekly intervals. RESULTS: Twenty......-free for >27 and >37 months, respectively. This result is particularly meaningful as all patients had progressive disease before treatment. Overall, DC vaccination was associated with a serial decline in regulatory T cells. Using an antibody array, we characterized plasma protein profiles in responding...

Optimizing parameters for clinical-scale production of high IL-12 secreting dendritic cells pulsed with oxidized whole tumor cell lysate

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Chiang Cheryl L-L

2011-11-01

Full Text Available Abstract Background Dendritic cells (DCs are the most potent antigen-presenting cell population for activating tumor-specific T cells. Due to the wide range of methods for generating DCs, there is no common protocol or defined set of criteria to validate the immunogenicity and function of DC vaccines. Methods Monocyte-derived DCs were generated during 4 days of culture with recombinant granulocyte-macrophage colony stimulating factor and interleukin-4, and pulsed with tumor lysate produced by hypochlorous acid oxidation of tumor cells. Different culture parameters for clinical-scale DC preparation were investigated, including: 1 culture media; 2 culture surface; 3 duration of activating DCs with lipopolysaccharide (LPS and interferon (IFN-gamma; 4 method of DC harvest; and 5 cryomedia and final DC product formulation. Results DCs cultured in CellGenix DC media containing 2% human AB serum expressed higher levels of maturation markers following lysate-loading and maturation compared to culturing with serum-free CellGenix DC media or AIM-V media, or 2% AB serum supplemented AIM-V media. Nunclon™Δ surface, but not Corning® tissue-culture treated surface and Corning® ultra-low attachment surface, were suitable for generating an optimal DC phenotype. Recombinant trypsin resulted in reduced major histocompatibility complex (MHC Class I and II expression on mature lysate-loaded DCs, however presentation of MHC Class I peptides by DCs was not impaired and cell viability was higher compared to cell scraping. Preservation of DCs with an infusible cryomedia containing Plasma-Lyte A, dextrose, sodium chloride injection, human serum albumin, and DMSO yielded higher cell viability compared to using human AB serum containing 10% DMSO. Finally, activating DCs for 16 hours with LPS and IFN-γ stimulated robust mixed leukocyte reactions (MLRs, and high IL-12p70 production in vitro that continued for 24 hours after the cryopreserved DCs were thawed and

Comparative evaluation of pyrogens tests in pharmaceutical products Avaliação comparativa de testes de pirogênios em produtos farmacêuticos

Directory of Open Access Journals (Sweden)

Rosimar L. Silveira

2004-06-01

Full Text Available A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta

The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study

Directory of Open Access Journals (Sweden)

Antonio M. Fea

2016-01-01

Full Text Available Purpose. To determine the effectiveness of autologous platelet lysate (APL eye drops in patients with primary Sjögren syndrome (SS dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM. Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI, best corrected visual acuity (BCVA, Schirmer test, fluorescein score, and breakup time (BUT. A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n=20 patients mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n=10 patients. The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p<0.05. Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

The Effect of Autologous Platelet Lysate Eye Drops: An In Vivo Confocal Microscopy Study.

Science.gov (United States)

Fea, Antonio M; Aragno, Vittoria; Testa, Valeria; Machetta, Federica; Parisi, Simone; D'Antico, Sergio; Spinetta, Roberta; Fusaro, Enrico; Grignolo, Federico M

2016-01-01

Purpose. To determine the effectiveness of autologous platelet lysate (APL) eye drops in patients with primary Sjögren syndrome (SS) dry eye, refractory to standard therapy, in comparison with patients treated with artificial tears. We focused on the effect of APL on cornea morphology with the in vivo confocal microscopy (IVCM). Methods. Patients were assigned to two groups: group A used autologous platelet lysate QID, and group B used preservative-free artificial tears QID, for 90 days. Ophthalmological assessments included ocular surface disease index (OSDI), best corrected visual acuity (BCVA), Schirmer test, fluorescein score, and breakup time (BUT). A subgroup of patients in group A underwent IVCM: corneal basal epithelium, subbasal nerves, Langerhans cells, anterior stroma activated keratocytes, and reflectivity were evaluated. Results. 60 eyes of 30 patients were enrolled; in group A (n = 20 patients) mean OSDI, fluorescein score, and BUT showed significant improvement compared with group B (n = 10 patients). The IVCM showed a significant increase in basal epithelium cells density and subbasal nerve plexus density and number and a decrease in Langerhans cells density (p < 0.05). Conclusion. APL was found effective in the treatment of SS dry eye. IVCM seems to be a useful tool to visualize cornea morphologic modifications.

Activation of NO and cytokines by low-molecular weight fractions of lysates from G+ and G bacteria

Czech Academy of Sciences Publication Activity Database

Kmoníčková, Eva; Dusilová, Adéla; Kostecká, Petra; Kverka, Miloslav; Zídek, Zdeněk

2013-01-01

Roč. 157, Suppl.1 (2013), S51-S55 ISSN 1213-8118 R&D Projects: GA ČR GAP303/12/0535 Institutional support: RVO:68378041 ; RVO:61388971 Keywords : bacteria * lysate * immunomodulation Subject RIV: FR - Pharmacology ; Medidal Chemistry; EC - Immunology (MBU-M) Impact factor: 1.661, year: 2013

Evaluation of a platelet lysate bilayered system for periodontal regeneration in a rat intrabony three-wall periodontal defect

NARCIS (Netherlands)

Babo, P.S.; Cai, X; Plachokova, A.S.; Reis, R.L.; Jansen, J.A.; Gomes, M.E.; Walboomers, X.F.

2018-01-01

With currently available therapies, full regeneration of lost periodontal tissues after periodontitis cannot be achieved. In this study, a combined compartmentalized system was tested, composed of (a) a platelet lysate (PL)-based construct, which was placed along the root aiming to regenerate the

Heat inactivation of a norovirus surrogate in cell culture lysate, abalone meat, and abalone viscera.

Science.gov (United States)

Park, Shin Young; Bae, San-Cheong; Ha, Sang-Do

2015-03-01

The current study examined the effects of temperature and heat treatment duration on murine norovirus-1 (MNV-1) from both viral cell culture lysate (7-8 log10 PFU) and experimentally contaminated abalone meat and viscera (5-6 log10 PFU) as a model of human norovirus (NoV). MNV-1 titers in cell culture lysate, abalone meat, and abalone viscera were gradually reduced to 1.93-4.55, 1.79-3.00, and 2.26-3.26 log10 PFU/ml, respectively, after treatment at 70 °C for 1-10 min. Treatment at 85 °C for 1-5 min gradually reduced MNV-1 titers in abalone meat to 2.71-4.15 log10 PFU/ml. MNV-1 titers in abalone viscera were gradually reduced to 2.91-3.46 log10 PFU/ml after treatment at 85 °C for 1-3 min. No significant difference (P > 0.05) was found in MNV-1 titers in the abalone meat and viscera among treatment groups (70 °C for 5 min, 70 °C for 3 min, and 85 °C for 1 min). Complete inactivation of MNV-1 in cell culture lysate was determined at 85 °C for ≥1 min and 100 °C for ≥0.5 min. Complete inactivation of MNV-1 in abalone was determined at 100 °C for ≥0.5 min for meat, and 85 °C for 5 min and 100 °C for ≥0.5 min for viscera. At treatments at 70 °C, the Td-values (3 log reduction time) were significantly lower (P abalone meat (6.07) and viscera (10.73). Td = 3 values were not significantly different (P > 0.05) between abalone meat (1.78) and abalone viscera (1.33) at treatments at 85 °C. This study suggests that 100 °C for ≥0.5 min could potentially be used to inactivate NoV in molluscan shellfishes, including viscera.

Decreased mechanical properties of heart valve tissue constructs cultured in platelet lysate as compared to fetal bovine serum

NARCIS (Netherlands)

Geemen, van D.; Riem Vis, P.W.; Soekhradj - Soechit, R.S.; Sluijter, J.P.G.; Liefde - van Beest, de M.; Kluin, J.; Bouten, C.V.C.

2011-01-01

In autologous heart valve tissue engineering, there is an ongoing search for alternatives of fetal bovine serum (FBS). Human platelet-lysate (PL) might be a promising substitute. In the present article, we aimed to examine the tissue formation, functionality, and mechanical properties of engineered

Strain-dependent augmentation of tight-junction barrier function in human primary epidermal keratinocytes by Lactobacillus and Bifidobacterium lysates.

Science.gov (United States)

Sultana, Reshma; McBain, Andrew J; O'Neill, Catherine A

2013-08-01

In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus rhamnosus GG. With the exception of L. fermentum (which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However, B. longum and L. rhamnosus GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by B. longum, but not L. rhamnosus GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.

Rapid and sensitive detection of multiple microRNAs in cell lysate by low-fouling surface plasmon resonance biosensor

Czech Academy of Sciences Publication Activity Database

Vaisocherová, Hana; Šípová, Hana; Víšová, Ivana; Bocková, Markéta; Špringer, Tomáš; Ermini, Maria Laura; Chadtová Song, Xue; Krejčík, Z.; Chrastinová, L.; Pastva, O.; Pimková, K.; Merkerová-Dostálová, M.; Dyr, J. E.; Homola, Jiří

2015-01-01

Roč. 70, August 05 (2015), s. 226-231 ISSN 0956-5663 R&D Projects: GA ČR(CZ) GBP205/12/G118 Institutional support: RVO:67985882 Keywords : Erythrocyte lysate * Polymer brushes * DNA array Subject RIV: JA - Electronics ; Optoelectronics, Electrical Engineering Impact factor: 7.476, year: 2015

Ambient endotoxin in PM10 and association with inflammatory activity, air pollutants, and meteorology, in Chitwan, Nepal.

Science.gov (United States)

Mahapatra, Parth Sarathi; Jain, Sumeet; Shrestha, Sujan; Senapati, Shantibhusan; Puppala, Siva Praveen

2018-03-15

Endotoxin associated with ambient PM (particulate matter) has been linked to adverse respiratory symptoms, but there have been few studies of ambient endotoxin and its association with co-pollutants and inflammation. Our aim was to measure endotoxin associated with ambient PM 10 (particulate matter with aerodynamic diametermeteorology, co-pollutants, and inflammatory activity. PM 10 concentrations were recorded and filter paper samples were collected using E-samplers; PM 1, PM 2.5 , black carbon (BC), methane (CH 4 ), and carbon monoxide (CO) were also measured. The Limulus amebocyte lysate (LAL) assay was used for endotoxin quantification and the nuclear factor kappa B (NFκB) activation assay to assess inflammatory activity. The mean concentration of PM 10 at the different locations ranged from 136 to 189μg/m 3 , and of endotoxin from 0.29 to 0.53EU/m 3 . Pollutant presence was positively correlated with endotoxin. Apart from relative humidity, meteorological variations had no significant impact on endotoxin concentration. NF-κB activity was negatively correlated with endotoxin concentration. To the best of our knowledge, this study provides the first measurements of ambient endotoxin associated with PM 10 in Nepal. Endotoxin and co-pollutants were positively associated indicating a similar source. Endotoxin was negatively correlated with inflammatory activity as a result of a time-limited forest fire event during the sampling period. Studies of co-pollutants suggested that the higher levels of endotoxin related to biomass burning were accompanied by increased levels of anti-inflammatory agents, which suppressed the endotoxin inflammatory effect. Copyright © 2017. Published by Elsevier B.V.

Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

OpenAIRE

Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

1994-01-01

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The...

A liquid chromatography/tandem mass spectrometry assay for the analysis of atomoxetine in human plasma and in vitro cellular samples

Science.gov (United States)

Appel, David I.; Brinda, Bryan; Markowitz, John S.; Newcorn, Jeffrey H.; Zhu, Hao-Jie

2012-01-01

A simple, rapid and sensitive method for quantification of atomoxetine by liquid chromatography- tandem mass spectrometry (LC-MS/MS) was developed. This assay represents the first LC-MS/MS quantification method for atomoxetine utilizing electrospray ionization. Deuterated atomoxetine (d3-atomoxetine) was adopted as the internal standard. Direct protein precipitation was utilized for sample preparation. This method was validated for both human plasma and in vitro cellular samples. The lower limit of quantification was 3 ng/ml and 10 nM for human plasma and cellular samples, respectively. The calibration curves were linear within the ranges of 3 ng/ml to 900 ng/ml and 10 nM to 10 μM for human plasma and cellular samples, respectively (r2 > 0.999). The intra- and inter-day assay accuracy and precision were evaluated using quality control samples at 3 different concentrations in both human plasma and cellular lysate. Sample run stability, assay selectivity, matrix effect, and recovery were also successfully demonstrated. The present assay is superior to previously published LC-MS and LC-MS/MS methods in terms of sensitivity or the simplicity of sample preparation. This assay is applicable to the analysis of atomoxetine in both human plasma and in vitro cellular samples. PMID:22275222

HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

Science.gov (United States)

Le Pape, Fiona; Cosnuau-Kemmat, Lucie; Richard, Gaëlle; Dubrana, Frédéric; Férec, Claude; Zal, Franck; Leize, Elisabeth; Delépine, Pascal

2017-04-01

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

DEFF Research Database (Denmark)

Damgaard, Dres; Senolt, Ladislav; Nielsen, Michael Friberg

2014-01-01

INTRODUCTION: Members of the peptidylarginine deiminase (PAD) family catalyse the posttranslational conversion of peptidylarginine to peptidylcitrulline. Citrullination of proteins is well described in rheumatoid arthritis (RA), and hypercitrullination of proteins may be related to inflammation...... in general. PAD activity has been demonstrated in various cell lysates, but so far not in synovial fluid. We aimed to develop an assay for detection of PAD activity, if any, in synovial fluid from RA patients. METHODS: An enzyme-linked immunosorbent assay using human fibrinogen as the immobilized substrate...... for citrullination and anti-citrullinated fibrinogen antibody as the detecting agent were used for measurement of PAD activity in synovial fluid samples from five RA patients. The concentrations of PAD2 and calcium were also determined. RESULTS: Approximately 150 times lower levels of recombinant human PAD2 (rhPAD2...

Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

Directory of Open Access Journals (Sweden)

Bryan A Piras

2016-01-01

Full Text Available With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development.

Platelet lysate as a novel serum-free media supplement for the culture of equine bone marrow-derived mesenchymal stem cells.

Science.gov (United States)

Naskou, Maria C; Sumner, Scarlett M; Chocallo, Anna; Kemelmakher, Hannah; Thoresen, Merrilee; Copland, Ian; Galipeau, Jacques; Peroni, John F

2018-03-22

Mesenchymal stem cells (MSCs) produced for clinical purposes rely on culture media containing fetal bovine serum (FBS) which is xenogeneic and has the potential to significantly alter the MSC phenotype, rendering these cells immunogenic. As a result of bovine-derived exogenous proteins expressed on the cell surface, MSCs may be recognized by the host immune system as non-self and be rejected. Platelet lysate (PL) may obviate some of these concerns and shows promising results in human medicine as a possible alternative to FBS. Our goal was to evaluate the use of equine platelet lysate (ePL) pooled from donor horses in place of FBS to culture equine MSCs. We hypothesized that ePL, produced following apheresis, will function as the sole media supplement to accelerate the expansion of equine bone marrow-derived MSCs without altering their phenotype and their immunomodulatory capacity. Platelet concentrate was obtained via plateletpheresis and ePL were produced via freeze-thaw and centrifugation cycles. Population doublings (PD) and doubling time (DT) of bone marrow-derived MSCs (n = 3) cultured with FBS or ePL media were calculated. Cell viability, immunophenotypic analysis, and trilineage differentiation capacity of MSCs were assessed accordingly. To assess the ability of MSCs to modulate inflammatory responses, E. coli lipopolysaccharide (LPS)-stimulated monocytes were cocultured with MSCs cultured in the two different media formulations, and cell culture supernatants were assayed for the production of tumor necrosis factor (TNF)-α. Our results showed that MSCs cultured in ePL media exhibited similar proliferation rates (PD and DT) compared with those cultured in FBS at individual time points. MSCs cultured in ePL showed a statistically significant increased viability following a single washing step, expressed similar levels of MSC markers compared to FBS, and were able to differentiate towards the three lineages. Finally, MSCs cultured in ePL efficiently suppressed

Increasing the flexibility of the LANCE cAMP detection kit.

Science.gov (United States)

Hunter, Morag Rose; Glass, Michelle

2015-01-01

The detection of cAMP signalling is a common endpoint in the study of G-protein coupled receptors. A number of commercially available kits enable easy detection of cAMP. These kits are based on competition for a cAMP binding site on an antibody or cAMP binding protein and as such have a limited dynamic range. Here, we describe the optimisation of the commercially-available LANCE cAMP detection kit (PerkinElmer) to enable detection in cell lysates. This kit has been designed for use with live cells, with detection reagents applied to cells without wash steps. The standard protocol therefore requires that all assay reagents are compatible with the antibody and the final fluorescent detection stage, limiting the range of assay media and test compounds that can be utilised. The entire experiment must be repeated if cAMP levels fall outside the limited dynamic range. Here we describe a modified protocol that enables the assay to be performed on cell lysates, thereby overcoming these limitations. In this modified protocol, cells are stimulated for a cAMP response in standard media/buffers, washed and then lysed. The cell lysate is then assayed using a modified protocol for the LANCE cAMP detection kit. Samples were tested for stability following a freeze-thaw cycle. The modified LANCE cAMP detection protocol gives a reproducible measurement of cAMP in cell lysate. Lysate samples remain stable when stored at -80°C. Separating the stimulation and detection phases of this cAMP assay allows a vast array of cell stimulation conditions to be tested. The lysate-modified protocol for the LANCE cAMP detection kit therefore increases the flexibility, versatility and convenience of the assay. As samples are insensitive to freeze-thaw, it enables retesting of samples under different dilution conditions to ensure that all samples remain within the dynamic range of the standard curve. Copyright © 2014 Elsevier Inc. All rights reserved.

Electrospun Gelatin–Chondroitin Sulfate Scaffolds Loaded with Platelet Lysate Promote Immature Cardiomyocyte Proliferation

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Francesca Saporito

2018-02-01

Full Text Available The aim of the present work was the development of heart patches based on gelatin (G and chondroitin sulfate (CS to be used as implants to improve heart recovery after corrective surgery for critical congenital heart defects (CHD. Patches were prepared by means of electrospinning to obtain nanofibrous scaffolds and they were loaded with platelet lysate (PL as a source of growth factors to further enhance the repair process. Scaffolds were characterized for morphology and mechanical properties and for the capability to support in vitro adhesion and proliferation of dermal fibroblasts in order to assess the system’s general biocompatibility. Adhesion and proliferation of endothelial cells and cardiac cells (cardiomyocytes and cardiac fibroblasts from rat fetuses onto PL-loaded patches was evaluated. Patches presented good elasticity and high stiffness suitable for in vivo adaptation to heart contraction. CS improved adhesion and proliferation of dermal fibroblasts, as proof of their biocompatibility. Moreover, they enhanced the adhesion and proliferation of endothelial cells, a crucial mediator of cardiac repair. Cell adhesion and proliferation could be related to elastic properties, which could favor cell motility. The presence of platelet lysate and CS was crucial for the adhesion and proliferation of cardiac cells and, in particular, of cardiomyocytes: G/CS scaffold embedded with PL appeared to selectively promote proliferation in cardiomyocytes but not cardiac fibroblasts. In conclusion, G/CS scaffold seems to be a promising system to assist myocardial-repair processes in young patient, preserving cardiomyocyte viability and preventing cardiac fibroblast proliferation, likely reducing subsequent uncontrolled collagen deposition by fibroblasts following repair.

[The lysate and recombinant antigens in ELISA-test-systems for diagnostic of herpes simplex].

Science.gov (United States)

Ganova, L A; Kovtoniuk, G V; Korshun, L N; Kiseleva, E K; Tereshchenko, M I; Vudmaska, M I; Moĭsa, L N; Shevchuk, V A; Spivak, N Ia

2014-08-01

The lysate and recombinant antigens of various production included informula of ELISA-test-systems were analyzed. The ELISA-test-systems are used for detection of IgG to Herpes simplex virus type I and II. For testing the panel of serums PTH 201 (BBI Inc.) were used. The samples of this panel contain antibodies to Herpes simplex virus type I and II in mixed titers. The 69 serums of donors were used too (17 samples had IgG to Herpes simplex virus type I, 23 samples to Herpes simplex virus type II and 29 samples had no antibodies to Herpes simplex virus). The diagnostic capacity of mixture of recombinant antigens gG1 Herpes simplex virus type I and gG2 Herpes simplex virus type II (The research-and-production complex "DiaprofMed") was comparable with mixture of lysate antigen Herpes simplex virus type I and II (Membrane) EIE Antigen ("Virion Ltd."). In the test-systems for differentiation of IgG to Herpes simplex virus type I the recombinant antigen gG1 Herpes simplex virus type I proved to be comparable with commercial analogue Herpes simplex virus-1 gG1M ("Viral Therapeutics Inc."'). At the same time, capacity to detect IgG to Herpes simplex virus type II in recombinant protein gG2 Herpes simplex virus type II is significantly higher than in its analogue Herpes simplex virus-2 gG2c ("Viral Therapeutics Inc.").

Feasibility and Efficiency of Human Bone Marrow Stromal Cell Culture with Allogeneic Platelet Lysate-Supplementation for Cell Therapy against Stroke

Directory of Open Access Journals (Sweden)

Chengbo Tan

2016-01-01

Full Text Available Currently, there is increasing interest in human bone marrow stromal cells (hBMSCs as regeneration therapy against cerebral stroke. The aim of the present study was to evaluate the feasibility and validity of hBMSC cultures with allogeneic platelet lysates (PLs. Platelet concentrates (PC were harvested from healthy volunteers and made into single donor-derived PL (sPL. The PL mixtures (mPL were made from three different sPL. Some growth factors and platelet cell surface antigens were detected by enzyme-linked immunosorbent assay (ELISA. The hBMSCs cultured with 10% PL were analyzed for their proliferative potential, surface markers, and karyotypes. The cells were incubated with superparamagnetic iron oxide (SPIO agents and injected into a pig brain. MRI and histological analysis were performed. Consequently, nine lots of sPL and three mPL were prepared. ELISA analysis showed that PL contained adequate growth factors and a particle of platelet surface antigens. Cell proliferation capacity of PLs was equivalent to or higher than that of fetal calf serum (FCS. No contradiction in cell surface markers and no chromosomal aberrations were found. The MRI detected the distribution of SPIO-labeled hBMSCs in the pig brain. In summary, the hBMSCs cultured with allogeneic PL are suitable for cell therapy against stroke.

Molecular and clinical characterization of a series of patients with childhood-onset lysosomal acid lipase deficiency. Retrospective investigations, follow-up and detection of two novel LIPA pathogenic variants.

Science.gov (United States)

Pisciotta, Livia; Tozzi, Giulia; Travaglini, Lorena; Taurisano, Roberta; Lucchi, Tiziano; Indolfi, Giuseppe; Papadia, Francesco; Di Rocco, Maja; D'Antiga, Lorenzo; Crock, Patricia; Vora, Komal; Nightingale, Scott; Michelakakis, Helen; Garoufi, Anastasia; Lykopoulou, Lilia; Bertolini, Stefano; Calandra, Sebastiano

2017-10-01

Childhood/Adult-onset Lysosomal Acid Lipase Deficiency (LAL-D) is a recessive disorder due to loss of function variants of LAL, the enzyme which hydrolyses cholesteryl esters, derived from internalized apoB containing lipoproteins. The disease is characterized by multi-organ involvement including the liver, spleen, intestine and cardiovascular system. The aim of this study was the clinical and molecular characterization of 14 (13 unrelated) previously unreported patients with childhood-onset LAL-D. Data collected included clinical and laboratory investigations, liver imaging, liver biopsy and LIPA gene analysis. The response to lipid-lowering medications, liver transplantation and enzyme replacement therapy (ERT) was reported for some patients. LAL-D was suspected at 4.4 ± 3.3 years of age for the presence of hepatomegaly, elevated serum transaminases and hypercholesterolemia, and was confirmed by liver biopsy/imaging and LAL assay. The follow up period ranged from 3 to 40 years (mean 7.8 ± 4.0 years in 13 cases). Patients treated with statins with or without ezetimibe showed 28% reduction of plasma LDL-cholesterol without a tangible effect on liver enzymes; some patients receiving ERT showed normalized lipoprotein profile and transaminase levels. The common c.894G > A variant was observed in homozygosity or compound heterozygosity in 10 patients. We found seven previously reported variants: p.(Trp140*), p.(Arg218*), p.(Gly266*), p.(Thr288Ile), p.(Leu294Ser), p.(His295Tyr) and p.(Gly342Arg) and two novel variants: p.(Asp345Asn), affecting the LAL catalytic triad, and c.229+3A > C, affecting splicing. Homozygosity for p.(Thr288Ile) or c.229+3A > C was associated with a severe phenotype. This study provides additional data on the features of childhood-onset LAL-D and describes two novel pathogenic variants of the LIPA gene. Copyright © 2017. Published by Elsevier B.V.

Differentiation of Rat bone marrow Mesenchymal stem cells into Adipocytes and Cardiomyocytes after treatment with platelet lysate.

Science.gov (United States)

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Barzegar, Kazem

2016-01-01

Mesenchymal stem cells (MSCs) are multipotential cells and their therapeutic potency is under intense investigation. Studying the effect of different induction factors on MSCs could increase our knowledge about the differentiation potency of these cells. One of the most important sources of these factors in mammalian body is platelet. Platelet lysate (PL) contains many growth factors and therefore, it can be used as a differentiation inducer. In the present study, the effect of PL on differentiation of rat bone marrow MSCs into cardiomyocytes was studied. To study the differentiation-inducing effect of PL, MSCs were treated with 2.5, 5 and 10% PL. Early results of this study showed that PL in high concentrations (10%) induces adipogenic differentiation of MSCs. Therefore, to evaluate differentiation to cardiomyocytes, MSCs were cultured in media containing lower levels of PL (2.5% and 5%) and then cardiomyogenic differentiation was induced by treatment with 5-azacytidine. Differentiation of MSCs was evaluated using direct observation of beating cells, immunostaining and real-time PCR techniques. The results of qPCR showed that treatment with PL alone increased the expression of cardiac alpha actinin (CAA) being predictable by earlier observation of beating cells in PL-treated groups. The results of staining assays against cardiac alpha actinin also showed that there were stained cells in PL-treated groups. The results of the present study showed that PL is a powerful induction factor for differentiation of MSCs into different cell lines such as cardiomyocytes and adipocytes.

Detection of endotoxins in radiopharmaceutical preparations. II. Comparison of the sensitivity of methods using the rabbit and the Limulus amoebocyte lysate for the detection of endotoxins

Energy Technology Data Exchange (ETDEWEB)

Bruneau, J; Cohen, Y; Merlin, L; Peysson, S

1986-01-01

The rise of the rabbit internal temperature after i.v. injection of an endotoxin solution is proportional to concentration. Gelation of Limulus amoebocyte, when in presence of an endotoxin solution, is also related to concentration. We compared the sensitivity of these two methods. With our experimental procedure, the rabbit is sensitive to a 0.40 ng/mL solution and the Limulus amoebocyte lysate to a 0.14 ng/mL solution. The rabbit sensitivity increase is related to the per kilogramme injected volume, whereas sensitivity is not related to the volume to check in the case of the lysate.

Comparative Analysis of Click Chemistry Mediated Activity-Based Protein Profiling in Cell Lysates

Directory of Open Access Journals (Sweden)

Yinliang Yang

2013-10-01

Full Text Available Activity-based protein profiling uses chemical probes that covalently attach to active enzyme targets. Probes with conventional tags have disadvantages, such as limited cell permeability or steric hindrance around the reactive group. A tandem labeling strategy with click chemistry is now widely used to study enzyme targets in situ and in vivo. Herein, the probes are reacted in live cells, whereas the ensuing detection by click chemistry takes place in cell lysates. We here make a comparison of the efficiency of the activity-based tandem labeling strategy by using Cu(I-catalyzed and strain-promoted click chemistry, different ligands and different lysis conditions.

Tailor-made purified human platelet lysate concentrated in neurotrophins for treatment of Parkinson's disease.

Science.gov (United States)

Chou, Ming-Li; Wu, Joe-Wei; Gouel, Flore; Jonneaux, Aurélie; Timmerman, Kelly; Renn, Ting-Yi; Laloux, Charlotte; Chang, Hung-Ming; Lin, Liang-Tzung; Devedjian, Jean-Christophe; Devos, David; Burnouf, Thierry

2017-10-01

Human platelet lysates (PLs), which contain multiple neurotrophins, have been proposed for treating neurodegenerative disorders, including Parkinson's disease (PD). However, current PLs suspended in plasma have high protein content and contain fibrinogen/fibrin and, following activation, also proteolytic and thrombogenic enzymes. Upon brain administration, such PLs may saturate the cerebrospinal fluid and exert neurotoxicity. We assessed whether purified PLs, concentrated in neurotrophins, protected dopaminergic neurons in PD models. Platelet concentrates were collected by apheresis and centrifuged to eliminate plasma and recover the platelets. Platelets were lysed by freeze-thaw cycles, and the 10-fold concentrated platelet pellet lysates (PPLs) were heat-treated (at 56 °C for 30 min). The heat-treated PPLs were low in total proteins, depleted in both plasma and platelet fibrinogen, and devoid of thrombogenic and proteolytic activities. They exerted very high neuroprotective activity when non-oncogenic, Lund human mesencephalic (LUHMES) cells that had differentiated into dopaminergic neurons were exposed to the MPP + neurotoxin. Heat treatment improved the neuroprotection and inactivated the neurotoxic blood-borne hepatitis C virus. PPL did not induce inflammation in BV2 microglial cells and inhibited COX-2 expression upon lipopolysaccharide exposure. Intranasal administration in mice revealed (a) diffusion of neurotrophins in the striatum and cortex, and (b) MPTP intoxication neuroprotection in the substantia nigra and striatum and the absence of neuroinflammation. These dedicated heat-treated PPLs can be a safe and valuable candidate for a therapeutic strategy for PD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Vaccination with melanoma lysate-pulsed dendritic cells, of patients with advanced colorectal carcinoma: report from a phase I study

DEFF Research Database (Denmark)

Burgdorf, S K; Fischer, A; Claesson, M H

2006-01-01

Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma...... contained 3-5 x 10(6) DCs. Five of the six patients received all five vaccines. The treatment was well tolerated in all patients without any observed vaccine-correlated adverse effects. Treatment with this DC-based cancer vaccine proved safe and non-toxic.......Immune therapy have shown new and exciting perspectives for cancer treatment. Aim of our study was to evaluate toxicity and possible adverse effects from vaccination of patients with advanced colorectal cancer with autologous dendritic cells (DC) pulsed with lysate from a newly developed melanoma...... and selected melanoma cell line enriched in expression of MAGE-A antigens and deficient in expression of melanoma differentiation antigens: tyrosinase, MART-1 and gp100. Vaccinations were administered intradermally on the proximal thigh with a total of five given vaccines at 2 weeks intervals. Each vaccine...

The effects of platelet lysate on maturation, fertilization and embryo development of NMRI mouse oocytes at germinal vesicle stage.

Science.gov (United States)

Pazoki, Hassan; Eimani, Hussein; Farokhi, Farah; Shahverdi, Abdol-Hossein; Tahaei, Leila Sadat

2016-04-01

Improving in vitro maturation could increase the rate of pregnancy from oocytes matured in vitro. Consequently, patients will be prevented from using gonadotropin with its related side effects. In this study, the maturation medium was enriched by platelet lysate (PL), then maturation and subsequent developments were monitored. Oocytes at germinal vesicle stage with cumulus cells (cumulus-oocyte complex) and without cumulus cells (denuded oocytes) were obtained from mature female mice. The maturation medium was enriched by 5 and 10 % PL and 5 % PL + 5 % fetal bovine serum (FBS) as experimental groups; the control groups' media consisted of 5 and 10 % FBS. After 18 h, the matured oocytes were collected and, after fertilization, subsequent development was monitored. The rates of maturation, fertilization and 2-cell embryo development for the denuded oocyte groups in experimental media 5 % PL and 5 % PL + 5 % FBS were significantly higher than those of the control groups ( P platelet lysate could improve the maturation rate in the absence of granulosa cells compared to media with FBS. This extract also had positive effects on fertilization and embryo development.

Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction.

Science.gov (United States)

VerHeul, Ross; Sweet, Craig; Thompson, David H

2018-03-26

Elastin-like polypeptides (ELP) are a well-known class of proteins that are being increasingly utilized in a variety of biomedical applications, due to their beneficial physicochemical properties. A unifying feature of ELP is their demonstration of a sequence tunable inverse transition temperature (Tt) that enables purification using a simple, straightforward process called inverse transition cycling (ITC). Despite the utility of ITC, the process is inherently limited to ELP with an experimentally accessible Tt. Since the underlying basis for the ELP Tt is related to its high overall hydrophobicity, we anticipated that ELP would be excellent candidates for purification by organic extraction. We report the first method for rapidly purifying ELP directly from whole E. coli cells or clarified lysates using pure organic solvents and solvent mixtures, followed by aqueous back extraction. Our results show that small ELP and a large ELP-fusion protein can be isolated in high yield from whole cells or cell lysates with greater than 95% purity in less than 30 min and with very low levels of LPS and DNA contamination.

Innate immune responses of equine monocytes cultured in equine platelet lysate.

Science.gov (United States)

Naskou, Maria C; Norton, Natalie A; Copland, Ian B; Galipeau, Jacques; Peroni, John F

2018-01-01

Platelet lysate (PL) has been extensively used for the laboratory expansion of human mesenchymal stem cells (MSC) in order to avoid fetal bovine serum (FBS) which has been associated with immune-mediated host reactions and transmission of bovine-origin microbial contaminants. Before suggesting the routine use of PL for MSC culture, we wanted to further investigate whether PL alone might trigger inflammatory responses when exposed to reactive white blood cells such as monocytes. Our objectives were to evaluate the inflammatory profile of equine monocytes cultured with equine PL (ePL) and to determine if ePL can modulate the expression of inflammatory cytokines in lipopolysaccharide (LPS)-stimulated monocytes. In a first experiment, equine monocytes were isolated and incubated with donor horse serum (DHS), FBS, six individual donors ePL or pooled ePL from all horses. In a second experiment, monocytes were stimulated with E. coli LPS in the presence of 1, 5 or 10% DHS and/or pooled ePL. After 6h of incubation, cell culture supernatants were assayed via ELISA for production of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and Interleukin 1β (IL-1β) as well as for the anti-inflammatory Interleukin 10 (IL-10). Equine monocytes incubated with pooled ePL produced significantly less TNF-α and significantly more IL-10 than monocytes incubated in FBS. A statistically significant difference was not identified for the production of IL-1β. The second experiment showed that pooled ePL added to LPS-stimulated equine monocytes resulted in a significant reduction in TNF-α and IL-1β production. IL-10 production was not significantly upregulated by the addition of ePL to LPS-stimulated monocytes. Finally, the addition of ePL to LPS-stimulated monocytes in the presence of various concentrations of DHS resulted to statistically significant decrease of TNF-α and IL-1β compared to the control groups. This is the first study to demonstrate that ePL suppresses

Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge

Science.gov (United States)

Shibusawa, Yoichi; Ito, Yoichiro

2014-01-01

This review describes protein purifications from cell culture medium or cell-lysate by high speed counter-current chromatography using the cross-axis coil planet centrifuge. Purifications were performed using aqueous two phase systems composed of polyethylene glycols and dextrans. PMID:25360182

Platelet lysate embedded scaffolds for skin regeneration.

Science.gov (United States)

Sandri, Giuseppina; Bonferoni, Maria Cristina; Rossi, Silvia; Ferrari, Franca; Mori, Michela; Cervio, Marila; Riva, Federica; Liakos, Ioannis; Athanassiou, Athanassia; Saporito, Francesca; Marini, Lara; Caramella, Carla

2015-04-01

The work presents the development of acellular scaffolds extemporaneously embedded with platelet lysate (PL), as an innovative approach in the field of tissue regeneration/reparation. PL embedded scaffolds should have a tridimensional architecture to support cell migration and growth, in order to restore skin integrity. For this reason, chondroitin sulfate (CS) was associated with sodium alginate (SA) to prepare highly porous systems. The developed scaffolds were characterized for chemical stability to γ-radiation, morphology, hydration and mechanical properties. Moreover, the capability of fibroblasts and endothelial cells to populate the scaffold was evaluated by means of proliferation test 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and confocal laser scanning microscopy study. The scaffolds, not altered by sterilization, were characterized by limited swelling and high flexibility, by foam-like structure with bubbles that formed a high surface area and irregular texture suitable for cell adhesion. Cell growth and scaffold population were evident on the bubble surface, where the cells appeared anchored to the scaffold structure. Scaffold network based on CS and SA demonstrated to be an effective support to enhance and to allow fibroblasts and endothelial cells (human umbilical vein endothelial cells, HUVEC) adhesion and proliferation. In particular, it could be hypothesized that cell adhesion was facilitated by the synergic effect of PL and CS. Although further in vivo evaluation is needed, on the basis of in vitro results, PL embedded scaffolds seem promising systems for skin wound healing.

Validation of an improved enzyme-linked immunosorbent assay for the diagnosis of trypanosomal antibodies in Ghanaian cattle

International Nuclear Information System (INIS)

Doku, C.K.; Seidu, I.B.M.

2000-01-01

The validation of an enzyme-linked immunosorbent assay (Ab-ELISA) for the detection of antibodies to pathogenic trypanosomes in cattle is described. Two hundred known negative sera obtained from the tsetse-free zone of Dori (Burkina Faso) were analyzed using microtitre plates pre-coated with crude antigen lysates of Trypanosoma congolense and T. vivax. A pre-test optimization was carried out and a percent positivity (PP) of 50% was chosen (specificity: >82%) for assaying field sera. A total of 440 serum samples collected from cattle in areas of known and unknown disease prevalence were assayed. For all animals the packed red cell volume (PCV) was determined and the buffy coat technique (BCT) and blood smears were examined to detect trypanosomes at the species level. A comparison of the BCT and Ab-ELISA results showed there was a much higher prevalence of antibodies to both species than the parasite prevalence as shown by the BCT (10 fold). The rate of agreement between BCT-positive and Ab-ELISA-positive samples for both species was low (<10%). No conclusion could be drawn from this finding because of the low number of known BCT positive cases that were identified. There was a better, albeit highly variable, agreement between BCT-negative and Ab-ELISA-negative samples (30-70%). Proposals for further improvement of the Ab-ELISA and prospects for the use of the assay in the monitoring of trypanosomosis control in Ghana are discussed. (author)

Performance of scintillation proximity assay (SPA) to measure the level of VEGFR 1 protein

International Nuclear Information System (INIS)

LEE, So-Young; LIM, Jae-Cheong; KIM, Jin-Joo

2014-01-01

Scintillation proximity assay is a one of radioimmunoassay and can be assayed without the washing or filtration procedures normally used to separate bound from free fractions. Due to its simplicity and high-throughput protocol, it is broadly applicable to immunology, receptor binding, monitoring receptor-ligand interactions and enzyme reactions. Briefly, an antibody or receptor is coated on SPA beads. When a radiolabeled antigen or ligand binds to the beads, the SPA beads stimulate to emit short range electrons. The 3 H and 125 I are commonly used for radiolabeling and the produced photons are detectable with a liquid scintillation counter (LSC). A binding affinity of unlabeled ligands can be determined by competitive reaction of the radiolabeled ligands. Bevacizumab is a recombinant humanized monoclonal antibody, and can stop or delay growth of tumors by inhibiting vascular endothelial growth factor (VEGF). Bevacizumab was approved by FDA for metastatic cancer such as colorectal cancers, ovarian cancers, breast cancers and glioblastoma multiform of the brain. Recently, Dan G. duda et al. was reported that the concentration of vascular endothelial growth factor receptor-1 (VEGFR-1) in plasma may potentially be used as a negative selection biomarker. In this study, we describe a method using scintillation proximity assay to detect the amounts of VEGFR-1 protein. This method is successfully used to measure the concentration of VEGFR-1 protein in human cell extracts. In summary, a simple and sensitive assay is developed for measuring the amount of VEGFR 1 protein in cancer cell lysate using SPA beads. The antibody coating on the beads and antigen binding are achieved in one mixing step

Performance of scintillation proximity assay (SPA) to measure the level of VEGFR 1 protein

Energy Technology Data Exchange (ETDEWEB)

LEE, So-Young; LIM, Jae-Cheong; KIM, Jin-Joo [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

2014-10-15

Scintillation proximity assay is a one of radioimmunoassay and can be assayed without the washing or filtration procedures normally used to separate bound from free fractions. Due to its simplicity and high-throughput protocol, it is broadly applicable to immunology, receptor binding, monitoring receptor-ligand interactions and enzyme reactions. Briefly, an antibody or receptor is coated on SPA beads. When a radiolabeled antigen or ligand binds to the beads, the SPA beads stimulate to emit short range electrons. The {sup 3}H and {sup 125}I are commonly used for radiolabeling and the produced photons are detectable with a liquid scintillation counter (LSC). A binding affinity of unlabeled ligands can be determined by competitive reaction of the radiolabeled ligands. Bevacizumab is a recombinant humanized monoclonal antibody, and can stop or delay growth of tumors by inhibiting vascular endothelial growth factor (VEGF). Bevacizumab was approved by FDA for metastatic cancer such as colorectal cancers, ovarian cancers, breast cancers and glioblastoma multiform of the brain. Recently, Dan G. duda et al. was reported that the concentration of vascular endothelial growth factor receptor-1 (VEGFR-1) in plasma may potentially be used as a negative selection biomarker. In this study, we describe a method using scintillation proximity assay to detect the amounts of VEGFR-1 protein. This method is successfully used to measure the concentration of VEGFR-1 protein in human cell extracts. In summary, a simple and sensitive assay is developed for measuring the amount of VEGFR 1 protein in cancer cell lysate using SPA beads. The antibody coating on the beads and antigen binding are achieved in one mixing step.

Purification of CD47-streptavidin fusion protein from bacterial lysate using biotin-agarose affinity chromatography.

Science.gov (United States)

Salehi, Nasrin; Peng, Ching-An

2016-07-08

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47-streptavidin fusion protein was expressed and purified because it can easily bind to biotin-tagged materials via the unique biotin-streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47-SA fusion gene. After bacteria transformation, the CD47-SA fusion protein was expressed by isopropyl-β-d-thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47-SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin-streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non-ionic detergent Triton X-100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47-SA fusion protein from the biotin agarose column. The purified CD47-SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949-958, 2016. © 2016 American Institute of Chemical Engineers.

Endotoxins in surgical instruments of hip arthroplasty

Directory of Open Access Journals (Sweden)

Vania Regina Goveia

2016-06-01

Full Text Available Abstract OBJECTIVE To investigate endotoxins in sterilized surgical instruments used in hip arthroplasties. METHOD A descriptive exploratory study conducted in a public teaching hospital. Six types of surgical instruments were selected, namely: acetabulum rasp, femoral rasp, femoral head remover, chisel box, flexible bone reamer and femoral head test. The selection was based on the analysis of the difficulty in removing bone and blood residues during cleaning. The sample was made up of 60 surgical instruments, which were tested for endotoxins in three different stages. The EndosafeTM Gel-Clot LAL (Limulus Amebocyte Lysate method was used. RESULT There was consistent gel formation with positive analysis in eight instruments, corresponding to 13.3%, being four femoral rasps and four bone reamers. CONCLUSION Endotoxins in quantity ≥0.125 UE/mL were detected in 13.3% of the instruments tested.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence.

Science.gov (United States)

Xie, Yicheng; Wahab, Laith; Gill, Jason J

2018-04-12

Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6%) of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence

Directory of Open Access Journals (Sweden)

Yicheng Xie

2018-04-01

Full Text Available Bacteriophages, which are the natural predators of bacteria, have re-emerged as an attractive alternative to combat antibiotic resistant bacteria. Phages are highly specific at the species and strain level and measurement of the phage host range plays an important role in utilizing the phage as antimicrobials. The most common method for phage host range determination has been to spot phage lysates on soft agar overlays and observe plaque formation. In this study, a liquid culture-based assay was developed in a 96-well microtiter plate format to measure the phage host range and virulence for a collection of 15 Salmonella phages against a panel of 20 Salmonella strains representing 11 serovars. This method was compared to a traditional spot method. The majority of the host range results from two methods were in agreement including in cases where a bacterial strain was insensitive to the phage. Each method produced a false-negative result in 19/300 (6% of the measured phage-host combinations when compared to the other method. The spot method tended to indicate greater phage sensitivity than the microtiter assay even though direct comparisons of the response magnitude between the two methods is difficult since they operate on different mechanisms. The microtiter plate assay was able to provide data on both the phage host range and virulence in greater resolution in a high-throughput format.

Clinical and Immunological Effects in Patients with Advanced Non-Small Cell Lung-Cancer after Vaccination with Dendritic Cells Exposed to an Allogeneic Tumor Cell Lysate*

DEFF Research Database (Denmark)

Engell-Noerregaard, Lotte; Kvistborg, Pia; Zocca, Mai-Britt

2013-01-01

Background: We evaluated the clinical and immunological effects of dendritic cell (DC) vaccination of patients with NSCLC. Autologous DCs were pulsed with a MAGE containing allogeneic melanoma cell lysate (MelCancerVac®, Dandrit Biotech, Copenhagen, Denmark). Imiquimod cream, proleukin and celeco......Background: We evaluated the clinical and immunological effects of dendritic cell (DC) vaccination of patients with NSCLC. Autologous DCs were pulsed with a MAGE containing allogeneic melanoma cell lysate (MelCancerVac®, Dandrit Biotech, Copenhagen, Denmark). Imiquimod cream, proleukin...... and celecoxib were used as adjuvants to the vaccines. The objective of the study was to evaluate specific T cell response in vitro by IFN EliSpot. Secondary objec- tives were overall survival, response and quality of life (QoL). Results: Twenty-two patients initiated the vaccination program consisting of ten...

Formation of lysinoalanine in egg white under alkali treatment.

Science.gov (United States)

Zhao, Yan; Luo, Xuying; Li, Jianke; Xu, Mingsheng; Tu, Yonggang

2016-03-01

To investigate the formation mechanism of lysinoalanine (LAL) in eggs during the alkali treatment process, NaOH was used for the direct alkali treatment of egg white, ovalbumin, and amino acids; in addition, the amount of LAL formed during the alkali treatment process was measured. The results showed that the alkali treatment resulted in the formation of LAL in the egg white. The LAL content increased with increasing pH and temperature, with the LAL content first increasing and then leveling off with increasing time. The amount of LAL formed in the ovalbumin under the alkali treatment condition accounted for approximately 50.51% to 58.68% of the amount of LAL formed in the egg white. Thus, the LAL formed in the ovalbumin was the main source for the LAL in the egg white during the alkali treatment process. Under the alkali treatment condition, free L-serine, L-cysteine, and L-cystine reacted with L-lysine to form LAL; therefore, they are the precursor amino acids of LAL formed in eggs during the alkali treatment process. © 2016 Poultry Science Association Inc.

Development of Novel Technetium-99m-Labeled Steroids as Estrogen-Responsive Breast Cancer Imaging Agents

Science.gov (United States)

2007-06-01

incubations performed at room temperature overnight with lysates of Escherichia coli in which the LBD of human ERα ( M250 -V595) is expressed [25,26...lysates of Escherichia coli in which the LBD of human ERα ( M250 –V595)(5) is expressed as described (6) The assay was performed overnight in phosphate

Autologous platelet lysate local injections for the treatment of refractory lateral epicondylitis.

Science.gov (United States)

Tan, Xun-xiang; Ju, Hai-yang; Yan, Wei; Jiang, Hong-jiang; Su, Jin-ping; Dong, Hua-jun; Wang, Ling-shuang; Zou, De-bao

2016-01-25

The purpose of this study was to investigate the efficacy and safety of autologous platelet lysate (APL) local injections in reducing pain and improving function in patients with refractory lateral epicondylitis. A total of 56 patients with refractory lateral epicondylitis were enrolled in this study. All the patients received three injections in one course of treatment. Subjective assessments include visual analog scale (VAS) pain score and Mayo elbow score before injection (baseline) and at 1, 6, and 12 months after injection. Significant differences were observed in VAS and Mayo scores at baseline and at 1, 6, and 12 months after injection. Overall, the injections of APL improved local symptoms and all the patients recovered to normal elbow function with 12 months follow-up. Local injections of APL resulted in favorable clinical outcomes for the treatment of lateral epicondylitis. APL could be clinically effective in the treatment of lateral epicondylitis.

The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates

International Nuclear Information System (INIS)

Jayaram, Jyothi; Youn, Soonjeon; Collisson, Ellen W.

2005-01-01

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid protein (N) may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. 32 P-orthophosphate labeling and Western blot analyses confirmed that N was the only viral protein that was phosphorylated. Pulse labeling with 32 P-orthophosphate indicated that the IBV N protein was phosphorylated in the virion, as well as at all times during infection in either chicken embryo kidney cells or Vero cells. Pulse-chase analyses followed by immunoprecipitation of IBV N proteins using rabbit anti-IBV N polyclonal antibody demonstrated that the phosphate on the N protein was stable for at least 1 h. Simultaneous labeling with 32 P-orthophosphate and 3 H-leucine identified a 3.5-fold increase in the 32 P: 3 H counts per minute (cpm) ratio of N in the virion as compared to the 32 P: 3 H cpm ratio of N in the cell lysates from chicken embryo kidney cells, whereas in Vero cells the 32 P: 3 H cpm ratio of N from the virion was 10.5-fold greater than the 32 P: 3 H cpm ratio of N from the cell lysates. These studies are consistent with the phosphorylation of the IBV N playing a role in assembly or maturation of the viral particle

Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells.

Science.gov (United States)

Wang, Chuan; Xiao, Ran; Cao, Yi-Lin; Yin, Hong-Yu

2017-09-09

Cryopreservation provides an effective technique to maintain the functional properties of human adipose-derived stem cells (ASCs). Dimethylsulfoxide (DMSO) and fetal bovine serum (FBS) are frequently used as cryoprotectants for this purpose. However, the use of DMSO can result in adverse effects and toxic reactions and FBS can introduce risks of viral, prion, zoonose contaminations and evoke immune responses after injection. It is therefore crucial to reduce DMSO concentrations and use serum-free solution in the cryopreservation process. Human platelet lysate (PL) is a promising candidate for use as an alternative to DMSO and FBS. Therefore, in this study, with an aim to identify a cryoprotective agent for ASC cryopreservation, we determined the viability, proliferation potential, phenotype, and differentiation potential of fresh ASCs and ASCs cryopreserved using different combinations of three cryoprotective agents: fetal bovine serum (FBS), dimethylsulfoxide (DMSO), and human platelet lysate (PL). The viability of the ASCs cryopreserved with 90% FBS and 10% DMSO, 95% FBS and 5% DMSO, and 97% PL and 3% DMSO was >80%, and the proliferation potentials, cell phenotypes, and differentiation potentials of these groups were similar to those of fresh ASCs. Together, our findings suggest that a combination of 97% PL and 3% DMSO is an ideal cryoprotective agent for the efficient cryopreservation of human ASCs. Copyright © 2017 Elsevier Inc. All rights reserved.

BACTERIAL LYSATES FOR TOPICAL APPLICATION IN PREVENTION AND TREATMENT OF CHRONIC TONSILLITIS AMONG CHILDREN

Directory of Open Access Journals (Sweden)

T.I. Garashchenko

2006-01-01

Full Text Available The authors have carried on the research of the influence Imudon exerts (during topical application on the run of chronic tonsillitis among 48 children aged between 5 and 10 years old, being dispensary registrants. The sublingual application of a medicine 6 pills daily within 20 days demonstrated the frequency reduction of chronic tonsillitis acerbations by 2.9 times, as well as the reduction of total need in systemic antibacterial treatment by 10 times. Apart from that, the frequency of S. pyogenes group a exposure reduced by 3 times. The researchers noticed the tendency to normalization of pharynx biocenosis. Thus, Imudon may be recommended for the daily courses of treatment to the people, suffering from chronic tonsillitis, palatine tonsil auxesis and recurrent tonsillo-pharyngites.Key words: chronic tonsillitis, children, prevention, bacterial lysates.

Pathogen-free, plasma-poor platelet lysate and expansion of human mesenchymal stem cells.

Science.gov (United States)

Iudicone, Paola; Fioravanti, Daniela; Bonanno, Giuseppina; Miceli, Michelina; Lavorino, Claudio; Totta, Pierangela; Frati, Luigi; Nuti, Marianna; Pierelli, Luca

2014-01-27

Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC. PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation. PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic

Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

National Research Council Canada - National Science Library

Johnson, Erik A; Daugherty, Kelly S

2006-01-01

... behavioral and protein changes due to the absence of guinea pig-specific antibodies. We have developed a procedure to determine the specificity of commercially available, non-guinea pig-specific antibodies in guinea pig lysates...

SH2-PLA: a sensitive in-solution approach for quantification of modular domain binding by proximity ligation and real-time PCR.

Science.gov (United States)

Thompson, Christopher M; Bloom, Lee R; Ogiue-Ikeda, Mari; Machida, Kazuya

2015-06-26

There is a great interest in studying phosphotyrosine dependent protein-protein interactions in tyrosine kinase pathways that play a critical role in many aspects of cellular function. We previously established SH2 profiling, a phosphoproteomic approach based on membrane binding assays that utilizes purified Src Homology 2 (SH2) domains as a molecular tool to profile the global tyrosine phosphorylation state of cells. However, in order to use this method to investigate SH2 binding sites on a specific target in cell lysate, additional procedures such as pull-down or immunoprecipitation which consume large amounts of sample are required. We have developed PLA-SH2, an alternative in-solution modular domain binding assay that takes advantage of Proximity Ligation Assay and real-time PCR. The SH2-PLA assay utilizes oligonucleotide-conjugated anti-GST and anti-EGFR antibodies recognizing a GST-SH2 probe and cellular EGFR, respectively. If the GST-SH2 and EGFR are in close proximity as a result of SH2-phosphotyrosine interactions, the two oligonucleotides are brought within a suitable distance for ligation to occur, allowing for efficient complex amplification via real-time PCR. The assay detected signal across at least 3 orders of magnitude of lysate input with a linear range spanning 1-2 orders and a low femtomole limit of detection for EGFR phosphotyrosine. SH2 binding kinetics determined by PLA-SH2 showed good agreement with established far-Western analyses for A431 and Cos1 cells stimulated with EGF at various times and doses. Further, we showed that PLA-SH2 can survey lung cancer tissues using 1 μl lysate without requiring phospho-enrichment. We showed for the first time that interactions between SH2 domain probes and EGFR in cell lysate can be determined in a microliter-scale assay using SH2-PLA. The obvious benefit of this method is that the low sample requirement allows detection of SH2 binding in samples which are difficult to analyze using traditional protein

Comparison of clinical grade human platelet lysates for cultivation of mesenchymal stromal cells from bone marrow and adipose tissue

DEFF Research Database (Denmark)

Juhl, Morten; Tratwal, Josefine; Follin, Bjarke

2016-01-01

be devoid of any animal derived components. We have evaluated whether human Platelet Lysate (hPL) could be an attractive alternative to animal supplements. METHODS: MSCs from bone marrow (BMSCs) and adipose tissue-derived stromal cells (ASCs) obtained from three donors were culture expanded in three...... culture conditions with 10% fetal bovine serum (FBS). Cell morphology, proliferation, phenotype, genomic stability, and differentiation potential were analyzed. RESULTS: Regardless of manufacturer, BMSCs and ASCs cultured in hPL media showed a significant increase in proliferation capacity compared to FBS...

Effect of basic alkali-pickling conditions on the production of lysinoalanine in preserved eggs.

Science.gov (United States)

Zhao, Yan; Luo, Xuying; Li, Jianke; Xu, Mingsheng; Tu, Yonggang

2015-09-01

During the pickling process, strong alkali causes significant lysinoalanine (LAL) formation in preserved eggs, which may reduce the nutritional value of the proteins and result in a potential hazard to human health. In this study, the impacts of the alkali treatment conditions on the production of LAL in preserved eggs were investigated. Preserved eggs were prepared using different times and temperatures, and alkali-pickling solutions with different types and concentrations of alkali and metal salts, and the corresponding LAL contents were measured. The results showed the following: during the pickling period of the preserved egg, the content of LAL in the egg white first rapidly increased and then slowly increased; the content of LAL in the egg yolk continued to increase significantly. During the aging period, the levels of LAL in both egg white and egg yolk slowly increased. The amounts of LAL in the preserved eggs were not significantly different at temperatures between 20 and 25ºC. At higher pickling temperatures, the LAL content in the preserved eggs increased. With the increase of alkali concentration in the alkali-pickling solution, the LAL content in the egg white and egg yolk showed an overall trend of an initial increase followed by a slight decrease. The content of LAL produced in preserved eggs treated with KOH was lower than in those treated with NaOH. NaCl and KCl produced no significant effects on the production of LAL in the preserved eggs. With increasing amounts of heavy metal salts, the LAL content in the preserved eggs first decreased and then increased. The LAL content generated in the CuSO4 group was lower than that in either the ZnSO4 or PbO groups. © 2015 Poultry Science Association Inc.

GMP-grade platelet lysate enhances proliferation and migration of tenon fibroblasts.

Science.gov (United States)

Carducci, Augusto; Scafetta, Gaia; Siciliano, Camilla; Carnevale, Roberto; Rosa, Paolo; Coccia, Andrea; Mangino, Giorgio; Bordin, Antonella; Vingolo, Enzo Maria; Pierelli, Luca; Lendaro, Eugenio; Ragona, Giuseppe; Frati, Giacomo; De Falco, Elena

2016-01-01

Tenon's fibroblasts (TFs), widely employed as in vitro model for many ophthalmological studies, are routinely cultured with FBS. Platelet Lysate (PL), a hemoderivate enriched with growth factors and cytokines has been largely tested in several clinical applications and as substitute of FBS in culture. Here, we investigate whether PL can exert biological effects on TF populations similarly to other cell types. Results show that PL significantly enhances cell proliferation and migration vs. FBS, without influencing cell size/granularity. Upregulation of EGF, VEGF, KDR, MMP2-9, FAK mRNA levels also occurs and phosphorylation of AKT but not of ERK1/2 is significantly enhanced. The inhibition of the PI3kinase/AKT pathway with the specific inhibitor wortmannin, decreases PL-induced cell migration but not proliferation. Condition supernatants containing PL show increased bioavailability of Nitric Oxide and reduced levels of 8-Iso-PGF2-alpha, correlating with cell proliferation and migration. Pro-angiogenic/inflammatory soluble factors (GRO, Angiogenin, EGF, I-309, PARC) are exclusively or greater expressed in media containing PL than FBS. GMP-grade PL preparations positively influence in vitro biological effects of TFs representing a suitable and safer alternative to FBS.

Toxic cyanobacteria and cyanotoxins in public hot springs in Saudi Arabia.

Science.gov (United States)

Mohamed, Zakaria A

2008-01-01

Toxic cyanobacteria are well reported in rivers, lakes and even marine environments, but the toxin production of cyanobacteria in hot springs is largely unexplored. Therefore, the present study investigated the presence of toxic cyanobacteria and cyanotoxins in public hot springs in Saudi Arabia. The results of an enzyme-linked immunosorbent assay (ELISA) revealed that Saudi spring cyanobacterial mats contained microcystins (MCYSTs) at concentrations ranging from 468 to 512.5 microg g(-1). The Limulus amebocyte lystae (LAL) assay detected lipopolysaccharide (LPS) endotoxins in these mats at concentrations ranging from 433.3 to 506.8 EU g(-1). MCYSTs and endotoxins were also detected in spring waters at levels of 5.7 microg l(-1) and 640 EU ml(-1), respectively, exceeding WHO's provisional guideline value for MCYST-LR in drinking-water. High-performance liquid chromatography (HPLC) analysis revealed that only Oscillatoria limosa and Synechococcus lividus can produce MCYSTs with a profile consisting of MCYST-RR and -LR. Based on the LAL assay, 12 out of 17 cyanobacterial species contained LPS at concentrations ranging from 0.93 to 21.06 EU g(-1). However, not all LPS of these species were toxic to mice. This study suggests that the hot springs in the world including Saudi Arabia should be screened for toxic cyanobacteria to avoid the exposure of people recreating and bathing in spring waters to cyanobacterial toxins.

Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA).

Science.gov (United States)

Narayan, Malathi; Seeley, Kent W; Jinwal, Umesh K

2016-06-01

Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA) on a mouse microglial (N9) cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier PXD003032.

Human platelet lysate stimulates high-passage and senescent human multipotent mesenchymal stromal cell growth and rejuvenation in vitro.

Science.gov (United States)

Griffiths, Sarah; Baraniak, Priya R; Copland, Ian B; Nerem, Robert M; McDevitt, Todd C

2013-12-01

Multipotent mesenchymal stromal cells (MSCs) are clinically useful because of their immunomodulatory and regenerative properties, but MSC therapies are limited by the loss of self-renewal and cell plasticity associated with ex vivo expansion culture and, on transplantation, increased immunogenicity from xenogen exposure during culture. Recently, pooled human platelet lysate (hPL) has been used as a culture supplement to promote MSC growth; however, the effects of hPL on MSCs after fetal bovine serum (FBS) exposure remain unknown. MSCs were cultured in medium containing FBS or hPL for up to 16 passages, and cell size, doubling time and immunophenotype were determined. MSC senescence was assessed by means of a fluorometric assay for endogenous β-galactosidase expression. MSCs cultured with FBS for different numbers of passages were switched to hPL conditions to evaluate the ability of hPL to "rescue" the proliferative capacity of MSCs. hPL culture resulted in more rapid cell proliferation at earlier passages (passage 5 or earlier) than remove FBS; by day 4, hPL (5%) yielded an MSC doubling time of 1.28 days compared with 1.52 days in 16% FBS. MSCs cultured first in FBS and switched to hPL proliferated more and demonstrated less β-galactosidase production and smaller cell sizes than remove MSCs continuously propagated in FBS. hPL enables rapid expansion of MSCs without adversely affecting immunophenotype. hPL culture of aged and senescent MSCs demonstrated cellular rejuvenation, reflected by decreased doubling time and smaller cell size. These results suggest that expansion of MSCs in hPL after FBS exposure can enhance cell phenotype and proliferative capacity. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Increased efficiency of exogenous messenger RNA translation in a Krebs ascites cell lysate.

Science.gov (United States)

Metafora, S; Terada, M; Dow, L W; Marks, P A; Bank, A

1972-05-01

Addition of a 0.5 M KCl wash fraction from rabbit reticulocyte ribosomes causes a 3- to 10-fold increase in the extent of translation of natural mRNAs by Krebs-cell lysates. In the presence of the wash fraction, 1 pmol of rabbit or mouse 10S RNA directs the incorporation of 80 pmol of leucine into rabbit globin. The addition of human 10S RNA results in the synthesis of equal amounts of human alpha and beta chains, identified by column chromatography. The stimulation by the wash fraction is almost completely dependent on added mammalian tRNA. In contrast to the wash fraction from rabbit reticulocytes, the wash fraction isolated from Krebs-cell ribosomes is inhibitory to both endogenous and exogenous mRNA translation. The stimulation by the wash fraction from rabbit ribosomes is not specific for globin mRNAs, but also increases endogenous, phage Qbeta, and viral RNA-directed protein synthesis.

Ligament Tissue Engineering Using a Novel Porous Polycaprolactone Fumarate Scaffold and Adipose Tissue-Derived Mesenchymal Stem Cells Grown in Platelet Lysate

Science.gov (United States)

Wagner, Eric R.; Bravo, Dalibel; Dadsetan, Mahrokh; Riester, Scott M.; Chase, Steven; Westendorf, Jennifer J.; Dietz, Allan B.; van Wijnen, Andre J.; Yaszemski, Michael J.

2015-01-01

Purpose: Surgical reconstruction of intra-articular ligament injuries is hampered by the poor regenerative potential of the tissue. We hypothesized that a novel composite polymer “neoligament” seeded with progenitor cells and growth factors would be effective in regenerating native ligamentous tissue. Methods: We synthesized a fumarate-derivative of polycaprolactone fumarate (PCLF) to create macro-porous scaffolds to allow cell–cell communication and nutrient flow. Clinical grade human adipose tissue-derived human mesenchymal stem cells (AMSCs) were cultured in 5% human platelet lysate (PL) and seeded on scaffolds using a dynamic bioreactor. Cell growth, viability, and differentiation were examined using metabolic assays and immunostaining for ligament-related markers (e.g., glycosaminoglycans [GAGs], alkaline phosphatase [ALP], collagens, and tenascin-C). Results: AMSCs seeded on three-dimensional (3D) PCLF scaffolds remain viable for at least 2 weeks with proliferating cells filling the pores. AMSC proliferation rates increased in PL compared to fetal bovine serum (FBS) (p < 0.05). Cells had a low baseline expression of ALP and GAG, but increased expression of total collagen when induced by the ligament and tenogenic growth factor fibroblast growth factor 2 (FGF-2), especially when cultured in the presence of PL (p < 0.01) instead of FBS (p < 0.05). FGF-2 and PL also significantly increased immunostaining of tenascin-C and collagen at 2 and 4 weeks compared with human fibroblasts. Summary: Our results demonstrate that AMSCs proliferate and eventually produce a collagen-rich extracellular matrix on porous PCLF scaffolds. This novel scaffold has potential in stem cell engineering and ligament regeneration. PMID:26413793

Microbial translocation is associated with increased monocyte activation and dementia in AIDS patients.

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Petronela Ancuta

2008-06-01

Full Text Available Elevated plasma lipopolysaccharide (LPS, an indicator of microbial translocation from the gut, is a likely cause of systemic immune activation in chronic HIV infection. LPS induces monocyte activation and trafficking into brain, which are key mechanisms in the pathogenesis of HIV-associated dementia (HAD. To determine whether high LPS levels are associated with increased monocyte activation and HAD, we obtained peripheral blood samples from AIDS patients and examined plasma LPS by Limulus amebocyte lysate (LAL assay, peripheral blood monocytes by FACS, and soluble markers of monocyte activation by ELISA. Purified monocytes were isolated by FACS sorting, and HIV DNA and RNA levels were quantified by real time PCR. Circulating monocytes expressed high levels of the activation markers CD69 and HLA-DR, and harbored low levels of HIV compared to CD4(+ T-cells. High plasma LPS levels were associated with increased plasma sCD14 and LPS-binding protein (LBP levels, and low endotoxin core antibody levels. LPS levels were higher in HAD patients compared to control groups, and were associated with HAD independently of plasma viral load and CD4 counts. LPS levels were higher in AIDS patients using intravenous heroin and/or ethanol, or with Hepatitis C virus (HCV co-infection, compared to control groups. These results suggest a role for elevated LPS levels in driving monocyte activation in AIDS, thereby contributing to the pathogenesis of HAD, and provide evidence that cofactors linked to substance abuse and HCV co-infection influence these processes.

Induction of IL-1 during hemodialysis: Transmembrane passage of intact endotoxins (LPS)

Energy Technology Data Exchange (ETDEWEB)

Laude-Sharp, M.; Caroff, M.; Simard, L.; Pusineri, C.; Kazatchkine, M.D.; Haeffner-Cavaillon, N. (INSERM U 28, Hopital Broussais, Paris (France))

1990-12-01

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes.

Induction of IL-1 during hemodialysis: Transmembrane passage of intact endotoxins (LPS)

International Nuclear Information System (INIS)

Laude-Sharp, M.; Caroff, M.; Simard, L.; Pusineri, C.; Kazatchkine, M.D.; Haeffner-Cavaillon, N.

1990-01-01

Circulating monocytes of patients undergoing chronic hemodialysis are triggered to produce interleukin-1 (IL-1) in vivo. Intradialytic induction of IL-1 is associated with complement activation in patients dialyzed with first-use cellulose membranes. Chronic stimulation of IL-1 production occurs because of an yet unidentified mechanism in patients dialyzed with high permeability membranes. The present study demonstrates that intact bacterial lipopolysaccharide (LPS) molecules may cross cuprophan, AN69 and polysulfone membranes under in vitro conditions simulating in vivo hemodialysis. The experiments used purified LPS from Neisseria meningitidis and LPS from Pseudomonas testosteroni, a bacterial strain grown out from a clinically used dialysate. LPS were purified to homogeneity and radiolabeled. Transmembrane passage of 3H-labeled LPS was observed within the first five minutes of dialysis. A total of 0.1 to 1% of 3H-labeled LPS were recovered in the dialysate compartment after one hour of dialysis. High amounts of LPS, representing 40 to 70% of the amount originally present in the dialysate, were absorbed onto high permeability membranes. Low amounts of LPS were absorbed onto cuprophan membranes. The amount of LPS absorbed decreased with the concentration of LPS in the dialysate. LPS recovered from the blood compartment exhibited the same molecular weight as that used to contaminate the dialysate. Biochemically detectable transmembrane passage of LPS was not associated with that of material detectable using the limulus amebocyte lysate (LAL) assay. An IL-1-inducing activity was, however, detected in the blood compartment upon dialysis with high permeability membranes, as previously found by others with cuprophan membranes

Lab-on-a-Chip: From Astrobiology to the International Space Station

Science.gov (United States)

Maule, Jake; Wainwright, Nor; Steele, Andrew; Gunter, Dan; Monaco, Lisa A.; Wells, Mark E.; Morris, Heather C.; Boudreaux, Mark E.

2008-01-01

The continual and long-term habitation of enclosed environments, such as Antarctic stations, nuclear submarines and space stations, raises unique engineering, medical and operational challenges. There is no easy way out and no easy way to get supplies in. This situation elevates the importance of monitoring technology that can rapidly detect events within the habitat that affect crew safety such as fire, release of toxic chemicals and hazardous microorganisms. Traditional methods to monitor microorganisms on the International Space Station (ISS) have consisted of culturing samples for 3-5 days and eventual sample return to Earth. To augment these culture methods with new, rapid molecular techniques, we developed the Lab-on-a-Chip Application Development - Portable Test System (LOCAD-PTS). The system consists of a hand-held spectrophotometer, a series of interchangeable cartridges and a surface sampling/dilution kit that enables crew to collect samples and detect a range of biological molecules, all within 15 minutes. LOCAD-PTS was launched to the ISS aboard Space Shuttle Discovery in December 2006, where it was operated for the first time during March-May 2007. The surfaces of five separate sites in the US Lab and Node 1 of ISS were analyzed for endotoxin, using cartridges that employ the Limulus Amebocyte Lysate (LAL) assay; results of these tests will be presented. LOCAD-PTS will remain permanently onboard ISS with new cartridges scheduled for launch in February and October of 2008 for the detection of fungi (Beta-glucan) and Gram-positive bacteria (lipoteichoic acid), respectively.

Sponge-Like Dressings Based on the Association of Chitosan and Sericin for the Treatment of Chronic Skin Ulcers. II. Loading of the Hemoderivative Platelet Lysate.

Science.gov (United States)

Mori, Michela; Rossi, Silvia; Ferrari, Franca; Bonferoni, Maria C; Sandri, Giuseppina; Riva, Federica; Tenci, Marika; Del Fante, Claudia; Nicoletti, Giovanni; Caramella, Carla

2016-03-01

Platelet lysate (PL) was loaded into dressings based on chitosan glutamate (CSG) low and high molecular weight, sericin (Ser), and glycine (Gly). A synergic effect of Ser and PL on fibroblast proliferation was proved in vitro. Two different PL loading approaches were considered: the first provided to prepare dressings by freeze-drying a mixture of PL and CSG/Gly/Ser solution, the second approach consisted in the extemporarily loading of PL in the CSG/Gly/Ser freeze-dried dressings. As for the first approach, PL loading did not produce any variation in dressing mechanical properties. Such dressings absorbed a high amount (about 8-fold of dry weight) of phosphate-buffered saline (fluid mimicking wound exudate), forming a gel with pseudoplastic and elastic properties. Platelet-derived growth factor AB assay indicated that neither freeze-drying nor the excipients alter PL growth factor content. As for the second approach, mechanical and rheological properties of the gel formed upon PL absorption enabled to choose a PL loading of about 90 μL/cm(2). Upon contact with fibroblasts, all PL loaded formulations increased the number not only of viable cells but also of those in the proliferative phase. Histological studies effected on human skin strips pointed out the positive effect of PL loaded dressings on dermal matrix reconstruction. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Gene expression profiling of chicken cecal tonsils and ileum following oral exposure to soluble and PLGA-encapsulated CpG ODN, and lysate of Campylobacter jejuni.

Science.gov (United States)

Taha-Abdelaziz, Khaled; Alkie, Tamiru Negash; Hodgins, Douglas C; Yitbarek, Alexander; Shojadoost, Bahram; Sharif, Shayan

2017-12-01

Campylobacter jejuni (C. jejuni) is a leading bacterial cause of food-borne illness in humans. Contaminated chicken meat is an important source of infection for humans. Chickens are not clinically affected by colonization, and immune responses following natural infection have limited effects on bacterial load in the gut. Induction of intestinal immune responses may possibly lead to a breakdown of the commensal relationship of chickens with Campylobacter. We have recently shown that soluble and poly D, L-lactic-co-glycolic acid (PLGA)-encapsulated CpG oligodeoxynucleotide (ODN) as well as C. jejuni lysate, are effective in reducing the intestinal burden of C. jejuni in chickens; however, the mechanisms behind this protection have yet to be determined. The present study was undertaken to investigate the mechanisms of host responses conferred by these treatments. Chickens were treated orally with soluble CpG ODN, or PLGA-encapsulated CpG ODN, or C. jejuni lysate, and expression of cytokines and antimicrobial peptides was evaluated in cecal tonsils and ileum using quantitative RT-PCR. Oral administration of soluble CpG ODN upregulated the expression of interferon (IFN)-γ, interleukin (IL)-1β, CXCLi2, transforming growth factor (TGF)-β4/1, IL-10 and IL-13, while treatment with PLGA-encapsulated CpG ODN upregulated the expression of IL-1β, CXCLi2, TGF-β4/1, IL-13, avian β-defensin (AvBD) 1, AvBD2 and cathelicidin 3 (CATHL-3). C. jejuni lysate upregulated the expression of IFN-γ, IL-1β, TGF-β4/1, IL-13, AvBD1, and CATHL-3. In conclusion, induction of cytokine and antimicrobial peptides expression in intestinal microenvironments may provide a means of reducing C. jejuni colonization in broiler chickens, a key step in reducing the incidence of campylobacteriosis in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

A novel platelet lysate hydrogel for endothelial cell and mesenchymal stem cell-directed neovascularization.

Science.gov (United States)

Robinson, Scott T; Douglas, Alison M; Chadid, Tatiana; Kuo, Katie; Rajabalan, Ajai; Li, Haiyan; Copland, Ian B; Barker, Thomas H; Galipeau, Jacques; Brewster, Luke P

2016-05-01

Mesenchymal stem cells (MSC) hold promise in promoting vascular regeneration of ischemic tissue in conditions like critical limb ischemia of the leg. However, this approach has been limited in part by poor cell retention and survival after delivery. New biomaterials offer an opportunity to localize cells to the desired tissue after delivery, but also to improve cell survival after delivery. Here we characterize the mechanical and microstructural properties of a novel hydrogel composed of pooled human platelet lysate (PL) and test its ability to promote MSC angiogenic activity using clinically relevant in vitro and in vivo models. This PL hydrogel had comparable storage and loss modulus and behaved as a viscoelastic solid similar to fibrin hydrogels despite having 1/4-1/10th the fibrin content of standard fibrin gels. Additionally, PL hydrogels enabled sustained release of endogenous PDGF-BB for up to 20days and were resistant to protease degradation. PL hydrogel stimulated pro-angiogenic activity by promoting human MSC growth and invasion in a 3D environment, and enhancing endothelial cell sprouting alone and in co-culture with MSCs. When delivered in vivo, the combination of PL and human MSCs improved local tissue perfusion after 8days compared to controls when assessed with laser Doppler perfusion imaging in a murine model of hind limb ischemia. These results support the use of a PL hydrogel as a scaffold for MSC delivery to promote vascular regeneration. Innovative strategies for improved retention and viability of mesenchymal stem cells (MSCs) are needed for cellular therapies. Human platelet lysate is a potent serum supplement that improves the expansion of MSCs. Here we characterize our novel PL hydrogel's desirable structural and biologic properties for human MSCs and endothelial cells. PL hydrogel can localize cells for retention in the desired tissue, improves cell viability, and augments MSCs' angiogenic activity. As a result of these unique traits, PL

Layer-by-layer assembled cell instructive nanocoatings containing platelet lysate.

Science.gov (United States)

Oliveira, Sara M; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L; Mano, João F

2015-04-01

Great efforts have been made to introduce growth factors (GFs) onto 2D/3D constructs in order to control cell behavior. Platelet lysate (PL) presents itself as a cost-effective source of multiple GFs and other proteins. The instruction given by a construct-PL combination will depend on how its instructive cues are presented to the cells. The content, stability and conformation of the GFs affect their instruction. Strategies for a controlled incorporation of PL are needed. Herein, PL was incorporated into nanocoatings by layer-by-layer assembling with polysaccharides presenting different sulfation degrees (SD) and charges. Heparin and several marine polysaccharides were tested to evaluate their PL and GF incorporation capability. The consequent effects of those multilayers on human adipose derived stem cells (hASCs) were assessed in short-term cultures. Both nature of the polysaccharide and SD were important properties that influenced the adsorption of PL, vascular endothelial growth factor (VEGF), fibroblast growth factor b (FGFb) and platelet derived growth factor (PDGF). The sulfated polysaccharides-PL multilayers showed to be efficient in the promotion of morphological changes, serum-free adhesion and proliferation of high passage hASCs (P > 5). These biomimetic multilayers promise to be versatile platforms to fabricate instructive devices allowing a tunable incorporation of PL. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of glutathione in supernatants and lysates of a human proximal tubular cell line from perfusion culture upon intoxication with cadmium chloride by HPLC and LC-ESI-MS

NARCIS (Netherlands)

Hahn, Hans; Huck, Christian W; Rainer, Matthias; Najam-ul-Haq, Muhammad; Bakry, Rania; Abberger, Thomas; Jennings, Paul; Pfaller, Walter; Bonn, Günther K

A simple and highly effective reversed-phase (RP) high-performance liquid chromatography (HPLC) method is described for analysing glutathione (GSH) and glutathione disulfide (GSSG) in out-flowing supernatants and lysates of perfusion cell cultures of human kidney cells (HK-2 cells) continuously

Cultivation and irradiation of human fibroblasts in a medium enriched with platelet lysate for obtaining feeder layer in epidermal cell culture; Cultivo e irradiacao de fibroblastos humanos em meio enriquecido com lisado de plaquetas para obtencao de camada de sustentacao em culturas de celulas da epiderme

Energy Technology Data Exchange (ETDEWEB)

Yoshito, Daniele

2011-07-01

For over 30 years, the use of culture medium, enriched with bovine serum, and murines fibroblasts, with the rate of proliferation controlled by irradiation or by share anticarcinogenic drugs, has been playing successfully its role in assisting in the development of keratinocytes in culture, for clinical purposes. However, currently there is a growing concern about the possibility of transmitting prions and animals viruses to transplanted patients. Taking into account this concern, the present work aims to cultivate human fibroblasts in a medium enriched with human platelets lysate and determine the irradiation dose of these cells, for obtaining feeder layer in epidermal cell culture. For carrying out the proposed objective, platelets lysis has standardized, this lysate was used for human fibroblasts cultivation and the irradiation dose enough to inhibit its duplication was evaluated. Human keratinocytes were cultivated in these feeder layers, in culture medium enriched with the lysate. With these results we conclude that the 10% platelets lysate promoted a better adhesion and proliferation of human fibroblasts and in all dose levels tested (60 to 300 Gy), these had their mitotic activity inactivated by ionizing irradiation, being that the feeder layers obtained with doses from 70 to 150 Gy were those that provided the best development of keratinocytes in medium containing 2.5% of human platelet lysate. Therefore, it was possible to standardize both the cultivation of human fibroblasts as its inactivation for use as feeder layer in culture of keratinocytes, so as to eliminate xenobiotics components. (author)

Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system.

Science.gov (United States)

Buntru, Matthias; Vogel, Simon; Spiegel, Holger; Schillberg, Stefan

2014-05-03

Cell-free protein synthesis is a rapid and efficient method for the production of recombinant proteins. Usage of prokaryotic cell-free extracts often leads to non-functional proteins. Eukaryotic counterparts such as wheat germ extract (WGE) and rabbit reticulocyte lysate (RLL) may improve solubility and promote the correct folding of eukaryotic multi-domain proteins that are difficult to express in bacteria. However, the preparation of WGEs is complex and time-consuming, whereas RLLs suffer from low yields. Here we report the development of a novel cell-free system based on tobacco Bright Yellow 2 (BY-2) cells harvested in the exponential growth phase. The highly-productive BY-2 lysate (BYL) can be prepared quickly within 4-5 h, compared to 4-5 d for WGE. The efficiency of the BYL was tested using three model proteins: enhanced yellow fluorescent protein (eYFP) and two versions of luciferase. The added mRNA was optimized by testing different 5' and 3' untranslated regions (UTRs). The protein yield in batch and dialysis reactions using BYL was much higher than that of a commercial Promega WGE preparation, achieving a maximum yield of 80 μg/mL of eYFP and 100 μg/mL of luciferase, compared to only 45 μg/mL of eYFP and 35 μg/mL of luciferase in WGEs. In dialysis reactions, the BYL yielded about 400 μg/mL eYFP, representing up to 50% more of the target protein than the Promega WGE, and equivalent to the amount using 5Prime WGE system. Due to the high yield and the short preparation time the BYL represents a remarkable improvement over current eukaryotic cell-free systems.

PDGF-AB rich-trombocyte lysate supplementation from breast cancer patients increased the proliferation of breast cancer stem cells

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Wiwi A. Kartolo

2018-05-01

Full Text Available Background: Thrombocytosis in breast cancer (BC patient was thought to play a role in the invasiveness of breast cancer stem cells (BCSCs. Modification of tumor microenvironment was proposed to increase the efficacy of anticancer therapy. This study was aimed to analyze the effect of platelet lysate (PL as well as its PDGF-AB content as a tumor microenvironment on (CD24-/CD44+ BCSC proliferation.Methods: This was an experimental study that treated culture of BCSCs with PL from breast cancer (BC patients or healthy donors. Venous blood from all subjects were subjected to prior hematology test and then processed to obtain platelet rich plasma  (PRP. Platelet counts in PRP were determined. PRP was processed to obtain PL. PDGF-AB contents in PL were measured. PL at concentrations of 0.01% (v/v was supplemented into DMEM-F12 medium and used for culturing BCSCs (CD24-/CD44+ cells. After 48 hours, total cell count, population doubling time (PDT, and cell viability were calculated and their correlation with platelet count and PDGF-AB levels were analyzed.Results: BC patients (n=5 had higher platelet counts and PDGF-AB levels in PL compared to healthy donors (n=15, (p=0.02. PL from BC patients could stimulate the proliferation of BCSCs higher than healthy donors (p<0.001 and showed lower PDT value (p=0.001. Cell proliferation and PDT showed strong correlation with PDGF-AB level. This observation suggests that PDGF-AB has a role on BCSCs proliferation. PL showed no effect on BCSCs viability.Conclusion: Breast cancer patient platelet lysate stimulated BCSC proliferation.

High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere

Science.gov (United States)

Luis, Aurélie; Colotte, Marthe; Tuffet, Sophie; Bonnet, Jacques

2017-01-01

Conventional storage of blood-derived fractions relies on cold. However, lately, ambient temperature preservation has been evaluated by several independent institutions that see economic and logistic advantages in getting rid of the cold chain. Here we validated a novel procedure for ambient temperature preservation of DNA in white blood cell and buffy coat lysates based on the confinement of the desiccated biospecimens under anoxic and anhydrous atmosphere in original hermetic minicapsules. For this validation we stored encapsulated samples either at ambient temperature or at several elevated temperatures to accelerate aging. We found that DNA extracted from stored samples was of good quality with a yield of extraction as expected. Degradation rates were estimated from the average fragment size of denatured DNA run on agarose gels and from qPCR reactions. At ambient temperature, these rates were too low to be measured but the degradation rate dependence on temperature followed Arrhenius’ law, making it possible to extrapolate degradation rates at 25°C. According to these values, the DNA stored in the encapsulated blood products would remain larger than 20 kb after one century at ambient temperature. At last, qPCR experiments demonstrated the compatibility of extracted DNA with routine DNA downstream analyses. Altogether, these results showed that this novel storage method provides an adequate environment for ambient temperature long term storage of high molecular weight DNA in dehydrated lysates of white blood cells and buffy coats. PMID:29190767

High DNA stability in white blood cells and buffy coat lysates stored at ambient temperature under anoxic and anhydrous atmosphere.

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Anne-Lise Fabre

Full Text Available Conventional storage of blood-derived fractions relies on cold. However, lately, ambient temperature preservation has been evaluated by several independent institutions that see economic and logistic advantages in getting rid of the cold chain. Here we validated a novel procedure for ambient temperature preservation of DNA in white blood cell and buffy coat lysates based on the confinement of the desiccated biospecimens under anoxic and anhydrous atmosphere in original hermetic minicapsules. For this validation we stored encapsulated samples either at ambient temperature or at several elevated temperatures to accelerate aging. We found that DNA extracted from stored samples was of good quality with a yield of extraction as expected. Degradation rates were estimated from the average fragment size of denatured DNA run on agarose gels and from qPCR reactions. At ambient temperature, these rates were too low to be measured but the degradation rate dependence on temperature followed Arrhenius' law, making it possible to extrapolate degradation rates at 25°C. According to these values, the DNA stored in the encapsulated blood products would remain larger than 20 kb after one century at ambient temperature. At last, qPCR experiments demonstrated the compatibility of extracted DNA with routine DNA downstream analyses. Altogether, these results showed that this novel storage method provides an adequate environment for ambient temperature long term storage of high molecular weight DNA in dehydrated lysates of white blood cells and buffy coats.

Analyzing Protein Changes in Guinea Pig Tissue Lysates Using Non-guinea Pig Specific Antibodies: Procedures for Western Blotting and Examples Using 16 Individual Antibodies for Common CNS Proteins

Science.gov (United States)

2006-05-01

guinea pig model does present a significant problem...trying to correlate behavioral and protein changes due to the absence of guinea pig -specific antibodies. We...have developed a procedure to determine the specificity of commercially available, non- guinea pig -specific antibodies in guinea pig lysates.

Data from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with Withaferin A (WA

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Malathi Narayan

2016-06-01

Full Text Available Mass spectrometry data collected in a study analyzing the effect of withaferin A (WA on a mouse microglial (N9 cell line is presented in this article. Data was collected from SILAC-based quantitative analysis of lysates from mouse microglial cells treated with either WA or DMSO vehicle control. This article reports all the proteins that were identified in this analysis. The data presented here is related to the published research article on the effect of WA on the differential regulation of proteins in mouse microglial cells [1]. Mass spectrometry data has also been deposited in the ProteomeXchange with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003032.

Adjuvant treatment with the bacterial lysate (OM-85 improves management of atopic dermatitis: A randomized study.

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Christine Bodemer

Full Text Available Environmental factors play a major role on atopic dermatitis (AD which shows a constant rise in prevalence in western countries over the last decades. The Hygiene Hypothesis suggesting an inverse relationship between incidence of infections and the increase in atopic diseases in these countries, is one of the working hypothesis proposed to explain this trend.This study tested the efficacy and safety of oral administration of the bacterial lysate OM-85 (Broncho-Vaxom®, Broncho-Munal®, Ommunal®, Paxoral®, Vaxoral®, in the treatment of established AD in children.Children aged 6 months to 7 years, with confirmed AD diagnosis, were randomized in a double-blind, placebo-controlled trial to receive, in addition to conventional treatment with emollients and topical corticosteroids, 3.5mg of the bacterial extract OM-85 or placebo daily for 9 months. The primary end-point was the difference between groups in the occurrence of new flares (NF during the study period, evaluated by Hazard Ratio (HR derived from conditional Cox proportional hazard regression models accounting for repeated events.Among the 179 randomized children, 170 were analysed, 88 in the OM-85 and 82 in the placebo group. As expected most children in both treatment groups experienced at least 1 NF during the study period (75 (85% patients in the OM-85 group and 72 (88% in the placebo group. Patients treated with OM-85 as adjuvant therapy had significantly fewer and delayed NFs (HR of repeated flares = 0.80; 95% confidence interval (CI: 0.67-0.96, also when potential confounding factors, as family history of atopy and corticosteroids use, were taken into account (HR = 0.82; 95% CI: 0.69-0.98. No major side effect was reported, with comparable and good tolerability for OM-85 and placebo.Results show an adjuvant therapeutic effect of a well standardized bacterial lysate OM-85 on established AD.

Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates.

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Flores-Jasso, C Fabián; Salomon, William E; Zamore, Phillip D

2013-02-01

Small interfering RNAs (siRNAs) direct Argonaute proteins, the core components of the RNA-induced silencing complex (RISC), to cleave complementary target RNAs. Here, we describe a method to purify active RISC containing a single, unique small RNA guide sequence. We begin by capturing RISC using a complementary 2'-O-methyl oligonucleotide tethered to beads. Unlike other methods that capture RISC but do not allow its recovery, our strategy purifies active, soluble RISC in good yield. The method takes advantage of the finding that RISC partially paired to a target through its siRNA guide dissociates more than 300 times faster than a fully paired siRNA in RISC. We use this strategy to purify fly Ago1- and Ago2-RISC, as well as mouse AGO2-RISC. The method can discriminate among RISCs programmed with different guide strands, making it possible to deplete and recover specific RISC populations. Endogenous microRNA:Argonaute complexes can also be purified from cell lysates. Our method scales readily and takes less than a day to complete.

Development and Application of an Indirect Enzyme-Linked Immunosorbent Assay Using Recombinant Mag1 for Serodiagnosis of Toxoplasma gondii In Dogs.

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Zhuo, Xunhui; Sun, Hongchao; Zhang, Zhi; Luo, Jiaqing; Shan, Ying; Du, Aifang

2017-06-01

Serologic tests are widely accepted and applied as means to detect anti- Toxoplasma gondii immunoglobulin G antibodies. In this study, recombinant matrix antigen (rMAG1) was induced by isopropyl-β-d-thiogalactoside and purified by nickel-nitrilotriacetic acid purification system. We then developed and optimized an indirect enzyme-linked immunosorbent assay (ELISA) through checkerboard assays using serial dilutions of antigens and sera to assess the potential use of rMAG1 in serologic detection of T. gondii infection in dogs. Serum samples from 93 domestic dogs were analyzed by western blot and rMAG1-ELISA. The results were compared with those obtained from an ELISA with the soluble Toxoplasma lysate antigens (TLA). We found that although yielding an excellent agreement (96.7%) with western blot data (κ = 0.9659), rMAG1-ELISA produced higher sensitivity (93.9% vs. 87.8%) and specificity (98.3% vs. 96.7%) than TLA-ELISA. In addition, receiver operating characteristic analysis also revealed that rMAG1-ELISA is in more agreement with western blot (area under the curve [AUC] = 0.985) relative to TLA-ELISA (AUC = 0.955). These results indicated that the rMAG1-ELISA established in this study provides a promising and reliable tool for serologic detection of T. gondii infection in dogs.

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques

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Recchia, Anna Grazia; Caruso, Nadia; Bossio, Sabrina; Pellicanò, Mariavaleria; De Stefano, Laura; Franzese, Stefania; Palummo, Angela; Abbadessa, Vincenzo; Lucia, Eugenio; Gentile, Massimo; Vigna, Ernesto; Caracciolo, Clementina; Agostino, Antolino; Galimberti, Sara; Levato, Luciano; Stagno, Fabio; Molica, Stefano; Martino, Bruno; Vigneri, Paolo; Di Raimondo, Francesco; Morabito, Fortunato

2015-01-01

Chronic Myeloid Leukemia (CML) is characterized by a balanced translocation juxtaposing the Abelson (ABL) and breakpoint cluster region (BCR) genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR) defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i) CML can be properly diagnosed at onset, (ii) follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1) when BCR-ABL1IS transcripts are between 1–10%, and (iii) rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients. PMID:26111048

Flow Cytometric Immunobead Assay for Detection of BCR-ABL1 Fusion Proteins in Chronic Myleoid Leukemia: Comparison with FISH and PCR Techniques.

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Anna Grazia Recchia

Full Text Available Chronic Myeloid Leukemia (CML is characterized by a balanced translocation juxtaposing the Abelson (ABL and breakpoint cluster region (BCR genes. The resulting BCR-ABL1 oncogene leads to increased proliferation and survival of leukemic cells. Successful treatment of CML has been accompanied by steady improvements in our capacity to accurately and sensitively monitor therapy response. Currently, measurement of BCR-ABL1 mRNA transcript levels by real-time quantitative PCR (RQ-PCR defines critical response endpoints. An antibody-based technique for BCR-ABL1 protein recognition could be an attractive alternative to RQ-PCR. To date, there have been no studies evaluating whether flow-cytometry based assays could be of clinical utility in evaluating residual disease in CML patients. Here we describe a flow-cytometry assay that detects the presence of BCR-ABL1 fusion proteins in CML lysates to determine the applicability, reliability, and specificity of this method for both diagnosis and monitoring of CML patients for initial response to therapy. We show that: i CML can be properly diagnosed at onset, (ii follow-up assessments show detectable fusion protein (i.e. relative mean fluorescent intensity, rMFI%>1 when BCR-ABL1IS transcripts are between 1-10%, and (iii rMFI% levels predict CCyR as defined by FISH analysis. Overall, the FCBA assay is a rapid technique, fully translatable to the routine management of CML patients.

Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications.

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Fortunato, Tiago M; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A; Pula, Giordano

2016-05-04

Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs.

A simple fluorescence based assay for quantification of human immunodeficiency virus particle release

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Heuser Anke-Mareil

2010-04-01

Full Text Available Abstract Background The assembly and release of human immunodeficiency virus (HIV particles from infected cells represent attractive, but not yet exploited targets for antiretroviral therapy. The availability of simple methods to measure the efficiency of these replication steps in tissue culture would facilitate the identification of host factors essential for these processes as well as the screening for lead compounds acting as specific inhibitors of particle formation. We describe here the development of a rapid cell based assay for quantification of human immunodeficiency virus type 1 (HIV-1 particle assembly and/or release. Results Using a fluorescently labelled HIV-derivative, which carries an eYFP domain within the main viral structural protein Gag in the complete viral protein context, the release of virus like particles could be monitored by directly measuring the fluorescence intensity of the tissue culture supernatant. Intracellular Gag was quantitated in parallel by direct fluorescence analysis of cell lysates, allowing us to normalize for Gag expression efficiency. The assay was validated by comparison with p24 capsid ELISA measurements, a standard method for quantifying HIV-1 particles. Optimization of conditions allowed the robust detection of particle amounts corresponding to 50 ng p24/ml in medium by fluorescence spectroscopy. Further adaptation to a multi-well format rendered the assay suitable for medium or high throughput screening of siRNA libraries to identify host cell factors involved in late stages of HIV replication, as well as for random screening approaches to search for potential inhibitors of HIV-1 assembly or release. Conclusions The fast and simple fluorescence based quantification of HIV particle release yielded reproducible results which were comparable to the well established ELISA measurements, while in addition allowing the parallel determination of intracellular Gag expression. The protocols described here

Equine platelet lysate as an alternative to fetal bovine serum in equine mesenchymal stromal cell culture - too much of a good thing?

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Russell, K A; Koch, T G

2016-03-01

Multipotent mesenchymal stromal cells (MSC) are often culture-expanded in vitro. Presently, expansion medium (EM) for MSC is supplemented with fetal bovine serum (FBS). However, increasing cost, variable composition and potential risks associated with bovine antigens call for alternatives. Platelet lysate (PL) has shown promise as an alternative supplement. To determine how equine umbilical cord blood (CB) MSC proliferate in EM enriched with PL or FBS at various concentrations. Randomised dose escalation study. Platelet concentrate was generated from 5 equine whole blood samples through a double centrifugation method and standardised to 1 × 10(12) platelets/l prior to a freeze/thaw cycle to produce PL. Pooled PL or pooled FBS was added to EM at concentrations of 5% to 60%. Proliferation of 4 equine CB-MSC cultures was determined after 4 days using a resazurin semiquantitative assay. Cord blood-MSC proliferated with a dose-dependent response with no significant difference found between PL and FBS up to a 30% concentration. Beyond 30%, proliferation fell in the PL-cultured cells, while continued dose-dependent proliferation was noted in the FBS-cultured cells. Despite reduced cell numbers in high PL concentrations, live/dead staining revealed that adherent cells remained viable. Expansion medium enriched with PL can support short-term equine CB-MSC proliferation at conventional culture concentrations. Based on the unexpected suppression of CB-MSC at higher PL concentrations, an in vivo dose study is indicated to investigate if combinational therapies of CB-MSC and platelet-rich plasma are associated with synergistic or antagonistic effect on CB-MSC function. © 2015 EVJ Ltd.

Human platelet lysate is a feasible candidate to replace fetal calf serum as medium supplement for blood vascular and lymphatic endothelial cells.

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Hofbauer, Pablo; Riedl, Sabrina; Witzeneder, Karin; Hildner, Florian; Wolbank, Susanne; Groeger, Marion; Gabriel, Christian; Redl, Heinz; Holnthoner, Wolfgang

2014-09-01

As angiogenic and lymphangiogenic key players, endothelial cells (ECs) are promising candidates for vascular regenerative therapies. To culture ECs in vitro, fetal calf serum (FCS) is most often used. However, some critical aspects of FCS usage, such as possible internalization of xenogeneic proteins and prions, must be considered. Therefore, the aim of this project was to determine if human platelet lysate (hPL) is a suitable alternative to FCS as medium supplement for the culture of blood vascular and lymphatic endothelial cells. The usability of hPL was tested by analysis of endothelial surface marker expression, metabolic activity and vasculogenic potential of outgrowth ECs (OECs), human umbilical vein ECs (HUVECs), and lymphatic ECs (LECs). Expression of EC markers CD31, VEGFR2, VE-cadherin and CD146 did not differ significantly between the EC types cultured in FCS or hPL. In addition, OECs, HUVECs and LECs formed tube-like structures on Matrigel when cultured in hPL and FCS. With the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid assays, we found that the metabolic activity of OECs and LECs was slightly decreased when hPL was used. However, HUVECs and LECs did not show a significant decrease in metabolic activity, and HUVECs showed a slightly higher activity at low seeding densities. The use of hPL on different EC types did not reveal any substantial negative effects on EC behavior. Thus, hPL appears to be a favorable candidate to replace FCS as a medium supplement in the culture of ECs. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Comparative analyses of industrial-scale human platelet lysate preparations.

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Pierce, Jan; Benedetti, Eric; Preslar, Amber; Jacobson, Pam; Jin, Ping; Stroncek, David F; Reems, Jo-Anna

2017-12-01

Efforts are underway to eliminate fetal bovine serum from mammalian cell cultures for clinical use. An emerging, viable replacement option for fetal bovine serum is human platelet lysate (PL) as either a plasma-based or serum-based product. Nine industrial-scale, serum-based PL manufacturing runs (i.e., lots) were performed, consisting of an average ± standard deviation volume of 24.6 ± 2.2 liters of pooled, platelet-rich plasma units that were obtained from apheresis donors. Manufactured lots were compared by evaluating various biochemical and functional test results. Comprehensive cytokine profiles of PL lots and product stability tests were performed. Global gene expression profiles of mesenchymal stromal cells (MSCs) cultured with plasma-based or serum-based PL were compared to MSCs cultured with fetal bovine serum. Electrolyte and protein levels were relatively consistent among all serum-based PL lots, with only slight variations in glucose and calcium levels. All nine lots were as good as or better than fetal bovine serum in expanding MSCs. Serum-based PL stored at -80°C remained stable over 2 years. Quantitative cytokine arrays showed similarities as well as dissimilarities in the proteins present in serum-based PL. Greater differences in MSC gene expression profiles were attributable to the starting cell source rather than with the use of either PL or fetal bovine serum as a culture supplement. Using a large-scale, standardized method, lot-to-lot variations were noted for industrial-scale preparations of serum-based PL products. However, all lots performed as well as or better than fetal bovine serum in supporting MSC growth. Together, these data indicate that off-the-shelf PL is a feasible substitute for fetal bovine serum in MSC cultures. © 2017 AABB.

Stimulation with lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae maximizes cross-reactivity of anti-fungal T cells.

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Deo, Shivashni S; Virassamy, Balaji; Halliday, Catriona; Clancy, Leighton; Chen, Sharon; Meyer, Wieland; Sorrell, Tania C; Gottlieb, David J

2016-01-01

Invasive fungal diseases caused by filamentous fungi and yeasts are significant causes of morbidity and mortality in immunosuppressed hematology patients. We previously published a method to expand Aspergillus fumigatus-specific T cells for clinical cell therapy. In the present study, we investigated expansion of T cells specific for other fungal pathogens and creation of a broadly reactive panfungal T-cell product. Fungal strains selected were those frequently observed in the clinical hematology setting and included Aspergillus, Candida, Fusarium, Rhizopus and Lomentospora/Scedosporium. Four T-cell cultures specific to each fungus were established. We selected lysates of Aspergillus terreus, Candida krusei and Rhizopus oryzae to expand panfungal T cells. Allelic restriction of anti-fungal activity was determined through the use of specific major histocompatibility complex class II-blocking antibodies. Individual T-cell cultures specific to each fungus could be expanded in vitro, generating predominantly CD4(+) T cells of which 8% to 20% were fungus-specific. We successfully expanded panfungal T cells from the peripheral blood (n = 8) and granulocyte-colony-stimulating factor-primed stem cell products (n = 3) of normal donors by using a combination of lysates from Aspergillus terreus, Candida krusei and Rhizopus oryzae. Anti-fungal activity was mediated through human leukocyte antigen (HLA)-DR alleles and was maintained when antigen-presenting cells from partially HLA-DRB1-matched donors were used to stimulate T cells. We demonstrate a method to manufacture panfungal T-cell products with specificity against a range of clinical fungal pathogens by use of the blood and stem cells of healthy donors as the starting material. The safety and efficacy of these products will need to be tested clinically. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Gut-targeted immunonutrition boosting natural killer cell activity using Saccharomyces boulardii lysates in immuno-compromised healthy elderly subjects.

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Naito, Yasuhiro; Marotta, Francesco; Kantah, Makoto K; Zerbinati, Nicola; Kushugulova, Almagul; Zhumadilov, Zhaxybay; Illuzzi, Nicola; Sapienza, Chiara; Takadanohara, Hiroshi; Kobayashi, Riyichi; Catanzaro, Roberto

2014-04-01

The aim of this study was to assess the immunomodulatory effect of KC-1317 (a symbiotic mixture containing Saccharomyces boulardii lysate in a cranberry, colostrum-derived lactoferrin, fragaria, and lactose mixture) supplementation in immune-compromised but otherwise healthy elderly subjects. A liquid formulation of KC-1317 was administered in a randomized controlled trial (RCT) fashion to healthy volunteers (65-79 years) previously selected for low natural killer (NK) cell activity, and this parameter was checked at the completion of the study. A significant improvement in NK cell activity of KC-1317 consumers was observed as compared to placebo at the end of 2 months. Although preliminary, these beneficial immune-modulatory effects of KC-1317 in aged individuals might indicate its employment within a wider age-management strategy.

Purification of two high molecular weight proteases from rabbit reticulocyte lysate

International Nuclear Information System (INIS)

Hough, R.; Pratt, G.; Rechsteiner, M.

1987-01-01

The authors have purified two large proteases from rabbit reticulocyte lysate. The enzymes are so similar in their chromatographic behavior that each is the only significant contaminant of the other during the final stages of purification. At pH 7.8, both hydrolyze 125 I-α-casein and 4-methylcoumaryl-7-amide (MCA) derivatives with tyrosine, phenylalanine or arginine at the P 1 position. The larger, ATP-dependent enzyme degrades ubiquitin-lysozyme conjugates, but it does not degrade unmodified lysozyme. Hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA by this enzyme is also stimulated two-fold in the presence of ATP. The protease has a molecular weight of 950,000 based on sedimentation, gel filtration and non-denaturing PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protease is composed of a number of subunits with molecular masses between 32 and 110 kDa. Densitometric analysis showed equivalent amounts of the two larger chains, and the presence of one copy of each in the native enzyme would be consistent with an M/sub r/ of 950,000. The smaller protease has a molecular weight of 700,000 and is composed of 8 to 10 subunits ranging from 21,000 to 32,000. It cleaves ubiquitin-lysozyme conjugates only slightly, and hydrolysis of conjugates or fluorogenic peptide substrates is not stimulated by ATP. This protease appears similar, if not identical, to the multicatalytic protease complex first purified by Wilk and Orlowski

Engineering Enriched Microenvironments with Gradients of Platelet Lysate in Hydrogel Fibers.

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Santo, Vítor E; Babo, Pedro; Amador, Miguel; Correia, Cláudia; Cunha, Bárbara; Coutinho, Daniela F; Neves, Nuno M; Mano, João F; Reis, Rui L; Gomes, Manuela E

2016-06-13

Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute toward morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photo-cross-linkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition, and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics, and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers toward the PL-enriched sections in comparison with the nonloaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions.

Heparin concentration is critical for cell culture with human platelet lysate.

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Hemeda, Hatim; Kalz, Jana; Walenda, Gudrun; Lohmann, Michael; Wagner, Wolfgang

2013-09-01

Culture media for mesenchymal stromal cells (MSCs) are generally supplemented with fetal bovine serum. Human platelet lysate (hPL) has been proven to be a very effective alternative without the risk of xenogeneic infections or immune reactions. In contrast to fetal bovine serum, hPL comprises plasma, and anticoagulants-usually unfractionated heparin (UFH)-need to be added to prevent gel formation. Cultures of MSCs in hPL media with various concentrations of UFH and enoxaparin, a low-molecular-weight heparin (LMWH), were systematically compared with regard to proliferation, fibroblastoid colony-forming unit frequency, immunophenotype and in vitro differentiation. At least 0.61 IU/mL UFH or 0.024 mg/mL LMWH was necessary for reliable prevention of coagulation of hPL pools used in this study. Higher concentrations impaired cellular proliferation in a dose-dependent manner even without benzyl alcohol, which is commonly added to heparins as a bacteriostatic agent. Colony-forming unit frequency was also reduced at higher heparin concentrations, particularly with LMWH, whereas no significant effect was observed on cellular morphology or immunophenotype. High concentrations of heparins reduced the in vitro differentiation toward adipogenic and osteogenic lineages. Heparin concentration is critical for culture of MSCs in hPL media; this is of particular relevance for cellular therapy where cell culture procedures need to be optimized and standardized. Copyright © 2013 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Immuno-detection of cleaved SNAP-25 from differentiated mouse embryonic stem cells provides a sensitive assay for determination of botulinum A toxin and antitoxin potency.

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Yadirgi, G; Stickings, P; Rajagopal, S; Liu, Y; Sesardic, D

2017-12-01

Botulinum toxin type A is a causative agent of human botulism. Due to high toxicity and ease of production it is classified by the Centres for Disease Control and Prevention as a category A bioterrorism agent. The same serotype, BoNT/A, is also the most widely used in pharmaceutical preparations for treatment of a diverse range of neuromuscular disorders. Traditionally, animals are used to confirm the presence and activity of toxin and to establish neutralizing capabilities of countermeasures in toxin neutralization tests. Cell based assays for BoNT/A have been reported as the most viable alternative to animal models, since they are capable of reflecting all key steps (binding, translocation, internalization and cleavage of intracellular substrate) involved in toxin activity. In this paper we report preliminary development of a simple immunochemical method for specifically detecting BoNT/A cleaved intracellular substrate, SNAP-25, in cell lysates of neurons derived from mouse embryonic stem cells. The assay offers sensitivity of better than 0.1LD50/ml (3fM) which is not matched by other functional assays, including the mouse bioassay, and provides serotype specificity for quantitative detection of BoNT/A and anti-BoNT/A antitoxin. Subject to formal validation, the method described here could potentially be used as a substitute for the mouse bioassay to measure potency and consistency of therapeutic products. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Combined fluorimetric caspase 3/7 assay and bradford protein determination for assessment of polycation-mediated cytotoxicity.

Science.gov (United States)

Larsen, Anna K; Hall, Arnaldur; Lundsgart, Henrik; Moghimi, S Moein

2013-01-01

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Pile oscillator measurements of thermal absorption cross sections of Al, Mg, Fe and Cu; Mesure des sections efficaces thermiques d'absorption de l'Al, du Mg, du Fe et du Cu par la methode d'oscillation

Energy Technology Data Exchange (ETDEWEB)

Carre, J C; Vidal, R [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1964-07-01

The phase oscillation technique used at ZOE has the property of reducing of a marked factor the effect of neutron scattering by the sample. The absorption cross sections of poorly absorbing and highly scattering materials have been measured; for neutrons of 2,200 m/s, the following values are obtained: 229 {+-} 3 mb for Al; 64.2 {+-} 1.5 mb for Mg, 2.53 {+-} 0.03 b for Fe and 3.74 {+-} 0.04 b for Cu. (authors) [French] La methode d'oscillation de phase utilisee a ZOE possede la propriete de diminuer d'un facteur important l'influence de la diffusion. Les sections efficaces de capture de materiaux peu capturants et tres diffusants, ont ete mesurees; pour des neutrons de 2200 m/s, on obtient pour l'Al: 229 {+-} 3mb, pour le Mg: 64,2 {+-} 1,5 mb, pour le Fe: 2,53 {+-} 0,03 b et pour le Cu: 3,74 {+-} 0,04 b. (auteurs)

Is buffer a good proxy for a crowded cell-like environment? A comparative NMR study of calmodulin side-chain dynamics in buffer and E. coli lysate.

Directory of Open Access Journals (Sweden)

Michael P Latham

Full Text Available Biophysical studies of protein structure and dynamics are typically performed in a highly controlled manner involving only the protein(s of interest. Comparatively fewer such studies have been carried out in the context of a cellular environment that typically involves many biomolecules, ions and metabolites. Recently, solution NMR spectroscopy, focusing primarily on backbone amide groups as reporters, has emerged as a powerful technique for investigating protein structure and dynamics in vivo and in crowded "cell-like" environments. Here we extend these studies through a comparative analysis of Ile, Leu, Val and Met methyl side-chain motions in apo, Ca(2+-bound and Ca(2+, peptide-bound calmodulin dissolved in aqueous buffer or in E. coli lysate. Deuterium spin relaxation experiments, sensitive to pico- to nano-second time-scale processes and Carr-Purcell-Meiboom-Gill relaxation dispersion experiments, reporting on millisecond dynamics, have been recorded. Both similarities and differences in motional properties are noted for calmodulin dissolved in buffer or in lysate. These results emphasize that while significant insights can be obtained through detailed "test-tube" studies, experiments performed under conditions that are "cell-like" are critical for obtaining a comprehensive understanding of protein motion in vivo and therefore for elucidating the relation between motion and function.

Immunological Characterization of Whole Tumour Lysate-Loaded Dendritic Cells for Cancer Immunotherapy

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Ottobrini, Luisa; Biasin, Mara; Borelli, Manuela; Lucignani, Giovanni; Trabattoni, Daria; Clerici, Mario

2016-01-01

Introduction Dendritic cells play a key role as initiators of T-cell responses, and even if tumour antigen-loaded dendritic cells can induce anti-tumour responses, their efficacy has been questioned, suggesting a need to enhance immunization strategies. Matherials & Methods We focused on the characterization of bone marrow-derived dendritic cells pulsed with whole tumour lysate (TAA-DC), as a source of known and unknown antigens, in a mouse model of breast cancer (MMTV-Ras). Dendritic cells were evaluated for antigen uptake and for the expression of MHC class I/II and costimulatory molecules and markers associated with maturation. Results Results showed that antigen-loaded dendritic cells are characterized by a phenotypically semi-mature/mature profile and by the upregulation of genes involved in antigen presentation and T-cell priming. Activated dendritic cells stimulated T-cell proliferation and induced the production of high concentrations of IL-12p70 and IFN-γ but only low levels of IL-10, indicating their ability to elicit a TH1-immune response. Furthermore, administration of Antigen loaded-Dendritic Cells in MMTV-Ras mice evoked a strong anti-tumour response in vivo as demonstrated by a general activation of immunocompetent cells and the release of TH1 cytokines. Conclusion Data herein could be useful in the design of antitumoral DC-based therapies, showing a specific activation of immune system against breast cancer. PMID:26795765

Micropollutant degradation via extracted native enzymes from activated sludge.

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Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

2016-05-15

A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

Comparison of Cellular Alterations in Fat Cells Harvested With Laser-Assisted Liposuction and Suction-Assisted Liposuction.

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Yildiz, Kemalettin; Taşli, Pakize Neslihan; Şahin, Fikrettin; Güneren, Ethem

2016-05-01

The aim of the present study was to evaluate the viability and proliferative capacity of adipose-derived stem cells obtained by laser-assisted liposuction (LAL). Fat tissue was obtained from 7 male patients treated surgically for gynecomastia. On one side, harvesting was made before LAL, while it was implemented after LAL on the contralateral side. Viability, cell surface antigens, pluripotency, and apoptosis were assessed and compared in these samples. Cells harvested before and after LAL did not exhibit any significant difference in terms of surface cell markers. Number of viable stem cells was lower initially after exposure to laser, while this difference was reversed at the end of 72 hours. Genetic indicators of cellular differentiation were similar in both groups. Apoptosis indicators were increased remarkably after laser exposure in the first 24 hours, but this increase was absent 72 hours after LAL procedure. The authors' results have promising clinical relevance since mesenchymal stem cells harvested during LAL have maintained appropriate cellular features to be used for autologous fat transfer and fat grafting.

A Reverse-phase Protein Microarray-based Screen Identifies Host Signaling Dynamics upon Burkholderia spp. Infection

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2015-07-27

total protein in each sample was quantified by Bradford assay (Bio-Rad). RAW264.7 cell lysate preparations were boiled for 10 min with NuPAGE LDS Sample...RPMA assays , cells were harvested, washed with PBS, and then lysed in a mixture of T-PER Reagent (Thermo Scientific) and 2X Tris-Glycine SDS sample... assay (RIPA) buffer (Thermo Scientific) containing complete protease inhibitor cocktail (Roche), and phosphatase inhibitors (Roche). The amount of

Platelet lysate as replacement for fetal bovine serum in mesenchymal stromal cell cultures.

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Bieback, Karen

2013-10-01

Mesenchymal stromal cells (MSC) emerged as highly attractive in cell-based regenerative medicine. Initially thought to provide cells capable of differentiation towards mesenchymal cell types (osteoblasts, chondrocytes, adipocytes etc.), by and by potent immunoregulatory and pro-regenerative activities have been discovered, broadening the field of potential applications from bone and cartilage regeneration to wound healing and treatment of autoimmune diseases. Due to the limited frequency in most tissue sources, ex vivo expansion of MSC is required compliant with good manufacturing practice (GMP) guidelines to yield clinically relevant cell doses. Though, still most manufacturing protocols use fetal bovine serum (FBS) as cell culture supplement to isolate and to expand MSC. However, the high lot-to-lot variability as well as risk of contamination and immunization call for xenogenic-free culture conditions. In terms of standardization, chemically defined media appear as the ultimate achievement. Since these media need to maintain all key cellular and therapy-relevant features of MSC, the development of chemically defined media is still - albeit highly investigated - only in its beginning. The current alternatives to FBS rely on human blood-derived components: plasma, serum, umbilical cord blood serum, and platelet derivatives like platelet lysate. Focusing on quality aspects, the latter will be addressed within this review.

Isolation of RNA for dot hybridization by heparin-DNase I treatment of whole cell lysate.

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Krawczyk, Z; Wu, C

1987-08-15

We have developed a new procedure for the rapid preparation of undegraded total RNA from cultured cells for specific quantitation by dot blotting analysis. Pelleted cells are resuspended in hypotonic solution containing a ribonuclease inhibitor and heparin and disrupted by freeze-thaw. Heparin is employed as an agent for nuclear lysis, dissociation of chromosomal protein, and release of mRNA from rough endoplasmic reticulum. We eliminate chromosomal DNA by digestion with DNase I and denature the RNA in the lysate with formaldehyde. After centrifugation to remove debris, the supernatant is used directly for dot blotting. All manipulations are performed in the same microfuge tube and recovery of RNA is quantitative. The procedure is especially useful for processing large numbers of samples. We illustrate its versatility by analysis of specific RNAs in Drosophila, rat, and human cell lines. In reconstruction experiments, less than 80 molecules per cell of a small RNA (beta-globin) can be detected under highly stringent hybridization conditions, using only moderately labeled double-stranded plasmid DNA probes and short film exposures.

An innovative immunosensor for ultrasensitive detection of breast cancer specific carbohydrate (CA 15-3) in unprocessed human plasma and MCF-7 breast cancer cell lysates using gold nanospear electrochemically assembled onto thiolated graphene quantum dots.

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Hasanzadeh, Mohammad; Tagi, Solmaz; Solhi, Elham; Mokhtarzadeh, Ahad; Shadjou, Nasrin; Eftekhari, Aziz; Mahboob, Soltanali

2018-04-03

The accurate quantification of the level of breast cancer specific protein CA 15-3 in serum is crucial for cancer prognosis. This work, a novel and sensitive label-free immunoassay based on gold nanospear (Au NSs) electrochemically assembled onto thiolated graphene quantum dots (CysA/GQDs) for the detection of CA 15-3 antibodies. The CysA/Au NSs/GQDs hybrid interface provides a large surface area for the effective immobilization of CA 15-3 antigens, as well as it ascertains the bioactivity and stability of immobilized CA 15-3 antigens. Field emission scanning electron microscope (FE-SEM), and EDS photoelectron spectroscopies were used to monitor the sensor fabrication. Also, cyclic voltammetry was used to quantify the extent of Au NSs' surface coverage by CA 15-3 antigens. Square wave voltammetry (SWV) was employed to investigate the immunosensor fabrication and to monitor the binding events between CA 15-3 antigens-antibodies. Under optimized experimental conditions, the immunosensor displayed good sensitivity and specificity. The CA 15-3 were detected in a concentration as low as 0.11U/mL with a linear range from 0.16-125U/mL. The high sensitivity of the immunosensor may derive from the high loading of CA 15-3 antibodies on CysA/Au NSs/GQDs hybrid interface which increases the number of binding events. The method was successfully applied assay of the CA 15-3 in unprocessed human plasma samples. Also, proposed immunosensor was applied to the assay of CA 15-3 malignant cell line lysates (human breast adenocarcinoma cell line-MCF-7). Copyright © 2018. Published by Elsevier B.V.

Platelet-Rich Fibrin Lysate Can Ameliorate Dysfunction of Chronically UVA-Irradiated Human Dermal Fibroblasts.

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Wirohadidjojo, Yohanes Widodo; Budiyanto, Arief; Soebono, Hardyanto

2016-09-01

To determine whether platelet-rich fibrin lysate (PRF-L) could restore the function of chronically ultraviolet-A (UVA)-irradiated human dermal fibroblasts (HDFs), we isolated and sub-cultured HDFs from six different human foreskins. HDFs were divided into two groups: those that received chronic UVA irradiation (total dosages of 10 J cm⁻²) and those that were not irradiated. We compared the proliferation rates, collagen deposition, and migration rates between the groups and between chronically UVA-irradiated HDFs in control and PRF-L-treated media. Our experiment showed that chronic UVA irradiation significantly decreased (p<0.05) the proliferation rates, migration rates, and collagen deposition of HDFs, compared to controls. Compared to control media, chronically UVA-irradiated HDFs in 50% PRF-L had significantly increased proliferation rates, migration rates, and collagen deposition (p<0.05), and the migration rates and collagen deposition of chronically UVA-irradiated HDFs in 50% PRF-L were equal to those of normal fibroblasts. Based on this experiment, we concluded that PRF-L is a good candidate material for treating UVA-induced photoaging of skin, although the best method for its clinical application remains to be determined.

Human Platelet Lysate versus Fetal Calf Serum: These Supplements Do Not Select for Different Mesenchymal Stromal Cells.

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Fernandez-Rebollo, Eduardo; Mentrup, Birgit; Ebert, Regina; Franzen, Julia; Abagnale, Giulio; Sieben, Torsten; Ostrowska, Alina; Hoffmann, Per; Roux, Pierre-François; Rath, Björn; Goodhardt, Michele; Lemaitre, Jean-Marc; Bischof, Oliver; Jakob, Franz; Wagner, Wolfgang

2017-07-11

Culture medium of mesenchymal stromal cells (MSCs) is usually supplemented with either human platelet lysate (HPL) or fetal calf serum (FCS). Many studies have demonstrated that proliferation and cellular morphology are affected by these supplements - it is therefore important to determine if they favor outgrowth of different subpopulations and thereby impact on the heterogeneous composition of MSCs. We have isolated and expanded human bone marrow-derived MSCs in parallel with HPL or FCS and demonstrated that HPL significantly increases proliferation and leads to dramatic differences in cellular morphology. Remarkably, global DNA-methylation profiles did not reveal any significant differences. Even at the transcriptomic level, there were only moderate changes in pairwise comparison. Furthermore, the effects on proliferation, cytoskeletal organization, and focal adhesions were reversible by interchanging to opposite culture conditions. These results indicate that cultivation of MSCs with HPL or FCS has no systematic bias for specific cell types.

Learning Pedagogy in Physics

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Harlow, Danielle B.; Swanson, Lauren H.; Dwyer, Hilary A.; Bianchini, Julie A.

2010-10-01

We report on an adapted version of the Physics and Everyday Thinking (PET) curriculum. A unique aspect of PET is its inclusion of special activities that focus on Learning about Learning (LAL) in which undergraduates analyze videos of children talking about science and explicitly consider the nature of science. To create a course that intentionally linked science content, children's ideas, and strategies for science instruction, we augmented the existing LAL activities with discussions about teaching, and added activities focused on LAL from companion curricula such as Physical Science and Everyday Thinking (PSET) and Learning Physical Science (LEPS). To compensate for the additional time on LAL, we reduced the content activities to only those that directly supported LAL activities. We found that students made significant gains on the CLASS and expressed beliefs about teaching consistent with the PET pedagogy.

Comparison of osteo/odontogenic differentiation of human adult dental pulp stem cells and stem cells from apical papilla in the presence of platelet lysate.

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Abuarqoub, Duaa; Awidi, Abdalla; Abuharfeil, Nizar

2015-10-01

Human dental pulp cells (DPSCs) and stem cells from apical papilla have been used for the repair of damaged tooth tissues. Human platelet lysate (PL) has been suggested as a substitute for fetal bovine serum (FBS) for large scale expansion of dental stem cells. However, biological effects and optimal concentrations of PL for proliferation and differentiation of human dental stem cells remain to be elucidated. DPSCs and SCAP cells were isolated from impacted third molars of young healthy donors, at the stage of root development and identified by markers using flow cytometry. For comparison the cells were cultured in media containing PL (1%, 5% and 10%) and FBS, with subsequent induction for osteogenic/odontogenic differentiation. The cultures were analyzed for; morphology, growth characteristics, mineralization potential (Alizarin Red method) and differentiation markers using ELISA and real time -polymerase chain reaction (qPCR). The proliferation rates of DPSCs and SCAP significantly increased when cells were treated with 5% PL (7X doubling time) as compared to FBS. 5% PL also enhanced mineralized differentiation of DPSCs and SCAP, as indicated by the measurement of alkaline phosphatase activity, osteocalcin and osteopontin, calcium deposition and q-PCR. Our findings suggest that using 5% platelet lysate, proliferation and osteo/odontogenesis of DPSCs and SCAP for a short period of time (15 days), was significantly improved. This may imply its use as an optimum concentration for expansion of dental stem cells in bone regeneration. Copyright © 2015 Elsevier Ltd. All rights reserved.

Pooled human platelet lysate versus fetal bovine serum—investigating the proliferation rate, chromosome stability and angiogenic potential of human adipose tissue-derived stem cells intended for clinical use

DEFF Research Database (Denmark)

Trojahn Kølle, Stig-Frederik; Oliveri, Roberto S; Glovinski, Peter V

2013-01-01

Because of an increasing focus on the use of adipose-derived stem cells (ASCs) in clinical trials, the culture conditions for these cells are being optimized. We compared the proliferation rates and chromosomal stability of ASCs that had been cultured in Dulbecco's modified Eagle's Medium (DMEM) ......) supplemented with either pooled human platelet lysate (pHPL) or clinical-grade fetal bovine serum (FBS) (DMEM(pHPL) versus DMEM(FBS))....

Chopra, Prof. Virender Lal

Indian Academy of Sciences (India)

Ethical Guidelines and Procedures document. Posted on 17 January 2017. A revised version of the document 'Scientific Values: Ethical Guidelines and Procedures' has been produced by the Academy's Panel on Scientific Values. For more details, see the Academy Committee on Scientific Values page.

Assaying Cellular Viability Using the Neutral Red Uptake Assay.

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Ates, Gamze; Vanhaecke, Tamara; Rogiers, Vera; Rodrigues, Robim M

2017-01-01

The neutral red uptake assay is a cell viability assay that allows in vitro quantification of xenobiotic-induced cytotoxicity. The assay relies on the ability of living cells to incorporate and bind neutral red, a weak cationic dye, in lysosomes. As such, cytotoxicity is expressed as a concentration-dependent reduction of the uptake of neutral red after exposure to the xenobiotic under investigation. The neutral red uptake assay is mainly used for hazard assessment in in vitro toxicology applications. This method has also been introduced in regulatory recommendations as part of 3T3-NRU-phototoxicity-assay, which was regulatory accepted in all EU member states in 2000 and in the OECD member states in 2004 as a test guideline (TG 432). The present protocol describes the neutral red uptake assay using the human hepatoma cell line HepG2, which is often employed as an alternative in vitro model for human hepatocytes. As an example, the cytotoxicity of acetaminophen and acetyl salicylic acid is assessed.

Comparison of Endotoxin Exposure Assessment by Bioaerosol Impinger and Filter-Sampling Methods

OpenAIRE

Duchaine, Caroline; Thorne, Peter S.; Mériaux, Anne; Grimard, Yan; Whitten, Paul; Cormier, Yvon

2001-01-01

Environmental assessment data collected in two prior occupational hygiene studies of swine barns and sawmills allowed the comparison of concurrent, triplicate, side-by-side endotoxin measurements using air sampling filters and bioaerosol impingers. Endotoxin concentrations in impinger solutions and filter eluates were assayed using the Limulus amebocyte lysate assay. In sawmills, impinger sampling yielded significantly higher endotoxin concentration measurements and lower variances than filte...

Capillary zone electrophoresis-multiple reaction monitoring from 100 pg of RAW 264.7 cell lysate digest.

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Sun, Liangliang; Li, Yihan; Champion, Matthew M; Zhu, Guijie; Wojcik, Roza; Dovichi, Norman J

2013-06-07

Capillary zone electrophoresis-multiple/single reaction monitoring (CZE-MRM/SRM), which employed an electrokinetically driven sheath-flow electrospray interface, was used for the rapid and highly sensitive detection of protein analytes in complex tryptic digests. MRM channels were developed against a commercial exponential mixture of bovine proteins. Five proteins spanning four orders of magnitude concentration range were confidently detected from only 2.5 ng of the digest mixture; the mass detection limits (S/N = 3) of two detected proteins, alpha-casein and glutamate dehydrogenase were about 600 zmol and 30 amol, respectively. This technique was then applied to a RAW 264.7 cell lysate digest. Three proteins were confidently and reproducibly detected from 100 pg of this digest. The sample amount corresponds to the approximate protein content from a single cell, which suggests that CZE-MRM may be a useful analytical tool in chemical cytometry. In addition to providing highly sensitive detection of proteins in complex mixtures, this system is highly rapid; migration time of the protein digests was less than 10 min.

Evaluation of a broad-ranging and convenient enzyme-linked immunosorbent assay using the lysate of infected cells with five serotypes of Orientia tsutsugamushi, a causative agent of scrub typhus.

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Ogawa, Motohiko; Satoh, Masaaki; Saijo, Masayuki; Ando, Shuji

2017-01-05

Scrub typhus is a mite-borne rickettsiosis caused by infection of Orientia tsutsugamushi, which is endemic to several Asia-Pacific Rim countries, including Japan. Although micro-indirect immunofluorescent assay (micro-IFA) is the standard method for the serological diagnosis of scrub typhus, enzyme-linked immunosorbent assay (ELISA) is considered to be more objective, by providing digitized results as opposed to being subject to the judgment of the evaluator as in micro-IFA. Therefore, the aim of this study was to develop a broad-ranging ELISA using the five major prevalent serotypes of O. tsutsugamushi in Japan as the antigens. Furthermore, in contrast to previous studies that used purified microorganisms via ultracentrifugation, we directly used the infected cells, and evaluated the diagnostic accuracy of this simplified method to that of micro-IFA. Evaluation of paired patient sera against the five serotypes showed that the accuracy of ELISA relative to micro-IFA was 87.4 and 79.5% for immunoglobulin (Ig)M and IgG assays, respectively, at the optimized cut-off value. Further evaluation of patient sera against the expected serotype of the infecting strain showed that the accuracy of ELISA compared to micro-IFA increased to 100 and 97.4% in the IgM and IgG assays, respectively. This suggests that use of the five prevalent serotypes contributed to the increase of the accuracy of ELISA. When applying the criteria of serological diagnosis for paired sera samples to ELISA, all 19 patients were diagnosed as positive; a ≥4-fold elevation of the antibody titer was observed in 15 of 19 patients that were positive, and very high antibody titers were observed in both paired sera samples of the remaining four patients. In addition, all samples of healthy subjects and patients with other types of rickettsiosis were diagnosed as negative using these criteria. Our results suggest the excellent performance of the new broad-ranging and convenient ELISA, which appears to be

Rapid Monitoring of Bacteria and Fungi aboard the International Space Station (ISS)

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Gunter, D.; Flores, G.; Effinger, M.; Maule, J.; Wainwright, N.; Steele, A.; Damon, M.; Wells, M.; Williams, S.; Morris, H.;

QMix® irrigant reduces lipopolysacharide (LPS levels in an in vitro model

Directory of Open Access Journals (Sweden)

Grasiela Longhi GRÜNDLING

2015-08-01

Full Text Available AbstractThe presence of endotoxin inside the root canal has been associated with periapical inflammation, bone resorption and symptomatic conditions.Objectives To determine, in vitro, the effect of QMix® and other three root canal irrigants in reducing the endotoxin content in root canals.Material and Methods Root canals of single-rooted teeth were prepared. Samples were detoxified with Co-60 irradiation and inoculated with E. coli LPS (24 h, at 37°C. After that period, samples were divided into 4 groups, according to the irrigation solution tested: QMix®, 17% EDTA, 2% chlorhexidine solution (CHX, and 3% sodium hypochlorite (NaOCl. LPS quantification was determined by Limulus Amebocyte Lysate (LAL assay. The initial counting of endotoxins for all samples, and the determination of LPS levels in non-contaminated teeth and in contaminated teeth exposed only to non-pyrogenic water, were used as controls.Results QMix® reduced LPS levels, with a median value of 1.11 endotoxins units (EU/mL (p<0.001. NaOCl (25.50 EU/mL, chlorhexidine (44.10 EU/mL and positive control group (26.80 EU/mL samples had similar results. Higher levels were found with EDTA (176.00 EU/mL when compared to positive control (p<0.001. There was no significant difference among EDTA, NaOCl and CHX groups. Negative control group (0.005 EU/mL had statistically significant lower levels of endotoxins when compared to all test groups (p<0.001.Conclusion QMix® decreased LPS levels when compared to the other groups (p<0.001. 3% NaOCl, 2% CHX and 17% EDTA were not able to significantly reduce the root canal endotoxins load.

Applicability of the Monocyte Activation Test (MAT) in the quality control of the 17DD yellow fever vaccine.

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de Mattos, Katherine Antunes; Navega, Elaine Cristina Azevedo; Silva, Vitor Fernandes; Almeida, Alessandra Santos; da Silva, Cristiane Caldeira; Presgrave, Octavio Augusto França; Junior, Daniel da Silva Guedes; Delgado, Isabella Fernandes

2018-03-01

The need for alternatives to animal use in pyrogen testing has been driven by the Three Rs concept. This has resulted in the inclusion of the monocyte activation test (MAT) in the European Pharmacopoeia, 2010. However, some technical and regulatory obstacles must be overcome to ensure the effective implementation of the MAT by the industry, especially for the testing of biological products. The yellow fever (YF) vaccine (17DD-YFV) was chosen for evaluation in this study, in view of: a) the 2016-2018 outbreak of YF in Brazil; b) the increase in demand for 17DD-YFV doses; c) the complex production process with live attenuated virus; d) the presence of possible test interference factors, such as residual process components (e.g. ovalbumin); and e) the need for the investigation of other pyrogens that are not detectable by the methods prescribed in the YF vaccine monograph. The product-specific testing was carried out by using cryopreserved and fresh whole blood, and IL-6 and IL-1β levels were used as the marker readouts. After assessing the applicability of the MAT on a 1:10 dilution of 17DD-YFV, endotoxin and non-endotoxin pyrogens were quantified in spiked batches, by using the lipopolysaccharide and lipoteichoic acid standards, respectively. The quantitative analysis demonstrated the correlation between the MAT and the Limulus amoebocyte lysate (LAL) assays, with respect to the limits of endotoxin recovery in spiked batches and the detection of no pyrogenic contamination in commercial batches of 17DD-YFV. The data demonstrated the applicability of the MAT for 17DD-YFV pyrogen testing, and as an alternative method that can contribute to biological quality control studies. 2018 FRAME.

Global phenotypic characterisation of human platelet lysate expanded MSCs by high-throughput flow cytometry.

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Reis, Monica; McDonald, David; Nicholson, Lindsay; Godthardt, Kathrin; Knobel, Sebastian; Dickinson, Anne M; Filby, Andrew; Wang, Xiao-Nong

2018-03-02

Mesenchymal stromal cells (MSCs) are a promising cell source to develop cell therapy for many diseases. Human platelet lysate (PLT) is increasingly used as an alternative to foetal calf serum (FCS) for clinical-scale MSC production. To date, the global surface protein expression of PLT-expended MSCs (MSC-PLT) is not known. To investigate this, paired MSC-PLT and MSC-FCS were analysed in parallel using high-throughput flow cytometry for the expression of 356 cell surface proteins. MSC-PLT showed differential surface protein expression compared to their MSC-FCS counterpart. Higher percentage of positive cells was observed in MSC-PLT for 48 surface proteins, of which 13 were significantly enriched on MSC-PLT. This finding was validated using multiparameter flow cytometry and further confirmed by quantitative staining intensity analysis. The enriched surface proteins are relevant to increased proliferation and migration capacity, as well as enhanced chondrogenic and osteogenic differentiation properties. In silico network analysis revealed that these enriched surface proteins are involved in three distinct networks that are associated with inflammatory responses, carbohydrate metabolism and cellular motility. This is the first study reporting differential cell surface protein expression between MSC-PLT and MSC-FSC. Further studies are required to uncover the impact of those enriched proteins on biological functions of MSC-PLT.

Valoración de endotoxinas bacterianas en el inyectable ácido zoledrónico mediante la prueba de lisado del amebocito de Limulus Assessment of bacterial endotoxins in Zoledronic acid injectable drug by using Limulus amebocyte lysate test

Directory of Open Access Journals (Sweden)

Nancy Burguet Lago

2012-09-01

Full Text Available Objetivo: valorar las endotoxinas bacterianas por la técnica del lisado del amebocito de Limulus para el producto inyectable ácido zoledrónico, por el método de gelificación. Métodos: el ensayo se realizó mediante dos pruebas: 1 confirmación de la sensibilidad del lisado etiquetado, para lo cual se preparó una curva estándar con diluciones seriadas dobles de endotoxina por cuadruplicado; y 2 el ensayo del producto de inhibición, en el que se prepararon diluciones seriadas dobles de endotoxina con agua apirogénica y con las muestras de los lotes a ensayar sin sobrepasar la máxima dilución válida. Se determinó el punto final y se calculó la media geométrica. Se definió la dilución de trabajo, la cual se validó por cuadruplicado en tres lotes consecutivos. Resultados: la sensibilidad del lisado resultó 0,03125 UE/mL. La máxima dilución válida fue de 112 UE/mL y la dilución de trabajo 1/100. La cantidad de endotoxinas bacterianas presentes en tres lotes del producto inyectable no sobrepasó el límite establecido, por lo que cumplió con las especificaciones de calidad establecidas para el ensayo. Conclusión: la estandarización de las condiciones del método por gelificación, hace que este resulte eficaz, confiable, rápido y de fácil ejecución, por lo que puede emplearse como ensayo de rutina en el control de la calidad del inyectable analizado.Objective: To asses the presence of bacterial endotoxins in Zoledronic Acid injectable drug by using the Limulus amebocyte lysate test, particularly by the gelling procedure. Methods: The assay was performed in two tests: the first was the confirmation of labeled lysate sensitivity by preparing a standard curve with serial double dilutions of endotoxins four times, and the second was the inhibition product test in which serial double dilutions of endotoxins were prepared with apyrogenic water and with samples from the batches to be tested, without exceeding the maximum valid

Random assay in radioimmunoassay: Feasibility and application compared with batch assay

Energy Technology Data Exchange (ETDEWEB)

Lee, Jung Min; Lee, Hwan Hee; Park, Sohyun; Kim, Tae Sung; Kim, Seok Ki [Dept. of Nuclear MedicineNational Cancer Center, Goyang (Korea, Republic of)

2016-12-15

The batch assay has been conventionally used for radioimmunoassay (RIA) because of its technical robustness and practical convenience. However, it has limitations in terms of the relative lag of report time due to the necessity of multiple assays in a small number of samples compared with the random assay technique. In this study, we aimed to verify whether the random assay technique can be applied in RIA and is feasible in daily practice. The coefficients of variation (CVs) of eight standard curves within a single kit were calculated in a CA-125 immunoradiometric assay (IRMA) for the reference of the practically ideal CV of the CA-125 kit. Ten standard curves of 10 kits from 2 prospectively collected lots (pLot) and 85 standard curves of 85 kits from 3 retrospectively collected lots (Lot) were obtained. Additionally, the raw measurement data of both 170 control references and 1123 patients' sera were collected retrospectively between December 2015 and January 2016. A standard curve of the first kit of each lot was used as a master standard curve for a random assay. The CVs of inter-kits were analyzed in each lot, respectively. All raw measurements were normalized by decay and radioactivity. The CA-125 values from control samples and patients' sera were compared using the original batch assay and random assay. In standard curve analysis, the CVs of inter-kits in pLots and Lots were comparable to those within a single kit. The CVs from the random assay with normalization were similar to those from the batch assay in the control samples (CVs % of low/high concentration; Lot1 2.71/1.91, Lot2 2.35/1.83, Lot3 2.83/2.08 vs. Lot1 2.05/1.21, Lot2 1.66/1.48, Lot3 2.41/2.14). The ICCs between the batch assay and random assay using patients' sera were satisfactory (Lot1 1.00, Lot2 0.999, Lot3 1.00). The random assay technique could be successfully applied to the conventional CA-125 IRMA kits. The random assay showed strong agreement with the batch assay. The

Optimized human platelet lysate as novel basis for a serum-, xeno-, and additive-free corneal endothelial cell and tissue culture.

Science.gov (United States)

Thieme, Daniel; Reuland, Lynn; Lindl, Toni; Kruse, Friedrich; Fuchsluger, Thomas

2018-02-01

The expansion of donor-derived corneal endothelial cells (ECs) is a promising approach for regenerative therapies in corneal diseases. To achieve the best Good Manufacturing Practice standard the entire cultivation process should be devoid of nonhuman components. However, so far, there is no suitable xeno-free protocol for clinical applications. We therefore introduce a processed variant of a platelet lysate for the use in corneal cell and tissue culture based on a Good Manufacturing Practice-grade thrombocyte concentrate. This processed human platelet lysate (phPL), free of any animal components and of anticoagulants such as heparin with a physiological ionic composition, was used to cultivate corneal ECs in vitro and ex vivo in comparison to standard cultivation with fetal calf serum (FCS). Human donor corneas were cut in quarters while 2 quarters of each cornea were incubated with the respective medium supplement. Three fields of view per quarter were taken into account for the analysis. Evaluation of phPL as a medium supplement in cell culture of immortalized EC showed a superior viability compared with FCS control with reduced cell proliferation. Furthermore, the viability during the expansion of primary cells is significantly (3-fold ±0.5) increased with phPL compared with FCS standard medium. Quartering donor corneas was traumatic for the endothelium and therefore resulted in increased EC loss. Interestingly, however, cultivation of the quartered pieces for 2 weeks in 0.1-mg/ml pHPL in Biochrome I showed a 21 (±10) % EC loss compared with 67 (±12) % EC loss when cultivated in 2% FCS in Biochrome I. The cell culture protocol with pHPL as FCS replacement seems to be superior to the standard FCS protocols with respect to EC survival. It offers a xeno-free and physiological environment for corneal endothelial cells. This alternative cultivation protocol could facilitate the use of EC for human corneal cell therapy. Copyright © 2017 John Wiley & Sons, Ltd.

Matrix effects of TRU [transuranic] assays using the SWEPP PAN assay system

International Nuclear Information System (INIS)

Smith, J.R.

1990-08-01

The Drum Assay System (DAS) at the Stored Waste Experimental Pilot Plant (SWEPP) is a second-generation active-passive neutron assay system. It has been used to assay over 5000 208-liter drums of transuranic waste from the Rocky Flats Plant (RFP). Data from these assays have been examined and compared with the assays performed at Rocky Flats, mainly utilize counting of 239 Pu gamma rays. For the most part the passive assays are in very good agreement with the Rocky Flats assays. The active assays are strongly correlated with the results of the other two methods, but require matrix-dependent correction factors beyond those provided by the system itself. A set of matrix-dependent correction factors has been developed from the study of the assay results. 3 refs., 4 figs., 3 tabs

Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

Energy Technology Data Exchange (ETDEWEB)

Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul [Seoul Metropolitan Government Seoul National University Boramae Medical Center, Seoul (Korea, Republic of); Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo [Seoul National University college of Medicine, Seoul (Korea, Republic of)

2009-08-15

Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2+-1.7%, 3.9+-2.1%, 7.1+-6.2%, 11.2+-7.2%. The CVs by random assay were 2.1+-1.7%, 4.8+-3.1%, 3.6+-4.8%, and 7.4+-6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

Comparison of Batch Assay and Random Assay Using Automatic Dispenser in Radioimmunoassay

International Nuclear Information System (INIS)

Moon, Seung Hwan; Jang, Su Jin; Kang, Ji Yeon; Lee, Dong Soo; Chung, June Key; Lee, Myung Chul; Lee, Ho Young; Shin, Sun Young; Min, Gyeong Sun; Lee, Hyun Joo

2009-01-01

Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2±1.7%, 3.9±2.1%, 7.1±6.2%, 11.2±7.2%. The CVs by random assay were 2.1±1.7%, 4.8±3.1%, 3.6±4.8%, and 7.4±6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay

Radioreceptor opioid assay

International Nuclear Information System (INIS)

Miller, R.J.; Chang, K.-J.

1981-01-01

A radioreceptor assay is described for assaying opioid drugs in biological fluids. The method enables the assay of total opioid activity, being specific for opioids as a class but lacking specificity within the class. A radio-iodinated opioid and the liquid test sample are incubated with an opiate receptor material. The percentage inhibition of the binding of the radio-iodinated compound to the opiate receptor is calculated and the opioid activity of the test liquid determined from a standard curve. Examples of preparing radio-iodinated opioids and assaying opioid activity are given. A test kit for the assay is described. Compared to other methods, this assay is cheap, easy and rapid. (U.K.)

PcaO Positively Regulates pcaHG of the β-Ketoadipate Pathway in Corynebacterium glutamicum▿

OpenAIRE

Zhao, Ke-Xin; Huang, Yan; Chen, Xi; Wang, Nan-Xi; Liu, Shuang-Jiang

2010-01-01

We identified a new regulator, PcaO, which is involved in regulation of the protocatechuate (PCA) branch of the β-ketoadipate pathway in Corynebacterium glutamicum. PcaO is an atypical large ATP-binding LuxR family (LAL)-type regulator and does not have a Walker A motif. A mutant of C. glutamicum in which pcaO was disrupted (RES167ΔpcaO) was unable to grow on PCA, and growth on PCA was restored by complementation with pcaO. Both an enzymatic assay of PCA 3,4-dioxygenase activity (encoded by p...

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

Science.gov (United States)

Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

2003-09-01

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Human platelet lysate supports the formation of robust human periodontal ligament cell sheets.

Science.gov (United States)

Tian, Bei-Min; Wu, Rui-Xin; Bi, Chun-Sheng; He, Xiao-Tao; Yin, Yuan; Chen, Fa-Ming

2018-04-01

The use of stem cell-derived sheets has become increasingly common in a wide variety of biomedical applications. Although substantial evidence has demonstrated that human platelet lysate (PL) can be used for therapeutic cell expansion, either as a substitute for or as a supplement to xenogeneic fetal bovine serum (FBS), its impact on cell sheet production remains largely unexplored. In this study, we manufactured periodontal ligament stem cell (PDLSC) sheets in vitro by incubating PDLSCs in sheet-induction media supplemented with various ratios of PL and FBS, i.e. 10% PL without FBS, 7.5% PL + 2.5% FBS, 5% PL + 5% FBS, 2.5% PL + 7.5% FBS or 10% FBS without PL. Cultures with the addition of all the designed supplements led to successful cell sheet production. In addition, all the resultant cellular materials exhibited similar expression profiles of matrix-related genes and proteins, such as collagen I, fibronectin and integrin β1. Interestingly, the cell components within sheets generated by media containing both PL and FBS exhibited improved osteogenic potential. Following in vivo transplantation, all sheets supported significant new bone formation. Our data suggest that robust PDLSC sheets can be produced by applying PL as either an alternative or an adjuvant to FBS. Further examination of the relevant influences of human PL that benefit cell behaviour and matrix production will pave the way towards optimized and standardized conditions for cell sheet production. Copyright © 2017 John Wiley & Sons, Ltd.

Endotoxin content in endodontically involved teeth. 1975.

Science.gov (United States)

Schein, Benjamin; Schilder, Herbert

2006-04-01

Fluid was aspirated from the root canals of 40 endodontically involved teeth. This fluid was assayed for endotoxin with the limulus lysate test. Pulpless teeth contained greater concentrations of endotoxin than those with vital pulps. Symptomatic teeth also contained more endotoxin than asymptomatic teeth.

Selection of non-destructive assay methods: Neutron counting or calorimetric assay?

International Nuclear Information System (INIS)

Cremers, T.L.; Wachter, J.R.

1994-01-01

The transition of DOE facilities from production to D ampersand D has lead to more measurements of product, waste, scrap, and other less attractive materials. Some of these materials are difficult to analyze by either neutron counting or calorimetric assay. To determine the most efficacious analysis method, variety of materials, impure salts and hydrofluorination residues have been assayed by both calorimetric assay and neutron counting. New data will be presented together with a review of published data. The precision and accuracy of these measurements are compared to chemistry values and are reported. The contribution of the gamma ray isotopic determination measurement to the overall error of the calorimetric assay or neutron assay is examined and discussed. Other factors affecting selection of the most appropriate non-destructive assay method are listed and considered

The influence of biological and environmental factors on metallothionein concentration in the blood.

Science.gov (United States)

Kowalska, Katarzyna; Bizoń, Anna; Zalewska, Marta; Milnerowicz, Halina

2015-01-01

The concentration of metallothionein (MT), a low-molecular-weight protein, is regulated by many factors, primarily metals (zinc, cadmium, copper), cytokines, glucocorticoides and free radicals. These factors are determined by such aspects of human biology as gender, pregnancy and age, as well as by environmental factors including the use of oral contraceptives and cigarette smoking, all which may affect MT levels in the body. The aim of this study was to investigate the influence of these biological and environmental factors on MT concentrations in erythrocyte lysate and in plasma. MT concentrations were determined by a two-step direct enzyme-linked immunosorbent assay. Evaluation of exposure to cigarette smoking was performed by checking cotinine levels in the plasma of subjects. The studies showed higher MT concentrations in both the erythrocyte lysate and plasma of women when compared to men. Furthermore, pregnancy causes an increase of MT concentration in plasma, while oral contraceptives cause an elevated concentration of MT in erythrocyte lysate. Age impacts plasma MT concentrations in men, whereas it does not affect concentrations of MT in erythrocyte lysate. Copyright © 2014 Elsevier GmbH. All rights reserved.

Influence of apical enlargement and complementary canal preparation with the Self-Adjusting File on endotoxin reduction in retreatment cases.

Science.gov (United States)

Silva, E J N L; Ferreira, V M; Silva, C C; Herrera, D R; De-Deus, G; Gomes, B P

2017-07-01

To compare the effectiveness of large apical preparations and complementary canal preparation with the Self-Adjusting File (SAF) in removing endotoxins from the root canal of teeth with apical periodontitis. Ten single-rooted and single-canaled teeth with post-treatment apical periodontitis were selected. Endotoxin samples were taken after removal of the root filling (S1), after chemomechanical preparation (CMP) using 2.5% NaOCl and an R25 file (S2), after CMP using 2.5% NaOCl and an R40 file (S3) and after complementary CMP using the SAF system (S4). Limulus amebocyte lysate (LAL) was used to measure endotoxin levels. The Friedman and Wilcoxon tests were used to compare endotoxin levels at each clinical intervention (P file was able to significantly reduce endotoxin levels (P file (P  0.05) following the use of the R40 instrument. Apical enlargement protocols were effective in significantly reducing endotoxin levels. Complementary preparation with the SAF system failed to eliminate residual endotoxin contents beyond those obtained with the R40 instrument. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Immune response in the lungs following oral immunization with bacterial lysates of respiratory pathogens.

Science.gov (United States)

Ruedl, C; Frühwirth, M; Wick, G; Wolf, H

1994-03-01

We have investigated the local immune response of the BALB/c mouse respiratory tract after oral immunization with a bacterial lysate of seven common respiratory pathogens. After two immunization on five consecutive days, we examined the immunoglobulin (immunoglobulin G [IgG], IgM, and IgA) secretion rates of cells isolated from the lungs and compared them with those of spleen cells of orally immunized and nonimmunized animals by using a new test system based on time-resolved fluorescence. The procedure followed the principle of the classical ELISPOT test with nitrocellulose-bottomed microtiter plates, but europium (Eu3+)-linked streptavidin rather than enzyme-conjugated streptavidin was used, with the advantage of quantifying secreted immunoglobulins instead of detecting single antibody-secreting cells. Lymphocytes isolated from the lungs of treated animals revealed significant increases in total and antigen-specific IgA synthesis compared with the rates of the controls, whereas IgG and IgM production rates showed no remarkable differences. In addition, the sera of treated mice revealed higher antigen-specific IgA titers but not increased IgM and IgG levels. We conclude that priming the gut-associated lymphoid tissue with bacterial antigens of pneumotropic microorganisms can elicit an enhanced IgA response in a distant mucosal effector site, such as the respiratory tract, according to the concept of a common mucosa-associated immune system.

The use of lumbar epidural injection of platelet lysate for treatment of radicular pain.

Science.gov (United States)

Centeno, Christopher; Markle, Jason; Dodson, Ehren; Stemper, Ian; Hyzy, Matthew; Williams, Christopher; Freeman, Michael

2017-11-25

Epidural steroid injections (ESI) are the most common pain management procedure performed in the US, however evidence of efficacy is limited. In addition, there is early evidence that the high dose of corticosteroids used can have systemic side effects. We describe the results of a case series evaluating the use of platelet lysate (PL) epidural injections for the treatment of lumbar radicular pain as an alternative to corticosteroids. Registry data was obtained for patients (N = 470) treated with PL epidural injections presenting with symptoms of lumbar radicular pain and MRI findings that were consistent with symptoms. Collected outcomes included numeric pain score (NPS), functional rating index (FRI), and a modified single assessment numeric evaluation (SANE) rating. Patients treated with PL epidurals reported significantly lower (p < .0001) NPS and FRI change scores at all time points compared to baseline. Post-treatment FRI change score means exceeded the minimal clinically important difference beyond 1 month. Average modified SANE ratings showed 49.7% improvement at 24 months post-treatment. Twenty-nine (6.3%) patients reported mild adverse events related to treatment. Patients treated with PL epidurals reported significant improvements in pain, exceeded the minimal clinically important difference (MCID) for FRI, and reported subjective improvement through 2-year follow-up. PL may be a promising substitute for corticosteroid.

Laser-Assisted Liposuction Using the Novel 1,444-nm Nd:YAG Laser for the Treatment of Gynecomastia: A Pilot Study.

Science.gov (United States)

Yoo, Kwang Ho; Bae, Jung Min; Won, Chae Young; Chung, Yu Seok; Goo, Boncheol; Rho, Yong Kwan; Kim, Gyong Moon; Lee, Jongwon; Ahn, Byeong Heon; Kim, Beom Joon

2015-01-01

Laser-assisted liposuction (LAL) is currently widely used to reduce localized fat. A novel Nd:YAG laser that uses a wavelength of 1,444 nm, which is better absorbed by fat, has recently been introduced. In this study, we investigated the efficacy of 1,444-nm Nd:YAG LAL for the treatment of gynecomastia. Thirteen Korean male patients (20-28 years, mean age 23 years) diagnosed with gynecomastia were enrolled in this study. All patients were treated by LAL with 1,444-nm Nd:YAG laser (100 µs pulse width, 40 Hz frequency, 300 mJ pulse energy and 12 W power with continuous emission) after tumescent anesthetic infiltration and were then evaluated. Outcome was assessed using the following 4 methods: (1) clinical assessment with photographs obtained before and 12 weeks after LAL treatment, (2) comparison of pre- and postoperative patient chest circumferences, (3) computed tomography (CT) scans to evaluate changes in breast thickness and (4) a patient satisfaction survey at the end of the study. After 12 weeks, most patients (84.5%) showed an improvement greater than 50%. Mean chest circumference was significantly reduced from 109.6 ± 8.2 to 101.2 ± 4.4 cm 12 weeks after LAL (p Gynecomastia can be safely treated with 1,444-nm Nd:YAG LAL to reduce fatty tissue and total breast volume. © 2015 S. Karger AG, Basel.

Development and implementation of a novel assay for L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) in cell lysates: L-2-HGDH deficiency in 15 patients with L-2-hydroxyglutaric aciduria

DEFF Research Database (Denmark)

Kranendijk, M; Salomons, G S; Gibson, K M

2009-01-01

L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphoc...

Immune Response Generated With the Administration of Autologous Dendritic Cells Pulsed With an Allogenic Tumoral Cell-Lines Lysate in Patients With Newly Diagnosed Diffuse Intrinsic Pontine Glioma

Directory of Open Access Journals (Sweden)

Daniel Benitez-Ribas

2018-04-01

Full Text Available Background and objectiveDiffuse intrinsic pontine glioma (DIPG is a lethal brainstem tumor in children. Dendritic cells (DCs have T-cell stimulatory capacity and, therefore, potential antitumor activity for disease control. DCs vaccines have been shown to reactivate tumor-specific T cells in both clinical and preclinical settings. We designed a phase Ib immunotherapy (IT clinical trial with the use of autologous dendritic cells (ADCs pulsed with an allogeneic tumors cell-lines lysate in patients with newly diagnosed DIPG after irradiation (radiation therapy.MethodsNine patients with newly diagnosed DIPG met enrollment criteria. Autologous dendritic cell vaccines (ADCV were prepared from monocytes obtained by leukapheresis. Five ADCV doses were administered intradermally during induction phase. In the absence of tumor progression, patients received three boosts of tumor lysate every 3 months during the maintenance phase.ResultsVaccine fabrication was feasible in all patients included in the study. Non-specific KLH (9/9 patients and specific (8/9 patients antitumor response was identified by immunologic studies in peripheral blood mononuclear cells (PBMC. Immunological responses were also confirmed in the T lymphocytes isolated from the cerebrospinal fluid (CSF of two patients. Vaccine administration resulted safe in all patients treated with this schema.ConclusionThese preliminary results demonstrate that ADCV preparation is feasible, safe, and generate a DIPG-specific immune response detected in PBMC and CSF. This strategy shows a promising backbone for future schemas of combination IT.

Correlation between the genotoxicity endpoints measured by two different genotoxicity assays: comet assay and CBMN assay

Directory of Open Access Journals (Sweden)

Carina Ladeira

2015-06-01

The results concerning of positive findings by micronuclei and non significant ones by comet assay, are corroborated by Deng et al. (2005 study performed in workers occupationally exposed to methotrexate, also a cytostatic drug. According to Cavallo et al. (2009, the comet assay seems to be more suitable for the prompt evaluation of the genotoxic effects, for instance, of polycyclic aromatic hydrocarbons mixtures containing volatile substances, whereas the micronucleus test seems more appropriate to evaluate the effects of exposure to antineoplastic agents. However, there are studies that observed an increase in both the comet assay and the micronucleus test in nurses handling antineoplastic drugs, although statistical significance was only seen in the comet assay, quite the opposite of our results (Maluf & Erdtmann, 2000; Laffon et al. 2005.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches.

Science.gov (United States)

Saury, Charlotte; Lardenois, Aurélie; Schleder, Cindy; Leroux, Isabelle; Lieubeau, Blandine; David, Laurent; Charrier, Marine; Guével, Laëtitia; Viau, Sabrina; Delorme, Bruno; Rouger, Karl

2018-05-02

Canine MuStem cells have demonstrated regenerative efficacy in a dog model of muscular dystrophy, and the recent characterization of human counterparts (hMuStem) has highlighted the therapeutic potential of this muscle-derived stem cell population. To date, these cells have only been generated in research-grade conditions. However, evaluation of the clinical efficacy of any such therapy will require the production of hMuStem cells in compliance with good manufacturing practices (GMPs). Because the current use of fetal bovine serum (FBS) to isolate and expand hMuStem cells raises several ethical, safety, and supply concerns, we assessed the use of two alternative xeno-free blood derivatives: human serum (HS) and a human platelet lysate (hPL). hMuStem cells were isolated and expanded in vitro in either HS-supplemented or hPL-supplemented media and the proliferation rate, clonogenicity, myogenic commitment potential, and oligopotency compared with that observed in FBS-supplemented medium. Flow cytometry and high-throughput 3'-digital gene expression RNA sequencing were used to characterize the phenotype and global gene expression pattern of hMuStem cells cultured with HS or hPL. HS-supplemented and hPL-supplemented media both supported the isolation and long-term proliferation of hMuStem cells. Compared with FBS-based medium, both supplements enhanced clonogenicity and allowed for a reduction in growth factor supplementation. Neither supplement altered the cell lineage pattern of hMuStem cells. In vitro differentiation assays revealed a decrease in myogenic commitment and in the fusion ability of hMuStem cells when cultured with hPL. In return, this reduction of myogenic potential in hPL-supplemented cultures was rapidly reversed by substitution of hPL with HS or fibrinogen-depleted hPL. Moreover, culture of hMuStem cells in hPL hydrogel and fibrinogen-depleted hPL demonstrated that myogenic differentiation potential is maintained in heparin-free hPL derivatives. Our

Inactivation of gram-negative bacteria in milk and banana juice by hen egg white and lambda lysozyme under high hydrostatic pressure.

Science.gov (United States)

Nakimbugwe, Dorothy; Masschalck, Barbara; Anim, Grace; Michiels, Chris W

2006-10-15

The effect of hen egg white lysozyme (HEWL) and bacteriophage lambda lysozyme (LaL) in combination with high pressure (HP) treatment on the inactivation of four gram-negative bacteria (Escherichia coli O157:H7, Shigella flexneri, Yersinia enterocolitica and Salmonella typhimurium), was studied in skim milk (pH 6.8; a(w) 0.997) and in banana juice (pH 3.8; a(w) 0.971). In the absence of lysozymes, S. flexneri was more sensitive to HP in milk than in banana juice, while the opposite was observed for the other three bacteria. In combination with HP treatment, LaL was more effective than HEWL on all bacteria in both milk and banana juice. Depending on the bacteria, inactivation levels in banana juice were increased from 0.4-2.7 log units by HP treatment alone to 3.6-6.5 log units in the presence of 224 U/ml LaL. Bacterial inactivation in milk was also enhanced by LaL but only by 0.5-2.1 log units. Under the experimental conditions used, LaL was more effective in banana juice than in milk, while the effectiveness of HEWL under the same conditions was not significantly affected by the food matrix. This effect could be ascribed to the low pH of the banana juice since LaL was also more effective on E. coli in buffer at pH 3.8 than at pH 6.8. Since neither LaL nor HEWL are enzymatically active at pH 3.8, we analysed bacterial lysis after HP treatment in the presence of these enzymes, and found that inactivation proceeds through a non-lytic mechanism at pH 3.8 and a lytic mechanism at pH 6.8. Based on these results, LaL may offer interesting perspectives for use as an extra hurdle in high pressure food preservation.

Platelet lysate induces chondrogenic differentiation of umbilical cord-derived mesenchymal stem cells.

Science.gov (United States)

Hassan, Ghmkin; Bahjat, Mohammad; Kasem, Issam; Soukkarieh, Chadi; Aljamali, Majd

2018-01-01

Articular cartilage has a poor capacity for self-repair, and thus still presents a major challenge in orthopedics. Mesenchymal stem cells (MSCs) are multipotent stem cells with the potential to differentiate into chondrocytes in the presence of transforming growth factor beta (TGF-β). Platelet lysate (PL) contains a relatively large number of growth factors, including TGF-β, and has been shown to ameliorate cartilage repair. Here, we investigated the ability of PL to direct chondrogenic differentiation of MSCs along with other standard differentiation components in a pellet culture system. We isolated and expanded MSCs from human umbilical cords using a PL-supplemented medium and characterized the cells based on immunophenotype and potential for differentiation to adipocytes and osteocytes. We further cultured MSCs as pellets in a chondrogenic-differentiation medium supplemented with PL. After 21 days, the pellets were processed for histological analysis and stained with alician blue and acridine orange. The expression of SOX9 was investigated using RT-PCR. MSCs maintained their stemness characteristics in the PL-supplemented medium. However, the distribution of cells in the pellets cultured in the PL-supplemented chondrogenic differentiation medium had a greater similarity to cartilage tissue-derived chondrocytes than to the negative control. The intense alician blue staining indicated an increased production of mucopolysaccharides in the differentiated pellets, which also showed elevated expression of SOX9 . Our data suggest that MSCs could be differentiated to chondrocytes in the presence of PL and absence of exogenous TGF-β. Further research needs to be conducted to understand the exact role and potential of PL in chondrogenic differentiation and chondrocyte regeneration.

DNA Electrochemistry Shows DNMT1 Methyltransferase Hyperactivity in Colorectal Tumors.

Science.gov (United States)

Furst, Ariel L; Barton, Jacqueline K

2015-07-23

DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and have found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by qPCR, DNMT1 protein abundance by western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation. Copyright © 2015 Elsevier Ltd. All rights reserved.

An analysis of employee exposure to organic dust at large-scale composting facilities

Science.gov (United States)

Sykes, P.; Allen, J. A.; Wildsmith, J. D.; Jones, K. P.

2009-02-01

The occupational health implications from exposure to dust, endotoxin and 1-3 β Glucan at commercial composting sites are uncertain. This study aims to establish employee exposure levels to inhalable and respirable dust, endotoxin and 1-3 β Glucan during various operational practices in the composting process. Personal samples were collected and the inhalable and respirable dust fractions were determined by gravimetric analysis. Endotoxin concentrations were determined using a Limulus Amebocyte Lysate assay (LAL). 1-3 β Glucan levels were estimated using a specific blocking agent to establish the contribution that these compounds gave to the original endotoxin assay. Employees' exposure to dust was found to be generally lower than the levels stipulated in the Control of Substances Hazardous to Health Regulations (COSHH) 2002 (as amended), (median inhalable fraction 1.08 mg/m3, min 0.25 mg/m3 max 10.80 mg/m3, median respirable fraction 0.05 mg/m3, min 0.02 mg/m3, max 1.49 mg/m3). Determination of the biological component of the dust showed that employees' exposures to endotoxin were elevated (median 31.5 EU/m3, min 2.00 EU/m3, max 1741.78 EU/m3), particularly when waste was agitated (median 175.0 EU/m3, min 2.03 EU/m3, max 1741.78 EU/m3). Eight out of 32 (25%) of the personal exposure data for endotoxin exceeded the 200 EU/m3 temporary legal limit adopted in the Netherlands and thirteen out of 32 (40.6%) exceeded the suggested 50 EU/m3 guidance level suggested to protect workers from respiratory health effects. A significant correlation was observed between employee inhalable dust exposure and personal endotoxin concentration (r = 0.728, phealth risks associated with endotoxin exposure at composting sites. Employee exposure levels and dose-response disease mechanisms are not well understood at this present time. Consequently, in light of this uncertainty, it is recommended that a precautionary approach be adopted in managing the potential health risks associated

Microbiology of aquatic environments: Characterizations of the microbiotas of municipal water supplies, the International Space Station Internal Active Thermal Control System's heat transport fluid, and US Space Shuttle drinking water

Science.gov (United States)

Bernardini, James Nicholas, III

An understanding of the microbiota within life support systems is essential for the prolonged presence of humans in space. This is because microbes may cause disease or induce biofouling and/or corrosion within spacecraft water systems. It is imperative that we develop effective high-throughput technologies for characterizing microbial populations that can eventually be used in the space environment. This dissertation describes testing and development of such methodologies, targeting both bacteria and viruses in water, and examines the bacterial and viral diversity within two spacecraft life support systems. The bacterial community of the International Space Station Internal Active Thermal Control System (IATCS) was examined using conventional culture-based and advanced molecular techniques including adenosine triphosphate (ATP) and Limulus Amebocyte Lysate (LAL) assays, direct microscopic examination, and analyses of 16S rRNA gene libraries from the community metagenome. The cultivable heterotrophs of the IATCS fluids ranged from below detection limit to 1.1x10 5/100 ml, and viable cells, measured by ATP, ranged from 1.4x10 3/100 ml to 7.7x105/100 ml. DNA extraction, cloning, sequencing, and bioinformatic analysis of the clones from 16S RNA gene libraries showed members of the firmicutes, alpha, beta, and gamma-proteobacteria to be present in the fluids. This persistent microbial bioburden and the presence of probable metal reducers, biofilm formers, and opportunistic pathogens illustrate the need for better characterization of bacterial communities present within spacecraft fluids. A new methodology was developed for detection of viruses in water using microarrays. Samples were concentrated by lyophilization, resuspended and filtered (0.22microm). Viral nucleic acids were then extracted, amplified, fluorescently labeled and hybridized onto a custom microarray with probes for ˜1000 known viruses. Numerous virus signatures were observed. Human Adenovirus C and

Microbead agglutination based assays

KAUST Repository

Kodzius, Rimantas

2013-01-21

We report a simple and rapid room temperature assay for point-of-care (POC) testing that is based on specific agglutination. Agglutination tests are based on aggregation of microbeads in the presence of a specific analyte thus enabling the macroscopic observation. Such tests are most often used to explore antibody-antigen reactions. Agglutination has been used for protein assays using a biotin/streptavidin system as well as a hybridization based assay. The agglutination systems are prone to selftermination of the linking analyte, prone to active site saturation and loss of agglomeration at high analyte concentrations. We investigated the molecular target/ligand interaction, explaining the common agglutination problems related to analyte self-termination, linkage of the analyte to the same bead instead of different microbeads. We classified the agglutination process into three kinds of assays: a two- component assay, a three-component assay and a stepped three- component assay. Although we compared these three kinds of assays for recognizing DNA and protein molecules, the assay can be used for virtually any molecule, including ions and metabolites. In total, the optimized assay permits detecting analytes with high sensitivity in a short time, 5 min, at room temperature. Such a system is appropriate for POC testing.

Evidence-Based Practice in Liposuction.

Science.gov (United States)

Collins, Patrick S; Moyer, Kurtis E

2018-01-24

The goal of this study is to examine the existing peer reviewed literature comparing modern adjunctive techniques in liposuction including laser-assisted liposuction (LAL) and ultrasound-assisted liposuction (UAL) to standard suction-assisted liposuction (SAL). We intend to interpret these findings into a literature-based clinical application to influence practice patterns. A literature review was conducted using a keyword search in PubMed. Keyword search items included liposuction, lipoplasty, suction assisted liposuction, ultrasound assisted liposuction, laser assisted liposuction, tumescent, liposuction comparison, liposuction review, and combinations therein. Exclusion criteria included articles with a primary focus on histologic effects of energy devices, primary animal models, primary opinion papers with no reference to available data, and industry-sponsored publications. Inclusion criteria included articles with direct comparison of liposuction modalities, randomized or blinded studies, and studies with objective outcomes. Twenty-five articles that met the inclusion criteria comparing SAL to UAL or LAL out of 9972 articles identified were obtained. The selected literature was assigned into 3 categories: evidence demonstrating an advantage of 1 modality (SAL, UAL, or LAL) over another, evidence that showed no benefit of 1 modality over another, and evidence that demonstrated risks of complications of 1 modality over another. The benefits of UAL and LAL over SAL include the following: (1) UAL over SAL in the treatment of gynecomastia, (2) LAL and UAL over SAL with decreased hemoglobin/hematocrit in high-volume lipoaspirates, and (3) LAL over SAL with skin tightening in select areas specifically the submental area. Otherwise, the literature demonstrates equivocal results among the described techniques with no clear benefit to set one apart from the other. There appears to be no demonstrable added benefit to the addition of either UAL or LAL that would urge a

Progress report 2001-2002; Rapport d'activite 2001-2002

Energy Technology Data Exchange (ETDEWEB)

NONE

2003-07-01

The document reviews the activities of the linear accelerator laboratory (LAL) for the years 2001 and 2002. Most part of these activities are active collaborations in international programs. Some experimental programs like Aleph (apparatus for Lep physics) and Delphi (detector with lepton, photon, and hadron identification) at the Lep (CERN) or Na48 (search for direct violation of the CP invariance) have entered their last stage. Others like H1 (electron-positron experiment at Hera), Babar (measurement of CP violation in the B{sup 0}B-bar{sup 0} system) at Slac (Stanford), or D0 (search for new particles at the Tevatron) at Fermilab, are fully operating and this document describes the contribution of the LAL for each of them. The preparation of experiments for the future LHC has represented the core of LAL's technical activities. This document reviews these activities and achievements for the Atlas (air toroid LHC apparatus system) and LHCB (large hadron collider beauty) experiments. The LAL is also involved in the definition of the future high energy linear electron-positron collider (FLC). The LAL contributes to some experiments of neutrino physics through Nemo (violation of the lepton charge) and Opera (neutrino oscillations) experiments and to various astro-particles experiments such as Eros (micro-lensing effect), Archeops and Planck (cosmology), Virgo (gravitation waves) and Auger (very high energy particles showers). Another important part of LAL's activities is teaching activity mainly inside the curriculum of Paris-sud university. (A.C.)

[Epidemiological, clinical, cytologic and immunophenotypic aspects of acute leukemia in children: the experience at the hematology laboratory of IBN SINA University Hospital Center].

Science.gov (United States)

Doumbia, Mariam; Uwingabiye, Jean; Bissan, Aboubacar; Rachid, Razine; Benkirane, Souad; Masrar, Azlarab

2016-01-01

The aim of this study was to describe epidemiological, cytologic and immunophenotypic aspects of acute leukemias (AL) in children diagnosed at IBN SINA University Hospital Center and to determine the concordance between cytology and immunophenotyping results. This is a cross-sectional study conducted in the hematology laboratory of IBN SINA University Hospital Center between June 2012 and May 2014. Among the 104 cases with diagnosed AL, 52% were boys with a sex-ratio H/F= 1.32, the average age was 5.7 years. The distribution of different types of AL was: lymphoid AL (LAL) (74%), myeloid (AML) (20.2%), biphenotypic AL (BAL) (65.8%). Among the LALs, 78% were classified as B LAL and 22% as T LAL. Clinical signs were mainly presented with tumor syndrome (73.1%), fever (61%) and hemorrhagic syndrome (50%). The most common blood count abnormalities were: thrombopenia (89.4%), anemia (86.5%), hyperleukocytosis (79.8%). The rate of peripheral and bone marrow blasts was statistically higher for LAL than for AML and BAL (p <0.001). The rate of relapse and mortality was 21.2% and 16. 3% respectively. Concordance rate between the results of cytology and of immunophenotyping was 92.7% for LAL and 82.6% for AML. Diagnosis of AL is always based primarily on cytology. Immunophenotyping allowed us to make a better distinction between acute leukemias. The management of paediatric AL is a major health problem which requires specialized care centers.

Progress report 2001-2002

International Nuclear Information System (INIS)

2003-01-01

The document reviews the activities of the linear accelerator laboratory (LAL) for the years 2001 and 2002. Most part of these activities are active collaborations in international programs. Some experimental programs like Aleph (apparatus for Lep physics) and Delphi (detector with lepton, photon, and hadron identification) at the Lep (CERN) or Na48 (search for direct violation of the CP invariance) have entered their last stage. Others like H1 (electron-positron experiment at Hera), Babar (measurement of CP violation in the B 0 B-bar 0 system) at Slac (Stanford), or D0 (search for new particles at the Tevatron) at Fermilab, are fully operating and this document describes the contribution of the LAL for each of them. The preparation of experiments for the future LHC has represented the core of LAL's technical activities. This document reviews these activities and achievements for the Atlas (air toroid LHC apparatus system) and LHCB (large hadron collider beauty) experiments. The LAL is also involved in the definition of the future high energy linear electron-positron collider (FLC). The LAL contributes to some experiments of neutrino physics through Nemo (violation of the lepton charge) and Opera (neutrino oscillations) experiments and to various astro-particles experiments such as Eros (micro-lensing effect), Archeops and Planck (cosmology), Virgo (gravitation waves) and Auger (very high energy particles showers). Another important part of LAL's activities is teaching activity mainly inside the curriculum of Paris-sud university. (A.C.)

Progress report 2001-2002; Rapport d'activite 2001-2002

Energy Technology Data Exchange (ETDEWEB)

NONE

2003-07-01

The document reviews the activities of the linear accelerator laboratory (LAL) for the years 2001 and 2002. Most part of these activities are active collaborations in international programs. Some experimental programs like Aleph (apparatus for Lep physics) and Delphi (detector with lepton, photon, and hadron identification) at the Lep (CERN) or Na48 (search for direct violation of the CP invariance) have entered their last stage. Others like H1 (electron-positron experiment at Hera), Babar (measurement of CP violation in the B{sup 0}B-bar{sup 0} system) at Slac (Stanford), or D0 (search for new particles at the Tevatron) at Fermilab, are fully operating and this document describes the contribution of the LAL for each of them. The preparation of experiments for the future LHC has represented the core of LAL's technical activities. This document reviews these activities and achievements for the Atlas (air toroid LHC apparatus system) and LHCB (large hadron collider beauty) experiments. The LAL is also involved in the definition of the future high energy linear electron-positron collider (FLC). The LAL contributes to some experiments of neutrino physics through Nemo (violation of the lepton charge) and Opera (neutrino oscillations) experiments and to various astro-particles experiments such as Eros (micro-lensing effect), Archeops and Planck (cosmology), Virgo (gravitation waves) and Auger (very high energy particles showers). Another important part of LAL's activities is teaching activity mainly inside the curriculum of Paris-sud university. (A.C.)

Disposable Amperometric Immunosensor for the Determination of Human P53 Protein in Cell Lysates Using Magnetic Micro-Carriers

Directory of Open Access Journals (Sweden)

María Pedrero

2016-11-01

Full Text Available An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53. The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode, upon addition of hydroquinone (HQ as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.

Development of an Injectable Calcium Phosphate/Hyaluronic Acid Microparticles System for Platelet Lysate Sustained Delivery Aiming Bone Regeneration.

Science.gov (United States)

Babo, Pedro S; Santo, Vítor E; Gomes, Manuela E; Reis, Rui L

2016-11-01

Despite the biocompatibility and osteoinductive properties of calcium phosphate (CaP) cements their low biodegradability hampers full bone regeneration. Herein the incorporation of CaP cement with hyaluronic acid (HAc) microparticles loaded with platelet lysate (PL) to improve the degradability and biological performance of the cements is proposed. Cement formulations incorporating increasing weight ratios of either empty HAc microparticles or microparticles loaded with PL (10 and 20 wt%) are developed as well as cements directly incorporating PL. The direct incorporation of PL improves the mechanical properties of the plain cement, reaching values similar to native bone. Morphological analysis shows homogeneous particle distribution and high interconnectivity between the HAc microparticles. The cements incorporating PL (with or without the HAc microparticles) present a sustained release of PL proteins for up to 8 d. The sustained release of PL modulates the expression of osteogenic markers in seeded human adipose tissue derived stem cells, thus suggesting the stimulatory role of this hybrid system toward osteogenic commitment and bone regeneration applications. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The 1988 to 1989 progress report of the Linear Accelerator Laboratory

International Nuclear Information System (INIS)

1990-01-01

The 1988 to 1989 activity report of the Laboratoire de l'Accelerateur Lineaire (LAL), at Orsay (France), is presented. The LAL research activities are performed in the particle physics field. The studies carried out on the top quark and neutrinos are underlined. The experiments underway and applying the following devices, are summarized: ALEPH, DELPHI, NA31, NA14 ' , UA2 ' , TAU-P, ASTERIX, HIGGS, CELLO and DM2. The planned experiments (Double Beta, Thermistole, H1), the developments, the LAL scientific activities and the list of published papers are included [fr

Human platelet lysate versus minoxidil stimulates hair growth by activating anagen promoting signaling pathways.

Science.gov (United States)

Dastan, Maryam; Najafzadeh, Nowruz; Abedelahi, Ali; Sarvi, Mohammadreza; Niapour, Ali

2016-12-01

Minoxidil and human platelet lysate (HPL) are commonly used to treat patients with hair loss. However, the roles of HPL versus minoxidil in hair follicle biology largely remain unknown. Here, we hypothesized that bulge and dermal papilla (DP) cells may express specific genes, including Kras, Erk, Akt, Shh and β-catenin after exposure to minoxidil or HPL. The mouse hair follicles were isolated on day 10 after depilation and bulge or DP regions were dissected. The bulge and DP cells were cultured for 14days in DMEM/F12 medium. Then, the cells were treated with 100μM minoxidil and 10% HPL for 10 days. Nuclear morphology was identified using DAPi staining. Reverse transcriptase and real-time polymerase chain reaction (PCR) analysis were also performed to examine the expression of Kras, Erk, Akt, Shh and β-catenin mRNA levels in the treated bulge and DP regions after organ culture. Here, we found that minoxidil influences bulge and DP cell survival (Pminoxidil treatment in both bulge and DP cells. HPL mediated Erk upregulation in both bulge and DP cells (Pminoxidil-treated bulge cells. In contrast, the expression of β-cateinin and Shh in the DP cells was not meaningfully increased after treatment with HPL. Our results suggest that minoxidil and HPL can promote hair growth by activating the main anagen inducing signaling pathways. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Aplicabilidade do Teste de Ativação de Monócitos (MAT no Brasil: importância da sua utilização como teste para detecção de pirogênios no controle da qualidade de produtos injetáveis | Applicability of the Monocyte Activation Test (MAT in Brazil: the importance of its use as a test for the detection of pyrogens in the quality control of injectable products

Directory of Open Access Journals (Sweden)

Cristiane Caldeira da Silva

2015-08-01

Full Text Available O MAT (sigla do inglês Monocyte Activation Test é considerado um potencial substituto do Teste de Pirogênios, entretanto: i. não foi avaliado para um número suficiente de produtos; ii. faltam dados que possam garantir sua capacidade em detectar pirogênios não endotoxinas; e iii. deve ser realizada a validação do método para cada classe de produtos. O objetivo foi identificar as monografias que requerem testes de pirogenicidade e propor os produtos que têm por base somente o teste de pirogênios como um ponto de partida para futuros estudos. As monografias específicas nas Farmacopeias Americana, Europeia e Brasileira que recomendam o Teste de Pirogênios ou Teste de Endotoxina Bacteriana ou LAL (sigla do inglês Limulus Amebocyte Lysate foram: Teste de Pirogênios: 20 monografias na Americana, 37 na Europeia e 28 na Brasileira. LAL: 619 monografias na Americana, 157 na Europeia e 41 na Brasileira. Somente quatro produtos requerem testes de pirogenicidade nas três farmacopeias analisadas. O Teste de Pirogênios e LAL são recomendados em seis monografias na Brasileira e 15 na Europeia. Na Brasileira, a maior parte dessas monografias é referente a produtos biológicos, sugerindo, assim, que estes devam ser os primeiros a ser testados, uma vez que são ensaiados em animais. ----------------------------------------------------------------------------------------------- Monocyte Activation Test (MAT is thought to be a good replacement for rabbit pyrogen test (RPT; however, MAT remains controversial. MAT was not adequately evaluated in a sufficient number of products, and there is no sufficient data that support the ability of MAT to detect non-endotoxin pyrogens. Furthermore, MAT was used subject to validation for each specific product. The aim of this study was to identify in main pharmacopoeias, whose monographs require pyrogenicity tests, and propose those products for which only the rabbit pyrogen test is required to be used as a

Assay-specific decision limits for two new automated parathyroid hormone and 25-hydroxyvitamin D assays.

Science.gov (United States)

Souberbielle, Jean-Claude; Fayol, Véronique; Sault, Corinne; Lawson-Body, Ethel; Kahan, André; Cormier, Catherine

2005-02-01

The recent development of nonradioactive automated assays for serum parathyroid hormone (PTH) and 25-hydroxyvitamin D (25OHD) has made measurement of these two hormones possible in many laboratories. In this study, we compared two new assays for PTH and 25OHD adapted on an automated analyzer, the LIAISON, with two manual immunoassays used worldwide. We studied 228 osteoporotic patients, 927 healthy individuals, 38 patients with primary hyperparathyroidism, and 167 hemodialyzed patients. Serum PTH was measured with the Allegro and the LIAISON assays, and 25OHD was measured with DiaSorin RIA and the LIAISON assay. Regression analysis was used to calculate decision thresholds for the LIAISON assays that were equivalent to those of the Allegro PTH and DiaSorin 25OHD assays. The 25OHD concentrations obtained with the LIAISON assay and the RIA in osteoporotic patients were well correlated (r = 0.83; P 50 nmol/L as eligible for the reference population for the LIAISON PTH assay. In this group, the 3rd-97th percentile interval for LIAISON PTH was 3-51 ng/L. Considering upper reference limits of 46 and 51 ng/L for the Allegro and LIAISON assays, respectively, the frequency of above-normal PTH concentrations in patients with primary hyperparathyroidism was similar in both assays. Regression analysis between serum PTH measured by the Allegro and LIAISON assays in 167 hemodialyzed patients and the corresponding Bland-Altman analysis of these data suggest that the LIAISON PTH assay tends to read higher than the Allegro assay at low concentrations but lower at high concentrations (>300 ng/L). Because clinical decision limits for both PTH and 25OHD should be assay specific, we propose equivalences between these assays and two manual assays used worldwide. These assay-specific decision limits should help potential users of the LIAISON PTH and 25OHD assays.

A versatile coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates.

Science.gov (United States)

Buntru, Matthias; Vogel, Simon; Stoff, Katrin; Spiegel, Holger; Schillberg, Stefan

2015-05-01

Cell-free protein synthesis is a powerful method for the high-throughput production of recombinant proteins, especially proteins that are difficult to express in living cells. Here we describe a coupled cell-free transcription-translation system based on tobacco BY-2 cell lysates (BYLs). Using a combination of fractional factorial designs and response surface models, we developed a cap-independent system that produces more than 250 μg/mL of functional enhanced yellow fluorescent protein (eYFP) and about 270 μg/mL of firefly luciferase using plasmid templates, and up to 180 μg/mL eYFP using linear templates (PCR products) in 18 h batch reactions. The BYL contains actively-translocating microsomal vesicles derived from the endoplasmic reticulum, promoting the formation of disulfide bonds, glycosylation and the cotranslational integration of membrane proteins. This was demonstrated by expressing a functional full-size antibody (∼ 150 μg/mL), the model enzyme glucose oxidase (GOx) (∼ 7.3 U/mL), and a transmembrane growth factor (∼ 25 μg/mL). Subsequent in vitro treatment of GOx with peptide-N-glycosidase F confirmed the presence of N-glycans. Our results show that the BYL can be used as a high-throughput expression and screening platform that is particularly suitable for complex and cytotoxic proteins. © 2014 Wiley Periodicals, Inc.

Endotoxins in surgical instruments of hip arthroplasty.

Science.gov (United States)

Goveia, Vania Regina; Mendoza, Isabel Yovana Quispe; Guimarães, Gilberto Lima; Ercole, Flavia Falci; Couto, Bráulio Roberto Gonçalves Marinho; Leite, Edna Marilea Meireles; Stoianoff, Maria Aparecida Resende; Ferreira, José Antonio Guimarães

2016-01-01

To investigate endotoxins in sterilized surgical instruments used in hip arthroplasties. A descriptive exploratory study conducted in a public teaching hospital. Six types of surgical instruments were selected, namely: acetabulum rasp, femoral rasp, femoral head remover, chisel box, flexible bone reamer and femoral head test. The selection was based on the analysis of the difficulty in removing bone and blood residues during cleaning. The sample was made up of 60 surgical instruments, which were tested for endotoxins in three different stages. The EndosafeTM Gel-Clot LAL (Limulus Amebocyte Lysate method) was used. There was consistent gel formation with positive analysis in eight instruments, corresponding to 13.3%, being four femoral rasps and four bone reamers. Endotoxins in quantity ≥0.125 UE/mL were detected in 13.3% of the instruments tested. Investigar endotoxinas em instrumentais cirúrgicos esterilizados empregados em artroplastias do quadril. Estudo exploratório, descritivo, desenvolvido em um hospital público de ensino. Foram selecionados seis tipos de instrumentais, a saber: raspa acetabular, raspa femural, saca-cabeça de fêmur, formão box, fresa de fêmur e cabeça de prova de fêmur. A seleção foi feita a partir da análise da dificuldade para a remoção de resíduos de sangue e osso durante a limpeza. A amostra foi constituída por 60 instrumentais cirúrgicos, que foram testados para endotoxinas em três momentos distintos. Foi utilizado o método de gel-clot pelo Limulus Amebócito Lisado (LAL) Endosafe(tm). Houve formação de gel consistente com análise positiva em oito instrumentais, o que corresponde a 13,3%, sendo quatro raspas de fêmur e quatro fresas de fêmur. Foram detectadas endotoxinas em quantidade ≥0,125 UE/mL em 13,3% dos instrumentais testados.

Biomonitoring of genotoxic risk in radar facility workers: comparison of the comet assay with micronucleus assay and chromatid breakage assay

International Nuclear Information System (INIS)

Garaj-Vrhovac, V.; Kopjar, N.

2003-01-01

Genotoxic risks of occupational exposure in a radar facility were evaluated by using alkaline comet assay, micronucleus assay and chromatid breakage assay on peripheral blood leukocytes in exposed subjects and corresponding controls. Results show that occupational exposure to microwave radiation correlates with an increase of genome damage in somatic cells. The levels of DNA damage in exposed subjects determined by using alkaline comet assay were increased compared to control and showed interindividual variations. Incidence of micronuclei was also significantly increased compared to baseline control values. After short exposure of cultured lymphocytes to bleomycin, cells of occupationally exposed subjects responded with high numbers of chromatid breaks. Although the level of chromosome damage generated by bleomycin varied greatly between individuals, in exposed subjects a significantly elevated number of chromatid breaks was observed. Our results support data reported in literature indicating that microwave radiation represents a potential DNA-damaging hazard. Alkaline comet assay is confirmed as a sensitive and highly reproducible technique for detection of primary DNA damage inflicted in somatic cells. Micronucleus assay was confirmed as reliable bio-markers of effect and chromatid breakage assay as sensitive bio-marker of individual cancer susceptibility. The results obtained also confirm the necessity to improve measures and to perform accurate health surveillance of individuals occupationally exposed to microwave radiation

Erythrocyte metallothionein as an index of zinc status in humans

International Nuclear Information System (INIS)

Grider, A.; Bailey, L.B.; Cousins, R.J.

1990-01-01

Metallothionein concentrations in erythrocyte lysates derived from human subjects were measured by an ELISA procedure. IgG obtained from serum of sheep injected with human metallothionein 1 was used in this competitive assay. Subjects were fed a semipurified zinc-deficient diet for an 8-day depletion period after 3 days of acclimation. Fasting plasma zinc concentrations were reduced ∼7%. Metallothionein in the erythrocyte lysates was significantly decreased to 59% of the initial level by the end of the depletion period. Supplementation of these depleted subjects with zinc did not increase erythrocyte metallothionein levels within 24 hr. Daily supplementation of control subjects with zinc increased erythrocyte metallothionein to a 7-fold maximum within 7 days. These levels were reduced by 61% within 14 days after zinc supplementation was terminated. Incubation of rat [ 35 S]metallothionein with human erythrocyte lysate showed a time-dependent increase in 35 S soluble in 20% trichloroacetic acid, indicating degradation of the labeled protein, presumably via protease activity in the lysate. It is proposed that zinc supplementation induces erythrocyte metallothionein during erythropoiesis and that low zinc intake decreases synthesis and/or accelerates degradation of the protein in reticulocytes/erythrocytes. Metallothionein levels in erythrocytes may provide a useful index upon which to assess zinc status in humans

Vene tank kui force majeure / Andrei Hvostov

Index Scriptorium Estoniae

Hvostov, Andrei, 1963-

2008-01-01

Autor on seisukohal, et eestlane ei taha oma ametnikke ülal pidada. Küsimus laieneb tema sõnul eksistentsiaalseks: kas eestlane tahab üldse oma riiki ülal pidada? Rahvas näeb riigis vaid küllusesarve, poliitikud oma ambitsioonide realiseerimise vahendit

Integrated culture platform based on a human platelet lysate supplement for the isolation and scalable manufacturing of umbilical cord matrix-derived mesenchymal stem/stromal cells.

Science.gov (United States)

de Soure, António M; Fernandes-Platzgummer, Ana; Moreira, Francisco; Lilaia, Carla; Liu, Shi-Hwei; Ku, Chen-Peng; Huang, Yi-Feng; Milligan, William; Cabral, Joaquim M S; da Silva, Cláudia L

2017-05-01

Umbilical cord matrix (UCM)-derived mesenchymal stem/stromal cells (MSCs) are promising therapeutic candidates for regenerative medicine settings. UCM MSCs have advantages over adult cells as these can be obtained through a non-invasive harvesting procedure and display a higher proliferative capacity. However, the high cell doses required in the clinical setting make large-scale manufacturing of UCM MSCs mandatory. A commercially available human platelet lysate-based culture supplement (UltraGRO TM , AventaCell BioMedical) (5%(v/v)) was tested to effectively isolate UCM MSCs and to expand these cells under (1) static conditions, using planar culture systems and (2) stirred culture using plastic microcarriers in a spinner flask. The MSC-like cells were isolated from UCM explant cultures after 11 ± 2 days. After five passages in static culture, UCM MSCs retained their immunophenotype and multilineage differentiation potential. The UCM MSCs cultured under static conditions using UltraGRO TM -supplemented medium expanded more rapidly compared with UCM MSCs expanded using a previously established protocol. Importantly, UCM MSCs were successfully expanded under dynamic conditions on plastic microcarriers using UltraGRO TM -supplemented medium in spinner flasks. Upon an initial 54% cell adhesion to the beads, UCM MSCs expanded by >13-fold after 5-6 days, maintaining their immunophenotype and multilineage differentiation ability. The present paper reports the establishment of an easily scalable integrated culture platform based on a human platelet lysate supplement for the effective isolation and expansion of UCM MSCs in a xenogeneic-free microcarrier-based system. This platform represents an important advance in obtaining safer and clinically meaningful MSC numbers for clinical translation. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Error Estimation for the Linearized Auto-Localization Algorithm

Directory of Open Access Journals (Sweden)

Fernando Seco

2012-02-01

Full Text Available The Linearized Auto-Localization (LAL algorithm estimates the position of beacon nodes in Local Positioning Systems (LPSs, using only the distance measurements to a mobile node whose position is also unknown. The LAL algorithm calculates the inter-beacon distances, used for the estimation of the beacons’ positions, from the linearized trilateration equations. In this paper we propose a method to estimate the propagation of the errors of the inter-beacon distances obtained with the LAL algorithm, based on a first order Taylor approximation of the equations. Since the method depends on such approximation, a confidence parameter τ is defined to measure the reliability of the estimated error. Field evaluations showed that by applying this information to an improved weighted-based auto-localization algorithm (WLAL, the standard deviation of the inter-beacon distances can be improved by more than 30% on average with respect to the original LAL method.

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

Science.gov (United States)

Altaie, Ala; Owston, Heather; Jones, Elena

2016-02-26

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its "potency" assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

Y chromosomal and sex effects on the behavioral stress response in the defensive burying test in wild house mice

NARCIS (Netherlands)

Sluyter, F; Korte, SM; Van Baal, GCM; De Ruiter, AJH; Van Oortmerssen, GA

1999-01-01

Genetically selected short attack latency (SAL) and long attack latency (LAL) male wild house mice behave differently in the defensive burying test. When challenged, SAL males respond actively with more time spent on defensive burying, whereas LAL males are more passive with more time remaining

Assay strategies and methods for phospholipases

International Nuclear Information System (INIS)

Reynolds, L.J.; Washburn, W.N.; Deems, R.A.; Dennis, E.A.

1991-01-01

Of the general considerations discussed, the two issues which are most important in choosing an assay are (1) what sensitivity is required to assay a particular enzyme and (2) whether the assay must be continuous. One can narrow the options further by considering substrate availability, enzyme specificity, assay convenience, or the presence of incompatible side reactions. In addition, the specific preference of a particular phospholipase for polar head group, micellar versus vesicular substrates, and anionic versus nonionic detergents may further restrict the options. Of the many assays described in this chapter, several have limited applicability or serious drawbacks and are not commonly employed. The most commonly used phospholipase assays are the radioactive TLC assay and the pH-stat assay. The TLC assay is probably the most accurate, sensitive assay available. These aspects often outweigh the disadvantages of being discontinuous, tedious, and expensive. The radioactive E. coli assay has become popular recently as an alternative to the TLC assay for the purification of the mammalian nonpancreatic phospholipases. The assay is less time consuming and less expensive than the TLC assay, but it is not appropriate when careful kinetics are required. Where less sensitivity is needed, or when a continuous assay is necessary, the pH-stat assay is often employed. With purified enzymes, when free thiol groups are not present, a spectrophotometric thiol assay can be used. This assay is ∼ as sensitive as the pH-stat assay but is more convenient and more reproducible, although the substrate is not available commercially. Despite the many assay choices available, the search continues for a convenient, generally applicable assay that is both sensitive and continuous

Dose-dependent utilisation of casein-linked lysinoalanine, N(epsilon)-fructoselysine and N(epsilon)-carboxymethyllysine in rats.

Science.gov (United States)

Somoza, Veronika; Wenzel, Elisabeth; Weiss, Carola; Clawin-Rädecker, Ingrid; Grübel, Nadine; Erbersdobler, Helmut F

2006-09-01

During the heat treatment of protein-containing foods, the amino acid lysine is most prone to undergo chemical reactions in the course of amino acid cross-linking or Maillard reactions. Among the reaction products formed, lysinoalanine (LAL), N(epsilon)-fructoselysine (FL) and N(epsilon)-carboxymethyllysine (CML) are those which serve as sensitive markers for the heat treatment applied. From a nutritional perspective, these compounds are ingested with the diet in considerable amounts but information about their metabolic transit and putative in vivo effects is scarce. In the present study, casein-linked LAL, FL and CML were administered to rats in two different doses for 10 days. Quantitation of LAL, FL and CML in plasma, tissue and faeces samples revealed that the kidneys are the predominant sites of accumulation and excretion. The maximum percent of dietary LAL, FL and CML excreted in the urine was 5.6, 5.2 and 29%, whereas the respective recoveries in the kidneys were 0.02, 26 and 1.4%. The plasma and tissue analyses revealed that the endogenous load of either compound is increased by its dietary intake. But the dose-dependent utilisation of dietary protein-linked LAL, FL and CML in rats has been demonstrated for the first time to vary substantially from each other.

Solid phase assays

International Nuclear Information System (INIS)

Reese, M.G.; Johnson, L.R.; Ransom, D.K.

1980-01-01

In a solid phase assay for quantitative determination of biological and other analytes, a sample such as serum is contacted with a receptor for the analyte being assayed, the receptor being supported on a solid support. No tracer for the analyte is added to the sample before contacting with the receptor; instead the tracer is contacted with the receptor after unbound analyte has been removed from the receptor. The assay can be otherwise performed in a conventional manner but can give greater sensitivity. (author)

Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells.

Directory of Open Access Journals (Sweden)

Michael Lohmann

Full Text Available The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS or other serum supplements such as human platelet lysate (HPL. In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1, but it was significantly higher with HPLs from younger donors (45 years. Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal. HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1 or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3 were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.

Donor Age of Human Platelet Lysate Affects Proliferation and Differentiation of Mesenchymal Stem Cells

Science.gov (United States)

Lohmann, Michael; Walenda, Gudrun; Hemeda, Hatim; Joussen, Sylvia; Drescher, Wolf; Jockenhoevel, Stefan; Hutschenreuter, Gabriele; Zenke, Martin; Wagner, Wolfgang

2012-01-01

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation. PMID:22662236

Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

Energy Technology Data Exchange (ETDEWEB)

Singer, Timothy M. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Lambert, Iain B. [Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, Ont., K1S 5B6 (Canada); Williams, Andrew [Biostatistics and Epidemiology Division, Safe Environments Programme, 6604B, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Douglas, George R. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada); Yauk, Carole L. [Mutagenesis Section, Environmental and Occupational Toxicology Division, Safe Environments Programme, 0803A, Health Canada, Ottawa, Ont., K1A 0K9 (Canada)]. E-mail: carole_yauk@hc-sc.gc.ca

2006-06-25

Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.

Detection of induced male germline mutation: Correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

International Nuclear Information System (INIS)

Singer, Timothy M.; Lambert, Iain B.; Williams, Andrew; Douglas, George R.; Yauk, Carole L.

2006-01-01

Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently-the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development

Baltic air wars intensify / Dorian Ziedonis

Index Scriptorium Estoniae

Ziedonis, Dorian

2008-01-01

Leedu lennufirma FlyLAL pidi maksma ära 40 000 euro suuruse trahvi, et firma lennuk saaks Riia lennuväljalt lahkuda. Varem oli FlyLAL nõudnud Riia Lennujaamalt kompensatsiooni, kuna lennujaam oli võimaldanud Läti lennufirmal airBaltic maksta väiksemaid lennujaamatasusid

Clinical Features of Lysosomal Acid Lipase Deficiency

NARCIS (Netherlands)

Burton, Barbara K.; Deegan, Patrick B.; Enns, Gregory M.; Guardamagna, Ornella; Horslen, Simon; Hovingh, Gerard K.; Lobritto, Steve J.; Malinova, Vera; McLin, Valerie A.; Raiman, Julian; Di Rocco, Maja; Santra, Saikat; Sharma, Reena; Sykut-Cegielska, Jolanta; Whitley, Chester B.; Eckert, Stephen; Valayannopoulos, Vassili; Quinn, Anthony G.

2015-01-01

The aim of this study was to characterize key clinical manifestations of lysosomal acid lipase deficiency (LAL D) in children and adults. Investigators reviewed medical records of LAL D patients ages ≥5 years, extracted historical data, and obtained prospective laboratory and imaging data on living

Evaluation of total PSA assay on vitros ECi and correlation with Kryptor-PSA assay.

Science.gov (United States)

Cassinat, B; Wacquet, M; Toubert, M E; Rain, J D; Schlageter, M H

2001-01-01

An increasing number of multiparametric immuno-analysers for PSA assays are available. As different immuno-assays may vary in their analytical quality and their accuracy for the follow-up of patients, expertise is necessary for each new assay. The PSA assay on the Vitros-ECi analyser has been evaluated and compared with the PSA assay from the Kryptor analyser. Variation coefficients were 0.91 to 1.98% for within-run assays, and 4.2% to 5.4% for interassay (PSA levels = 0.8 microgram/L to 33.6 micrograms/L). Dilution tests showed 93 to 136% recovery until 70 micrograms/L PSA. Functional sensitivity was estimated at 0.03 microgram/L. Equimolarity of the test was confirmed. Correlation of PSA levels measured with Vitros-ECi and Kryptor analysers displayed a correlation coefficient r2 of 0.9716. The half-lives and doubling times of PSA were similar using both methods. Vitros-ECi PSA assay meets the major criteria for the management of prostate cancer patients.

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African Journals Online (AJOL)

Proff.Adewunmi

76 modifications (Feng et al. 2012a). Briefly, tissue lysate was prepared using RIPA lysis buffer, and supernatant was collected after centrifugation. (12,000 rpm for 5 min, 4°C). Then, the protein concentration of each sample supernatant was determined using protein assay kit. The protein was separated via SDS-PAGE and ...

Hormone assay

International Nuclear Information System (INIS)

Eisentraut, A.M.

1977-01-01

An improved radioimmunoassay is described for measuring total triiodothyronine or total thyroxine levels in a sample of serum containing free endogenous thyroid hormone and endogenous thyroid hormone bound to thyroid hormone binding protein. The thyroid hormone is released from the protein by adding hydrochloric acid to the serum. The pH of the separated thyroid hormone and thyroid hormone binding protein is raised in the absence of a blocking agent without interference from the endogenous protein. 125 I-labelled thyroid hormone and thyroid hormone antibodies are added to the mixture, allowing the labelled and unlabelled thyroid hormone and the thyroid hormone antibody to bind competitively. This results in free thyroid hormone being separated from antibody bound thyroid hormone and thus the unknown quantity of thyroid hormone may be determined. A thyroid hormone test assay kit is described for this radioimmunoassay. It provides a 'single tube' assay which does not require blocking agents for endogenous protein interference nor an external solid phase sorption step for the separation of bound and free hormone after the competitive binding step; it also requires a minimum number of manipulative steps. Examples of the assay are given to illustrate the reproducibility, linearity and specificity of the assay. (UK)

The Pilot Staffing Conundrum: A Delphi Study

Science.gov (United States)

2009-06-01

Project, AFIT/ GMO /LAL/98J-2. School of Logistics and Acquisition Management, Air Force Institute of Technology (AU), Wright Patterson AFB, OH, June...Kafer, John H. Relationship of Airline Pilot Demand and Air Force Pilot Retention. Graduate Research Project, AFIT/ GMO /LAL/98J-11. School of Logistics

Sadhana | Indian Academy of Sciences

Indian Academy of Sciences (India)

Home; Journals; Sadhana. BASANT LAL SHARMA. Articles written in Sadhana. Volume 42 Issue 5 May 2017 pp 713-728. Continuum limit of discrete Sommerfeld problems on square lattice · BASANT LAL SHARMA · More Details Abstract Fulltext PDF. A low-frequency approximation of the discrete Sommerfeld diffraction ...

Calcium alginate particles for the combined delivery of platelet lysate and vancomycin hydrochloride in chronic skin ulcers.

Science.gov (United States)

Mori, Michela; Rossi, Silvia; Bonferoni, Maria Cristina; Ferrari, Franca; Sandri, Giuseppina; Riva, Federica; Del Fante, Claudia; Perotti, Cesare; Caramella, Carla

2014-01-30

The aim of the present work was the development of a powder formulation for the combined delivery of platelet lysate and of a model antibiotic drug, vancomycin hydrochloride (VCM), in chronic skin ulcers. Calcium alginate particles were prepared by freeze-drying beads obtained by ionic gelation method. The experimental conditions adopted permitted the complete loading of VCM and of PDGF AB, the growth factor chosen as representative of those contained in PL. Such particles where able to absorb PBS (mimicking wound exudate), to form a gel and to modulate the release of VCM and of PDGF AB. They are characterized by enhancement properties of human fibroblast proliferation due to PL presence. In particular, PL, when loaded in alginate particles, was able not only to increase the number of viable cells, but also the number of cells in proliferative phase. Such properties were comparable to those of fresh PL indicating the capability of calcium alginate particles to load PL bioactive substances without altering their activity. The formulation developed is characterized by an easier and a less painful administration with respect to traditional gauzes and semisolid preparations and permits the loading in the same dosage form of active substances of different nature avoiding eventual incompatibility problems. Copyright © 2013 Elsevier B.V. All rights reserved.

Long-term safety and efficacy of autologous platelet lysate drops for treatment of ocular GvHD.

Science.gov (United States)

Pezzotta, S; Del Fante, C; Scudeller, L; Rossi, G C; Perotti, C; Bianchi, P E; Antoniazzi, E

2017-01-01

Current ocular GvHD (oGvHD) treatments are suboptimal. We investigated the safety and efficacy of long-term continuous treatment with autologous platelet lysate (PL) drops in patients with oGvHD Dry Eye Syndrome (DES) score 2-3 refractory to topical conventional therapy. Ophthalmic evaluation was performed at 6 month intervals. Symptoms were assessed using the Glaucoma Symptom Scale (GSS). Patients were defined 'responders' when showing a reduction at least one grade on National Institutes of Health Eye Score from baseline at the 6 month visit. Thirty-one patients were included, and 16 (51%) completed 36 months of follow-up (range 6.5-72.7). At 6 months all patients were classified as responders: median GSS symptom score decreased from 70 to 41 (33 at 36 months), median GSS function score reduced from 68 to 46 (33 at 36 months) (all P<0.001). Median Tear Break Up Time improved from 3 to 6 s after 6 months and was maintained over time. All signs improved at 6 and 36 months (clinical and statistical significance). No severe adverse events occurred. Long-term treatment with PL drops is secure and effective for oGvHD and can be an efficient therapy option from initial stages of oGvHD to prevent permanent ocular impairment and improving quality of life.

Development and validation of a production process of platelet lysate for autologous use.

Science.gov (United States)

Plöderl, Karin; Strasser, Cornelia; Hennerbichler, Simone; Peterbauer-Scherb, Anja; Gabriel, Christian

2011-01-01

Growth factors (GF) contained in platelets are a potential source to improve wound healing by the stimulation and acceleration of soft tissue and bone healing. This resulted in the idea that autologous platelet-rich plasma or platelet lysate (PL) containing high levels of GF might improve healing processes. Today platelet products are already applied in bone and maxillofacial surgery. In recent years, cosmetic surgery and facial rejuvenation procedures are growing steadily. New methods including platelet products aiming to induce non-surgical reduction of wrinkles upon topical injection and to minimize surgical risks in general are developed. Several point-of-care devices are already available on the market. However, the amount of PL obtained by these kits is far too high for certain applications in cosmetic surgery and they offer no possibility of storing the remaining material in a sterile manner. Therefore we developed a procedure for the sterile production of smaller amounts of PL in a closed system that can also be split into several products for repeated administration. The closed system was determined to be a bag system designed for an autologous blood donation of 100 ml whole blood. We set a special focus on the validation of the production procedure, mainly regarding sterility and platelet recovery. For validation 22 healthy volunteers were asked for a blood donation, which was centrifuged twice to obtain concentrated platelets (CP). A freeze-thaw cycle caused lysis of the CP to get approximately 8.48 ± 1.36 ml PL. We yielded satisfying results of 100% sterility and a platelet recovery of 36.92% ± 18.71%. We therefore conclude that the PL obtained is ready for studies comparing it with traditional treatments.

Effects of laser-assisted lipolysis on nipple-areola complex.

Science.gov (United States)

Sönmez Ergün, Selma; Kayan, Reşit Burak; Güleş, Mustafa Ekrem; Kuzu, İsmail Melih

2017-08-01

Gynecomastia, as a most common benign condition, represents itself as the enlargement of the male breast and also nipple-areola complex as the severity of the condition increases. With this study, we aimed to clarify the effects of 980-nm diode laser on nipple-areola complex (NAC). Although numerous open techniques have been described to correct gynecomastia, nowadays trends have shifted to minimally invasive techniques such as laser-assisted lipolysis (LAL). A total of 25 patients with bilateral gynecomastia treated with LAL by using a 980-nm diode laser. The resultant contour and reduced size of the complex were satisfactory. LAL leads to significant reduction of the size of NAC.

Use of activity-based probes to develop high throughput screening assays that can be performed in complex cell extracts.

Directory of Open Access Journals (Sweden)

Edgar Deu

2010-08-01

Full Text Available High throughput screening (HTS is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities.Here, we described a method to use activity-based probes (ABPs to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z'>0.8 that are suitable for use in screening large collections of small molecules (i.e >300,000 for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates.We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic.

Characterization of biological aerosol exposure risks from automobile air conditioning system.

Science.gov (United States)

Li, Jing; Li, Mingzhen; Shen, Fangxia; Zou, Zhuanglei; Yao, Maosheng; Wu, Chang-yu

2013-09-17

Although use of automobile air conditioning (AC) was shown to reduce in-vehicle particle levels, the characterization of its microbial aerosol exposure risks is lacking. Here, both AC and engine filter dust samples were collected from 30 automobiles in four different geographical locations in China. Biological contents (bacteria, fungi, and endotoxin) were studied using culturing, high-throughput gene sequence, and Limulus amebocyte lysate (LAL) methods. In-vehicle viable bioaerosol concentrations were directly monitored using an ultraviolet aerodynamic particle sizer (UVAPS) before and after use of AC for 5, 10, and 15 min. Regardless of locations, the vehicle AC filter dusts were found to be laden with high levels of bacteria (up to 26,150 CFU/mg), fungi (up to 1287 CFU/mg), and endotoxin (up to 5527 EU/mg). More than 400 unique bacterial species, including human opportunistic pathogens, were detected in the filter dusts. In addition, allergenic fungal species were also found abundant. Surprisingly, unexpected fluorescent peaks around 2.5 μm were observed during the first 5 min use of AC, which was attributed to the reaerosolization of those filter-borne microbial agents. The information obtained here can assist in minimizing or preventing the respiratory allergy or infection risk from the use of automobile AC system.

Masking of endotoxin in surfactant samples: Effects on Limulus-based detection systems.

Science.gov (United States)

Reich, Johannes; Lang, Pierre; Grallert, Holger; Motschmann, Hubert

2016-09-01

Over the last few decades Limulus Amebocyte Lysate (LAL) has been the most sensitive method for the detection of endotoxins (Lipopolysaccharides) and is well accepted in a broad field of applications. Recently, Low Endotoxin Recovery (LER) in biopharmaceutical drug products has been noticed, whereby the detection of potential endotoxin contaminations is not ensured. Notably, most of these drug products contain surfactants, which can have crucial effects on the detectability of endotoxin. In order to analyze the driving forces of LER, endotoxin detection in samples containing nonionic surfactants in various buffer systems was investigated. The results show that the process of LER is kinetically controlled and temperature-dependent. Furthermore, only the simultaneous presence of nonionic surfactants and components capable of forming metal complexes resulted in LER. In addition, capacity experiments show that even hazardous amounts of endotoxin can remain undetectable within such formulation compositions. In conclusion, the LER phenomenon is caused by endotoxin masking and not by test interference. In this process, the supramolecular structure of endotoxin is altered and exhibits only a limited susceptibility in binding to the Factor C of Limulus-based detection systems. We propose a two-step mechanism of endotoxin masking by complex forming agents and nonionic surfactants. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

The Comet Assay: Tails of the (Unexpected. Use of the comet assay in pharmaceutical development.

Directory of Open Access Journals (Sweden)

Bas-jan Van Der Leede

2015-08-01

Full Text Available In genotoxicity testing of pharmaceuticals the rodent alkaline comet assay is being increasingly used as a second in vivo assay in addition to the in vivo micronucleus assay to mitigate in vitro positive results as recommended by regulatory guidance. In this presentation we want to give insight into the circumstances in vivo comet assay is deployed in a Genetic Toxicology Department of a pharmaceutical company. As the in vivo comet assay is a salvage assay, it means that some events have occurred in an in vitro assay and that the compound (or metabolite responsible for this signal is potentially deselected for further development. More than often the decision to perform an in vivo comet assay is at a very early stage in development and the first time that the compound will be tested in vivo at high/toxic dose levels. As almost no toxicokinetic data and tissue distribution data are available a careful design with maximizes the chances for successful mitigation is necessary. Decisions on acute or repeated dosing need to be made and arrangements for combining the in vivo comet assay with the in vivo micronucleus assay are to be considered. Often synthesis methods need to be scaled up fast to provide the required amount of compound and information on suitable formulations needs to be in place. As exposure data is crucial for interpretation of results, analytical methods need to be brought in place rapidly. An experienced multi skilled and communicative team needs to be available to deploy successfully this kind of assays at an early stage of development. We will present a few scenarios on study conduct and demonstrate how this assay can make a difference for the further development of a new drug.

The ribosome-inactivating, antiproliferative and teratogenic activities and immunoreactivities of a protein from seeds of Luffa aegyptiaca (Cucurbitaceae).

Science.gov (United States)

Ng, T B; Chan, W Y; Yeung, H W

1993-05-01

1. The protein isolated from Luffa aegyptiaca seeds was capable of inhibiting protein synthesis in a rabbit reticulocyte lysate system and [3H]thymidine uptake by mouse melanoma (B16) cells. 2. It also adversely affected the development of mouse embryos in culture. 3. In enzyme-linked immunosorbent assay it reacted with antisera raised against other ribosome-inactivating proteins.

Radioreceptor assays: plasma membrane receptors and assays for polypeptide and glycoprotein hormones

International Nuclear Information System (INIS)

Schulster, D.

1977-01-01

Receptors for peptide, protein and glycoprotein hormones, and the catecholamines are located on the plasma membranes of their target cells. Preparations of the receptors may be used as specific, high-affinity binding agents for these hormones in assay methodology akin to that for radioimmunoassay. A particular advantage of the radioreceptor assay is that it has a specificity directed towards the biologically active region of the hormone, rather than to some immunologically active region that may have little (or no) involvement in the expression of hormonal activity. Methods for hormone receptor preparation vary greatly, and range from the use of intact cells (as the source of hormone receptor) to the use of purified or solubilized membrane receptors. Receptors isolated from plasma membranes have proved to be of variable stability, and may be damaged during preparation and/or storage. Moreover, since they are present in relatively low concentration in the cell, their preparation in sufficient quantity for use in a radioreceptor assay may present technical problems. In general, there is good correlation between radioreceptor assays and in-vitro bioassays; differences between results from radioreceptor assays and radioimmunoassays are similar to those noted between in-vitro bioassays and radioimmunoassays. The sensitivity of the method is such that normal plasma concentrations of various hormones have been assayed by this technique. (author)

Relationship between the radioisotopic footpad assay and other immunological assays in tumor bearing rats

International Nuclear Information System (INIS)

Mizushima, Yutaka; Takeichi, Noritoshi; Minami, Akio; Kasai, Masaharu; Itaya, Toshiyuki

1981-01-01

KMT-17, a fibrosarcoma induced by 3-methylcholanthrene in a WKA rat, is a sensitive tumor to various kinds of immunological assays and is a suitable model tumor for the study of the immune status in tumor bearing hosts. The antitumor immune response of KMT-17 bearing rats was studied by a radioisotopic footpad assay (FPA) in comparison with other in vivo and in vitro assays. Delayed hypersensitivity to tumor antigens measured by the FPA was observed from the 8th day after transplantation of KMT-17 cells, reached a peak on the 12 - 15th day, and then declined in the late stage on the 17th day. The kinetics of the FPA correlated well with those of an in vivo Winn assay and of an in vitro lymphocyte cytotoxicity assay ( 51 Cr-release assay). The appearance of an antitumor antibody detected by a complement dependent cytotoxicity test also correlated well with the kinetics of the FPA. A growth inhibition assay (GIA) for non-specific cell-mediated immunity also showed similar kinetics to that of the FPA. The delayed hypersensitivity footpad reaction to tumor cell extracts measured by this FPA was tumor-specific. These results suggest that the FPA is a simple and reliable in vivo assay for evaluating antitumor immunity in tumor bearing hosts. (author)

Improved detection limits of bacterial endotoxins using new type of planar interdigital sensors

KAUST Repository

Syaifudin, A. R Mohd

2012-10-01

New types of planar interdigital sensors were fabricated by photolithography and etching techniques on a Silicon/Silicon Dioxide (Si/SiO2) wafer (single side polished). The sensors were then coated with APTES (3-aminopropyltrietoxysilane) a cross linker used to bind Polymyxin B (PmB) molecules on electrodes surface. PmB is an antimicrobial peptide produced by the Gram-positive bacterium-Bacillus which has specific binding properties to Lipopolysaccharide (LPS). This paper will discuss the fabrication process, coating and immobilization procedures and analysis of sensors\\' performance based on Impedance Spectroscopy method. The sensor sensitivity was compared to standard ToxinSensor Chromogenic LAL Endotoxin Assay Kit for verification. © 2012 IEEE.

Liquor oligoclonal bands assay: interpretation, correlation with other laboratory assays and importance for diagnostics of neurological disorders

OpenAIRE

Bagdonas, Dovydas

2017-01-01

Aim: to analyse the possible relationship between liquor IgG oligoclonal bands assay and other laboratory assays in neurological patients. Objectives: to determine the frequency of oligoclonal bands in neurological patients; to compare the results between serum and liquor laboratory assays in dependence of oligoclonal bands assay results; to evaluate the relationships between oligoclonal bands assay and serological-immunological assays for infectious diseases, gender, age and neurological ...

Scaling down the size and increasing the throughput of glycosyltransferase assays: activity changes on stem cell differentiation.

Science.gov (United States)

Patil, Shilpa A; Chandrasekaran, E V; Matta, Khushi L; Parikh, Abhirath; Tzanakakis, Emmanuel S; Neelamegham, Sriram

2012-06-15

Glycosyltransferases (glycoTs) catalyze the transfer of monosaccharides from nucleotide-sugars to carbohydrate-, lipid-, and protein-based acceptors. We examined strategies to scale down and increase the throughput of glycoT enzymatic assays because traditional methods require large reaction volumes and complex chromatography. Approaches tested used (i) microarray pin printing, an appropriate method when glycoT activity was high; (ii) microwells and microcentrifuge tubes, a suitable method for studies with cell lysates when enzyme activity was moderate; and (iii) C(18) pipette tips and solvent extraction, a method that enriched reaction product when the extent of reaction was low. In all cases, reverse-phase thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified the reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrated an increase in overall β(1,3)galactosyltransferase and α(2,3)sialyltransferase activity and a decrease in α(1,3)fucosyltransferases when these cells differentiate toward cardiomyocytes. Enzymatic and lectin binding data suggest a transition from Lewis(x)-type structures in mESCs to sialylated Galβ1,3GalNAc-type glycans on differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated toward cardiomyocytes. Overall, simple, rapid, quantitative, and scalable glycoT activity analysis methods are presented. These use a range of natural and synthetic acceptors for the analysis of complex biological specimens that have limited availability. Copyright © 2012 Elsevier Inc. All rights reserved.

Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

International Nuclear Information System (INIS)

Perçin, Işık; Karakoç, Veyis; Akgöl, Sinan; Aksöz, Erol; Denizli, Adil

2012-01-01

The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m 2 /g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 μg/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 °C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: ► Magnetic nanoparticles have several advantages over conventional adsorbents. ► MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. ► pDNA adsorption amount was 154 mg/g. ► Fifty-fold capacity increase was obtained when compared to conventional matrices.

Validated hemoglobin-depletion approach for red blood cell lysate proteome analysis by means of 2D PAGE and Orbitrap MS.

Science.gov (United States)

Walpurgis, Katja; Kohler, Maxie; Thomas, Andreas; Wenzel, Folker; Geyer, Hans; Schänzer, Wilhelm; Thevis, Mario

2012-08-01

The analysis of the cytosolic red blood cell (RBC) proteome is negatively affected by the high intracellular amount of hemoglobin complicating the detection of low-abundant cytosolic proteins. In this study, an alternative approach for the preparation of hemoglobin-depleted RBC lysates is presented, which was established in combination with downstream 2D PAGE analysis and Orbitrap MS. Hemoglobin removal was accomplished by using HemoVoid(TM) depletion reagent, which enabled a very efficient enrichment of low-abundant proteins by simultaneously reducing the hemoglobin concentration of the sample. After defining selected sample preparation protocol characteristics including specificity/selectivity, precision and linearity, a 2D reference map (pH 4-7) of the cytosolic RBC proteome was generated and a total of 189 different proteins were identified. Thus, the presented approach proved to be highly suitable to prepare reproducible high-resolution 2D protein maps of the RBC cytosol and provides a helpful tool for future studies investigating disease- or storage-induced changes of the cytosolic RBC proteome. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Endogenous Locus Reporter Assays.

Science.gov (United States)

Liu, Yaping; Hermes, Jeffrey; Li, Jing; Tudor, Matthew

2018-01-01

Reporter gene assays are widely used in high-throughput screening (HTS) to identify compounds that modulate gene expression. Traditionally a reporter gene assay is built by cloning an endogenous promoter sequence or synthetic response elements in the regulatory region of a reporter gene to monitor transcriptional activity of a specific biological process (exogenous reporter assay). In contrast, an endogenous locus reporter has a reporter gene inserted in the endogenous gene locus that allows the reporter gene to be expressed under the control of the same regulatory elements as the endogenous gene, thus more accurately reflecting the changes seen in the regulation of the actual gene. In this chapter, we introduce some of the considerations behind building a reporter gene assay for high-throughput compound screening and describe the methods we have utilized to establish 1536-well format endogenous locus reporter and exogenous reporter assays for the screening of compounds that modulate Myc pathway activity.

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

Science.gov (United States)

Avanzini, Maria Antonietta; Bernardo, Maria Ester; Cometa, Angela Maria; Perotti, Cesare; Zaffaroni, Nadia; Novara, Francesca; Visai, Livia; Moretta, Antonia; Del Fante, Claudia; Villa, Raffaella; Ball, Lynne M.; Fibbe, Willem E.; Maccario, Rita; Locatelli, Franco

2009-01-01

Background Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. Design and Methods We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. Results The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16ink4a protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-γ secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E2-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA

Evaluation of human platelet lysate versus fetal bovine serum for culture of mesenchymal stromal cells.

Science.gov (United States)

Hemeda, Hatim; Giebel, Bernd; Wagner, Wolfgang

2014-02-01

Culture media for therapeutic cell preparations-such as mesenchymal stromal cells (MSCs)-usually comprise serum additives. Traditionally, fetal bovine serum is supplemented in basic research and in most clinical trials. Within the past years, many laboratories adapted their culture conditions to human platelet lysate (hPL), which further stimulates proliferation and expansion of MSCs. Particularly with regard to clinical application, human alternatives for fetal bovine serum are clearly to be preferred. hPL is generated from human platelet units by disruption of the platelet membrane, which is commonly performed by repeated freeze and thaw cycles. Such culture supplements are notoriously ill-defined, and many parameters contribute to batch-to-batch variation in hPL such as different amounts of plasma, a broad range of growth factors and donor-specific effects. The plasma components of hPL necessitate addition of anticoagulants such as heparins to prevent gelatinization of hPL medium, and their concentration must be standardized. Labels for description of hPL-such as "xenogen-free," "animal-free" and "serum free"-are not used consistently in the literature and may be misleading if not critically assessed. Further analysis of the precise composition of relevant growth factors, attachment factors, microRNAs and exosomes will pave the way for optimized and defined culture conditions. The use of hPL has several advantages and disadvantages: they must be taken into account because the choice of cell culture additive has major impact on cell preparations. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Umbilical Cord Blood Platelet Lysate as Serum Substitute in Expansion of Human Mesenchymal Stem Cells.

Science.gov (United States)

Shirzad, Negin; Bordbar, Sima; Goodarzi, Alireza; Mohammad, Monire; Khosravani, Pardis; Sayahpour, Froughazam; Baghaban Eslaminejad, Mohamadreza; Ebrahimi, Marzieh

2017-10-01

The diverse clinical applications for human mesenchymal stem cells (hMSCs) in cellular therapy and regenerative medicine warrant increased focus on developing adequate culture supplements devoid of animal-derived products. In the present study, we have investigated the feasibility of umbilical cord blood-platelet lysate (UCB-PL) as a standard substitute for fetal bovine serum (FBS) and human peripheral blood-PL (PB-PL). In this experimental study, platelet concentrates (PC) from UCB and human PB donors were frozen, melted, and sterilized to obtain PL. Quality control included platelet cell counts, sterility testing (viral and microbial), total protein concentrations, growth factor levels, and PL stability. The effects of UCB-PL and PB-PL on hMSCs proliferation and differentiation into osteocytes, chondrocytes, and adipocytes were studied and the results compared with FBS. UCB-PL contained high levels of protein content, platelet-derived growth factor- AB (PDGF-AB), and transforming growth factor (TGF) compared to PB-PL. All growth factors were stable for at least nine months post-storage at -70˚C. hMSCs proliferation enhanced following treatment with UCB-PL. With all three supplements, hMSCs could differentiate into all three lineages. PB-PL and UCB-PL both were potent in hMSCs proliferation. However, PB promoted osteoblastic differentiation and UCB-PL induced chondrogenic differentiation. Because of availability, ease of use and feasible standardization of UCB-PL, we have suggested that UCB-PL be used as an alternative to FBS and PB-PL for the cultivation and expansion of hMSCs in cellular therapy. Copyright© by Royan Institute. All rights reserved.

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

Science.gov (United States)

Tasev, Dimitar; van Wijhe, Michiel H; Weijers, Ester M; van Hinsbergh, Victor W M; Koolwijk, Pieter

2015-01-01

Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs) as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs. The findings presented in this study indicate that human platelet lysate (PL) as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS). Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL) retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator) or uPAR (uPA receptor) by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential. The presented isolation method and subsequent cell expansion in platelet lysate

Long-Term Expansion in Platelet Lysate Increases Growth of Peripheral Blood-Derived Endothelial-Colony Forming Cells and Their Growth Factor-Induced Sprouting Capacity.

Directory of Open Access Journals (Sweden)

Dimitar Tasev

Full Text Available Efficient implementation of peripheral blood-derived endothelial-colony cells (PB-ECFCs as a therapeutical tool requires isolation and generation of a sufficient number of cells in ex vivo conditions devoid of animal-derived products. At present, little is known how the isolation and expansion procedure in xenogeneic-free conditions affects the therapeutical capacity of PB-ECFCs.The findings presented in this study indicate that human platelet lysate (PL as a serum substitute yields twice more colonies per mL blood compared to the conventional isolation with fetal bovine serum (FBS. Isolated ECFCs displayed a higher proliferative ability in PL supplemented medium than cells in FBS medium during 30 days expansion. The cells at 18 cumulative population doubling levels (CPDL retained their proliferative capacity, showed higher sprouting ability in fibrin matrices upon stimulation with FGF-2 and VEGF-A than the cells at 6 CPDL, and displayed low β-galactosidase activity. The increased sprouting of PB-ECFCs at 18 CPDL was accompanied by an intrinsic activation of the uPA/uPAR fibrinolytic system. Induced deficiency of uPA (urokinase-type plasminogen activator or uPAR (uPA receptor by siRNA technology completely abolished the angiogenic ability of PB-ECFCs in fibrin matrices. During the serial expansion, the gene induction of the markers associated with inflammatory activation such as VCAM-1 and ICAM-1 did not occur or only to limited extent. While further propagation up to 31 CPDL proceeded at a comparable rate, a marked upregulation of inflammatory markers occurred in all donors accompanied by a further increase of uPA/uPAR gene induction. The observed induction of inflammatory genes at later stages of long-term propagation of PB-ECFCs underpins the necessity to determine the right time-point for harvesting of sufficient number of cells with preserved therapeutical potential.The presented isolation method and subsequent cell expansion in platelet

Direct 125I-radioligand assays for serum progesterone compared with assays involving extraction of serum

International Nuclear Information System (INIS)

Ratcliffe, W.A.; Corrie, J.E.T.; Dalziel, A.H.; Macpherson, J.S.

1982-01-01

Two direct radioimmunoassays for progesterone in 50 μL of unextracted serum or plasma with assays involving extraction of serum were compared. The direct assays include the use of either danazol at pH 7.4 or 8-anilino-1-naphthalenesulfonic acid at pH 4.0 to displace progesterone from serum binding-proteins. Progesterone is then assayed by using an antiserum to a progesterone 11α-hemisuccinyl conjugate and the radioligand 125 I-labeled progesterone 11α-glucuronyl tyramine, with separation by double-antibody techniques. Direct assays with either displacing agent gave good analytical recovery of progesterone added to human serum, and progesterone values for patients' specimens correlated well (r > 0.96) with results of assays involving extraction of serum. Precision was similar with each displacing agent over the working range 2.5-100 nmol/L and superior to that of extraction assays. We conclude that these direct assays of progesterone are analytically valid and more robust, precise, and technically convenient than many conventional methods involving extraction of serum

Absolute nuclear material assay

Science.gov (United States)

Prasad, Manoj K [Pleasanton, CA; Snyderman, Neal J [Berkeley, CA; Rowland, Mark S [Alamo, CA

2010-07-13

A method of absolute nuclear material assay of an unknown source comprising counting neutrons from the unknown source and providing an absolute nuclear material assay utilizing a model to optimally compare to the measured count distributions. In one embodiment, the step of providing an absolute nuclear material assay comprises utilizing a random sampling of analytically computed fission chain distributions to generate a continuous time-evolving sequence of event-counts by spreading the fission chain distribution in time.

Assay method and compositions

International Nuclear Information System (INIS)

1977-01-01

Methods are described for measuring catecholamine levels in human and animal body fluids and tissues using the catechol-O-methyl-transferase (COMT) radioassay. The assay involves incubating the biological sample with COMT and the tritiated methyl donor, S-adenosyl-L-methionine( 3 H)-methyl. The O-methylated ( 3 H) epinephrine and/or norepinephrine are extracted and oxidised to vanillin- 3 H which in turn is extracted and its radioactivity counted. When analysing dopamine levels the assay is extended by vanillin- 3 H and raising the pH of the aqueous periodate phase from which O-methylated ( 3 H) dopamine is extracted and counted. The assay may be modified depending on whether measurements of undifferentiated total endogenous catecholamine levels or differential analyses of the catecholamine levels are being performed. The sensitivity of the assay can be as low as 5 picograms for norepinephrine and epinephrine and 12 picograms for dopamine. The assemblance of the essential components of the assay into a kit for use in laboratories is also described. (U.K.)

Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation

Directory of Open Access Journals (Sweden)

Pasquale Marrazzo

2016-01-01

Full Text Available Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs. The perspective role of dental pulp stem cells (DPSCs in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1% was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H2O2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

Highly Efficient In Vitro Reparative Behaviour of Dental Pulp Stem Cells Cultured with Standardised Platelet Lysate Supplementation.

Science.gov (United States)

Marrazzo, Pasquale; Paduano, Francesco; Palmieri, Francesca; Marrelli, Massimo; Tatullo, Marco

2016-01-01

Dental pulp is an accessible source of multipotent mesenchymal stromal cells (MSCs). The perspective role of dental pulp stem cells (DPSCs) in regenerative medicine demands an in vitro expansion and in vivo delivery which must deal with the safety issues about animal serum, usually required in cell culture practice. Human platelet lysate (PL) contains autologous growth factors and has been considered as valuable alternative to fetal bovine serum (FBS) in cell cultures. The optimum concentration to be added of such supplement is highly dependent on its preparation whose variability limits comparability of results. By in vitro experiments, we aimed to evaluate a standardised formulation of pooled PL. A low selected concentration of PL (1%) was able to support the growth and maintain the viability of the DPSCs. The use of PL in cell cultures did not impair cell surface signature typically expressed by MSCs and even upregulated the transcription of Sox2. Interestingly, DPSCs cultured in presence of PL exhibited a higher healing rate after injury and are less susceptible to toxicity mediated by exogenous H 2 O 2 than those cultured with FBS. Moreover, PL addition was shown as a suitable option for protocols promoting osteogenic and chondrogenic differentiation of DPSCs. Taken together, our results indicated that PL is a valid substitute of FBS to culture and differentiate DPSCs for clinical-grade use.

Emory University: High-Throughput Protein-Protein Interaction Dataset for Lung Cancer-Associated Genes | Office of Cancer Genomics

Science.gov (United States)

To discover novel PPI signaling hubs for lung cancer, CTD2 Center at Emory utilized large-scale genomics datasets and literature to compile a set of lung cancer-associated genes. A library of expression vectors were generated for these genes and utilized for detecting pairwise PPIs with cell lysate-based TR-FRET assays in high-throughput screening format. Read the abstract.

Poly(hydroxyethyl methacrylate) based magnetic nanoparticles for plasmid DNA purification from Escherichia coli lysate

Energy Technology Data Exchange (ETDEWEB)

Percin, Is Latin-Small-Letter-Dotless-I k [Department of Biology, Hacettepe University, Ankara (Turkey); Karakoc, Veyis [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey); Akgoel, Sinan [Department of Biochemistry, Ege University, Izmir (Turkey); Aksoez, Erol [Department of Biology, Hacettepe University, Ankara (Turkey); Denizli, Adil, E-mail: denizli@hacettepe.edu.tr [Department of Chemistry, Biochemistry Division, Hacettepe University, Ankara (Turkey)

2012-07-01

The aim of this study is to prepare poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine) [PHEMAH] magnetic nanoparticles for plasmid DNA (pDNA) purification from Escherichia coli (E. coli) cell lysate. Magnetic nanoparticles were produced by surfactant free emulsion polymerization. mPHEMAH nanoparticles were characterized by elemental analysis, Fourier transform infrared spectroscopy (FTIR), atomic force microscopy (AFM), vibrating sample magnetometer (VSM), electron spin resonance (ESR), thermogravimetric analyses (TGA) and transmission electron microscopy (TEM). Surface area, average particle size and size distribution were also performed. Specific surface area of the mPHEMAH nanoparticles was found to be 1180 m{sup 2}/g. Elemental analysis of MAH for nitrogen was estimated as 0.18 mmol/g polymer. The amount of pDNA adsorbed onto the mPHEMAH nanoparticles first increased and then reached a saturation value at around 1.0 mg/mL of pDNA concentration. Compared with the mPHEMA nanoparticles (50 {mu}g/g polymer), the pDNA adsorption capacity of the mPHEMAH nanoparticles (154 mg/g polymer) was improved significantly due to the MAH incorporation into the polymeric matrix. The maximum pDNA adsorption was achieved at 25 Degree-Sign C. The overall recovery of pDNA was calculated as 92%. The mPHEMAH nanoparticles could be used six times without decreasing the pDNA adsorption capacity significantly. The results indicate that the PHEMAH nanoparticles promise high selectivity for pDNA. - Highlights: Black-Right-Pointing-Pointer Magnetic nanoparticles have several advantages over conventional adsorbents. Black-Right-Pointing-Pointer MAH acted as the pseudospecific ligand, ligand immobilization step was eliminated. Black-Right-Pointing-Pointer pDNA adsorption amount was 154 mg/g. Black-Right-Pointing-Pointer Fifty-fold capacity increase was obtained when compared to conventional matrices.

Mineralization by mesenchymal stromal cells is variously modulated depending on commercial platelet lysate preparations.

Science.gov (United States)

Boraldi, Federica; Burns, Jorge S; Bartolomeo, Angelica; Dominici, Massimo; Quaglino, Daniela

2018-03-01

Numerous cellular models have been developed to investigate calcification for regenerative medicine applications and for the identification of therapeutic targets in various complications associated with age-related diseases. However, results have often been contradictory due to specific culture conditions, cell type ontogeny and aging status. Human platelet lysate (hPL) has been recently investigated as valuable alternative to fetal bovine serum (FBS) in cell culture and bone regeneration. A parallel comparison of how all these multiple factors may converge to influence mineralization has yet to be reported. To compare mineralization of human mesenchymal cell types known to differ in extracellular matrix calcification potency, bone marrow-derived mesenchymal stromal cells and dermal fibroblasts from neonatal and adult donors, at both low and high passages, were investigated in an ex vivo experimental model by supplementing the osteogenic induction medium with FBS or with hPL. Four commercial hPL preparations were profiled by liquid chromatography/electrospray ionization quadrupole time-of-flight spectrometry, and mineralization was visualized by von Kossa staining and quantified by morphometric evaluations after 9, 14 and 21 days of culture. Data demonstrate that (i) commercial hPL preparations differ according to mass spectra profiles, (ii) hPL variously influences mineral deposition depending on cell line and possibly on platelet product preparation methods, (iii) donor age modifies mineral deposition in the presence of the same hPL and (iv) reduced in vitro proliferative capacity affects osteogenic induction and response to hPL. Despite the standardized procedures applied to obtain commercial hPL, this study highlights the divergent effects of different preparations and emphasizes the importance of cellular ontology, donor age and cell proliferative capacity to optimize the osteogenic induction capabilities of mesenchymal stromal cells and design more effective

Expert system for transuranic waste assay

Energy Technology Data Exchange (ETDEWEB)

Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

1989-01-01

Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs.

Expert system for transuranic waste assay

International Nuclear Information System (INIS)

Zoolalian, M.L.; Gibbs, A.; Kuhns, J.D.

1989-01-01

Transuranic wastes are generated at the Savannah River Site (SRS) as a result of routine production of nuclear materials. These wastes contain Pu-238 and Pu-239 and are placed into lined 55-gallon waste drums. The drums are placed on monitored storage pads pending shipment to the Waste Isolation Pilot Plant in New Mexico. A passive-active neutron (PAN) assay system is used to determine the mass of the radioactive material within the waste drums. Assay results are used to classify the wastes as either low-level or transuranic (TRU). During assays, the PAN assay system communicates with an IBM-AT computer. A Fortran computer program, called NEUT, controls and performs all data analyses. Unassisted, the NEUT program cannot adequately interpret assay results. To eliminate this limitation, an expert system shell was used to write a new algorithm, called the Transuranic Expert System (TRUX), to drive the NEUT program and add decision making capabilities for analysis of the assay results. The TRUX knowledge base was formulated by consulting with human experts in the field of neutron assay, by direct experimentation on the PAN assay system, and by observing operations on a daily basis. TRUX, with its improved ability to interpret assay results, has eliminated the need for close supervision by a human expert, allowing skilled technicians to operate the PAN assay system. 4 refs., 1 fig., 4 tabs

First results with a radioreceptor-assay (TRAK-Assay) for TSH-receptor-autoantibodies

International Nuclear Information System (INIS)

Becker, W.; Reiners, C.; Boerner, W.

1983-01-01

A new radioreceptor-assay (TRAK-assay) for autoantibodies against TSH-receptors was tested in 48 untreated thyrotoxic patients (26 regional autonomies, 22 toxic diffuse goiters). None of the 26 patients with regional autonomy showed positive autoantibody-titers. 4 patients with toxic diffuse goiter and thyrotoxic exophthalmos were TRAK-positive. Positive titers of microsomal and thyreoglobulin autoantibodies could be seen in 8 of 9 patients with positive TRAK-titers. In accordance with the conventional methods for detecting thyroid-stimulating immunoglobulins the new TRAK-assay seems to be suited for differentiating between immunogenic toxic diffuse goiter (Graves' disease) and goiter with disseminated autonomy as well as for prediction of relapse. (orig.) [de

Comprehensive analysis of sperm DNA fragmentation by five different assays: TUNEL assay, SCSA, SCD test and alkaline and neutral Comet assay.

Science.gov (United States)

Ribas-Maynou, J; García-Peiró, A; Fernández-Encinas, A; Abad, C; Amengual, M J; Prada, E; Navarro, J; Benet, J

2013-09-01

Sperm DNA fragmentation (SDF) is becoming an important test to assess male infertility. Several different tests are available, but no consensus has yet been reached as to which tests are most predictive of infertility. Few publications have reported a comprehensive analysis comparing these methods within the same population. The objective of this study was to analyze the differences between the five most common methodologies, to study their correlations and to establish their cut-off values, sensitivity and specificity in predicting male infertility. We found differences in SDF between fertile donors and infertile patients in TUNEL, SCSA, SCD and alkaline Comet assays, but none with the neutral Comet assay. The alkaline COMET assay was the best in predicting male infertility followed by TUNEL, SCD and SCSA, whereas the neutral COMET assay had no predictive power. For our patient population, threshold values for infertility were 20.05% for TUNEL assay, 18.90% for SCSA, 22.75% for the SCD test, 45.37% for alkaline Comet and 34.37% for neutral Comet. This work establishes in a comprehensive study that the all techniques except neutral Comet are useful to distinguish fertile and infertile men. © 2013 American Society of Andrology and European Academy of Andrology.

The effects of platelet lysate patches on the activity of tendon-derived cells.

Science.gov (United States)

Costa-Almeida, Raquel; Franco, Albina R; Pesqueira, Tamagno; Oliveira, Mariana B; Babo, Pedro S; Leonor, Isabel B; Mano, João F; Reis, Rui L; Gomes, Manuela E

2018-03-01

Platelet-derived biomaterials are widely explored as cost-effective sources of therapeutic factors, holding a strong potential for endogenous regenerative medicine. Particularly for tendon repair, treatment approaches that shift the injury environment are explored to accelerate tendon regeneration. Herein, genipin-crosslinked platelet lysate (PL) patches are proposed for the delivery of human-derived therapeutic factors in patch augmentation strategies aiming at tendon repair. Developed PL patches exhibited a controlled release profile of PL proteins, including bFGF and PDGF-BB. Additionally, PL patches exhibited an antibacterial effect by preventing the adhesion, proliferation and biofilm formation by S. aureus, a common pathogen in orthopaedic surgical site infections. Furthermore, these patches supported the activity of human tendon-derived cells (hTDCs). Cells were able to proliferate over time and an up-regulation of tenogenic genes (SCX, COL1A1 and TNC) was observed, suggesting that PL patches may modify the behavior of hTDCs. Accordingly, hTDCs deposited tendon-related extracellular matrix proteins, namely collagen type I and tenascin C. In summary, PL patches can act as a reservoir of biomolecules derived from PL and support the activity of native tendon cells, being proposed as bioinstructive patches for tendon regeneration. Platelet-derived biomaterials hold great interest for the delivery of therapeutic factors for applications in endogenous regenerative medicine. In the particular case of tendon repair, patch augmentation strategies aiming at shifting the injury environment are explored to improve tendon regeneration. In this study, PL patches were developed with remarkable features, including the controlled release of growth factors and antibacterial efficacy. Remarkably, PL patches supported the activity of native tendon cells by up-regulating tenogenic genes and enabling the deposition of ECM proteins. This patch holds great potential towards

Development and validation of a HPLC method for the assay of dapivirine in cell-based and tissue permeability experiments.

Science.gov (United States)

das Neves, José; Sarmento, Bruno; Amiji, Mansoor; Bahia, Maria Fernanda

2012-12-12

Dapivirine, a non-nucleoside reverse transcriptase inhibitor, is being currently used for the development of potential anti-HIV microbicide formulations and delivery systems. A new high-performance liquid chromatography (HPLC) method with UV detection was developed for the assay of this drug in different biological matrices, namely cell lysates, receptor media from permeability experiments and homogenates of mucosal tissues. The method used a reversed-phase C18 column with a mobile phase composed of trifluoroacetic acid solution (0.1%, v/v) and acetonitrile in a gradient mode. Injection volume was 50μL and the flow rate 1mL/min. The total run time was 12min and UV detection was performed at 290nm for dapivirine and the internal standard (IS) diphenylamine. A Box-Behnken experimental design was used to study different experimental variables of the method, namely the ratio of the mobile phase components and the gradient time, and their influence in responses such as the retention factor, tailing factor, and theoretical plates for dapivirine and the IS, as well as the peak resolution between both compounds. The optimized method was further validated and its usefulness assessed for in vitro and ex vivo experiments using dapivirine or dapivirine-loaded nanoparticles. The method showed to be selective, linear, accurate and precise in the range of 0.02-1.5μg/mL. Other chromatographic parameters, namely carry-over, lower limit of quantification (0.02μg/mL), limit of detection (0.006μg/mL), recovery (equal or higher than 90.7%), and sample stability at different storage conditions, were also determined and found adequate for the intended purposes. The method was successfully used for cell uptake assays and permeability studies across cell monolayers and pig genital mucosal tissues. Overall, the proposed method provides a simple, versatile and reliable way for studying the behavior of dapivirine in different biological matrices and assessing its potential as an anti

[Effect of thyroid hormones on the histotopography of lectin receptors in the rat salivary gland].

Science.gov (United States)

Lutsik, A D; Iashchenko, A M; Detiuk, E S

1987-04-01

Using lectin-peroxidase technique, the influence of hypo- and hyperthyroidism on histotopography of glycoconjugates has been investigated in rat submandibular gland. The following lectins were used: peanut agglutinin (PNA), wheat germ agglutinin (WGA), Laburnum anagyroides lectin (LAL) and concanavalin A (con A). It has been demonstrated that hyperthyroidism is accompanied by the loss of con A, WGA and LAL receptor sites. Hypothyrodism enhanced con A binding to granular duct cells with a parallel reduction in WGA and LAL binding to these or other duct cells. Hypothyroidism as well as hyperthyroidism markedly enhanced PNA binding to duct epitheliocytes with redistribution of these lectin binding sites from the luminal surface of salivary ducts into the cytoplasm of duct cells. Possible interpretations of the observed phenomena are discussed.

Radioimmunoassay of measles virus hemagglutinin protein G

International Nuclear Information System (INIS)

Lund, G.A.; Salmi, A.A.

1982-01-01

Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 μg of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose-response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production. Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1-4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells. (Auth.)

Radioimmunoassay of measles virus hemagglutinin protein G

Energy Technology Data Exchange (ETDEWEB)

Lund, G A; Salmi, A A [Turku Univ. (Finland)

1982-08-01

Guinea pig and rabbit antisera from animals immunized with purified measles virus hemagglutinin (G) protein were used to establish a solid-phase four-layer radioimmunoassay for quantitative measurement of the G protein. The sensitivity of the assay was 2 ng of purified G protein, and 200 ..mu..g of protein from uninfected Vero cells neither decreased the sensitivity nor reacted non-specifically in the assay. Radioimmunoassay standard dose-response curves were established and unknown values interpolated from these using the logit program of a desktop computer. Using this procedure, a measles virus growth curve in infected Vero cells was determined by measurement of G protein production. Under these same conditions, hemagglutination was not sensitive enough to detect early hemagglutinin production. Viral antigens in canine distemper virus, Newcastle disease virus, parainfluenza viruses 1-4, simian virus 5, and respiratory syncytial virus-infected cell lysates did not cross-react in the radioimmunoassay. A small degree of cross-reactivity was detected with mumps viral antigens, both with Vero cell-derived (wild-type strain) and egg-derived (Enders strain) purified virus preparations and with a cell lysate antigen prepared from wild-type mumps virus-infected Vero cells.

Safeguards and Non-destructive Assay

International Nuclear Information System (INIS)

Carchon, R.; Bruggeman, M.

2001-01-01

SCK-CEN's programme on safeguards and non-destructive assay includes: (1) various activities to assure nuclear materials accountancy; (2) contributes to the implementation of Integrated Safeguards measures in Belgium and to assist the IAEA through the Belgian Support Programme; (3) renders services to internal and external customers in the field of safeguards; (4) improves passive neutron coincidence counting techniques for waste assay and safeguards verification measurements by R and D on correlation algorithms implemented via software or dedicated hardware; (5) improves gamma assay techniques for waste assay by implementing advanced scanning techniques and different correlation algorithms; and (6) develops numerical calibration techniques. Major achievements in these areas in 2000 are reported

Platelet lysate supports the in vitro expansion of human periodontal ligament stem cells for cytotherapeutic use.

Science.gov (United States)

Wu, Rui-Xin; Yu, Yang; Yin, Yuan; Zhang, Xi-Yu; Gao, Li-Na; Chen, Fa-Ming

2017-08-01

Human platelet lysate (PL) produced under optimal conditions of standardization and safety has been increasingly suggested as the future 'gold standard' supplement to replace fetal bovine serum (FBS) for the ex vivo propagation of mesenchymal stem cells for translational medicine and cell therapy applications. However, the multifaceted effects of PL on tissue-specific stem cells remain largely unexplored. In the present study, we investigated the stem cell behaviours of human periodontal ligament stem cells (PDLSCs) in media with or without PL. Our data indicate that human PL, either as an adjuvant for culture media or as a substitute for FBS, supports the proliferation and expansion of human PDLSCs derived from either 'young' or 'old' donors to the same extent as FBS, without interfering with their immunomodulatory capacities. Although PL appears to inhibit the in vitro differentiation of 'young' or 'old' PDLSCs, their decreased osteogenic potential may be restored to similar or higher levels compared with FBS-expanded cells. PL- and FBS-expanded PDLSCs exhibited a similar potential to form mineralized nodules and expressed similar levels of osteogenic genes. Our data indicate that large clinically relevant quantities of PDLSCs may be yielded by the use of human PL; however, further analysis of its precise composition and function will pave the way for determining optimized, defined culture conditions. In addition to the potential increase in patient safety, our findings highlight the need for further research to develop the potential of PL-expanded PDLSCs for clinical use. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluation of Peripheral Blood and Cord Blood Platelet Lysates in Isolation and Expansion of Multipotent Mesenchymal Stromal Cells

Directory of Open Access Journals (Sweden)

Ioanna Christou

2018-02-01

Full Text Available Background: Multipotent Mesenchymal Stromal Cells (MSCs are used in tissue engineering and regenerative medicine. The in vitro isolation and expansion of MSCs involve the use of foetal bovine serum (FBS. However, many concerns have been raised regarding the safety of this product. In this study, alternative additives derived either from peripheral or cord blood were tested as an FBS replacement. Methods: Platelet lysates (PL from peripheral and cord blood were used for the expansion of MSCs. The levels of growth factors in peripheral blood (PB and cord blood (CB PLs were determined using the Multiple Reaction Monitoring (MRM. Finally, the cell doubling time (CDT, tri-lineage differentiation and phenotypic characterization of the MSCs expanded with FBS and PLs were determined. Results: MSCs treated with culture media containing FBS and PB-PL, were successfully isolated and expanded, whereas MSCs treated with CB-PL could not be maintained in culture. Furthermore, the MRM analysis yielded differences in growth factor levels between PB-PL and CB-PL. In addition, the MSCs were successfully expanded with FBS and PB-PL and exhibited tri-lineage differentiation and stable phenotypic characteristics. Conclusion: PB-PL could be used as an alternative additive for the production of MSCs culture medium applied to xenogeneic-free expansion and maintenance of MSCs in large scale clinical studies.

Radioactive wastes assay technique and equipment

International Nuclear Information System (INIS)

Lee, K. M.; Hong, D. S; Kim, T. K.; Bae, S. M.; Shon, J. S.; Hong, K. P.

2004-12-01

The waste inventory records such as the activities and radio- nuclides contained in the waste packages are to be submitted with the radioactive wastes packages for the final disposal. The nearly around 10,000 drums of waste stocked in KAERI now should be assayed for the preparation of the waste inventory records too. For the successive execution of the waste assay, the investigation into the present waste assay techniques and equipment are to be taken first. Also the installation of the waste assay equipment through the comprehensive design, manufacturing and procurement should be proceeded timely. As the characteristics of the KAERI-stocked wastes are very different from that of the nuclear power plant and those have no regular waste streams, the application of the in-direct waste assay method using the scaling factors are not effective for the KAERI-generated wastes. Considering for the versal conveniency including the accuracy over the wide range of waste forms and the combination of assay time and sensitivity, the TGS(Tomographic Gamma Scanner) is appropriate as for the KAERI -generated radioactive waste assay equipment

Radioactive waste package assay facility. Volume 1. Application of assay technology

International Nuclear Information System (INIS)

Findlay, D.J.S.; Green, T.H.; Molesworth, T.V.; Staniforth, D.; Strachan, N.R.; Rogers, J.D.; Wise, M.O.; Forrest, K.R.

1992-01-01

This report, in three volumes, covers the work carried out by Taylor Woodrow Construction Ltd., and two major sub-contractors: Harwell Laboratory (AEA Technology) and Siemens Plessey Controls Ltd., on the development of a radioactive waste package assay facility, for cemented 500 litre intermediate level waste drums. In volume 1, the reasons for assay are considered together with the various techniques that can be used, and the information that can be obtained. The practical problems associated with the use of the various techniques in an integrated assay facility are identified, and the key parameters defined. Engineering and operational features are examined and provisional designs proposed for facilities at three throughput levels: 15,000, 750 and 30 drums per year respectively. The capital and operating costs for such facilities have been estimated. A number of recommendations are made for further work. 16 refs., 14 figs., 13 tabs

A designated centre for people with disabilities operated by Brothers of Charity Southern Services, Cork

LENUS (Irish Health Repository)

Kamel, Dalia

2012-11-01

Being a significant health problem that affects patients in various age groups, breast cancer has been extensively studied to date. Recently, molecular breast cancer classification has advanced significantly with the availability of genomic profiling technologies. Proteomic technologies have also advanced from traditional protein assays including enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry to more comprehensive approaches including mass spectrometry and reverse phase protein lysate arrays (RPPA). The purpose of this manuscript is to review the current protein markers that influence breast cancer prediction and prognosis and to focus on novel advances in proteomic classification of breast cancer.

A designated centre for people with disabilities operated by Camphill Communities of Ireland - Wexford

LENUS (Irish Health Repository)

Kamel, Dalia

2012-11-01

Being a significant health problem that affects patients in various age groups, breast cancer has been extensively studied to date. Recently, molecular breast cancer classification has advanced significantly with the availability of genomic profiling technologies. Proteomic technologies have also advanced from traditional protein assays including enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry to more comprehensive approaches including mass spectrometry and reverse phase protein lysate arrays (RPPA). The purpose of this manuscript is to review the current protein markers that influence breast cancer prediction and prognosis and to focus on novel advances in proteomic classification of breast cancer.

Proteomic classification of breast cancer.

LENUS (Irish Health Repository)

Kamel, Dalia

2012-11-01

Being a significant health problem that affects patients in various age groups, breast cancer has been extensively studied to date. Recently, molecular breast cancer classification has advanced significantly with the availability of genomic profiling technologies. Proteomic technologies have also advanced from traditional protein assays including enzyme-linked immunosorbent assay, immunoblotting and immunohistochemistry to more comprehensive approaches including mass spectrometry and reverse phase protein lysate arrays (RPPA). The purpose of this manuscript is to review the current protein markers that influence breast cancer prediction and prognosis and to focus on novel advances in proteomic classification of breast cancer.

Automation of the dicentric chromosome assay and related assays

International Nuclear Information System (INIS)

Balajee, Adayabalam S.; Dainiak, Nicholas

2016-01-01

Dicentric Chromosome Assay (DCA) is considered to be the 'gold standard' for personalized dose assessment in humans after accidental or incidental radiation exposure. Although this technique is superior to other cytogenetic assays in terms of specificity and sensitivity, its potential application to radiation mass casualty scenarios is highly restricted because DCA is time consuming and labor intensive when performed manually. Therefore, it is imperative to develop high throughput automation techniques to make DCA suitable for radiological triage scenarios. At the Cytogenetic Biodosimetry Laboratory in Oak Ridge, efforts are underway to develop high throughput automation of DCA. Current status on development of various automated cytogenetic techniques in meeting the biodosimetry needs of radiological/nuclear incident(s) will be discussed

Radiometric assays for glycerol, glucose, and glycogen

International Nuclear Information System (INIS)

Bradley, D.C.; Kaslow, H.R.

1989-01-01

We have developed radiometric assays for small quantities of glycerol, glucose and glycogen, based on a technique described by Thorner and Paulus for the measurement of glycerokinase activity. In the glycerol assay, glycerol is phosphorylated with [32P]ATP and glycerokinase, residual [32P]ATP is hydrolyzed by heating in acid, and free [32P]phosphate is removed by precipitation with ammonium molybdate and triethylamine. Standard dose-response curves were linear from 50 to 3000 pmol glycerol with less than 3% SD in triplicate measurements. Of the substances tested for interference, only dihydroxyacetone gave a slight false positive signal at high concentration. When used to measure glycerol concentrations in serum and in media from incubated adipose tissue, the radiometric glycerol assay correlated well with a commonly used spectrophotometric assay. The radiometric glucose assay is similar to the glycerol assay, except that glucokinase is used instead of glycerokinase. Dose response was linear from 5 to 3000 pmol glucose with less than 3% SD in triplicate measurements. Glucosamine and N-acetylglucosamine gave false positive signals when equimolar to glucose. When glucose concentrations in serum were measured, the radiometric glucose assay agreed well with hexokinase/glucose-6-phosphate dehydrogenase (H/GDH)-based and glucose oxidase/H2O2-based glucose assays. The radiometric method for glycogen measurement incorporates previously described isolation and digestion techniques, followed by the radiometric assay of free glucose. When used to measure glycogen in mouse epididymal fat pads, the radiometric glycogen assay correlated well with the H/GDH-based glycogen assay. All three radiometric assays offer several practical advantages over spectral assays

Harmonization of radiobiological assays: why and how?

International Nuclear Information System (INIS)

Prasanna, Pataje G.

2014-01-01

The International Atomic Energy Agency has made available a technical manual for cytogenetic biodosimetry assays (dicentric chromosome aberration (DCA) and cytokinesis-block micronucleus (CBMN) assays) used for radiation dose assessment in radiation accidents. The International Standardization Organization, which develops standards and guidelines, also provides an avenue for laboratory accreditation, has developed guidelines and recommendations for performing cytogenetic biodosimetry assays. Harmonization of DCA and CBMN assays, has improved their accuracy. Double-blinded inter-laboratory comparison studies involving several networks have further validated DCA and CBMN assays and improved the confidence in their potential use for radiation dose assessment in mass casualties. This kind of international harmonization is lacking for pre-clinical radiobiology assays. The widely used pre-clinical assays that are relatively important to set stage for clinical trials include clonogenic assays, flow-cytometry assays, apoptotic assays, and tumor regression and growth delay assays. However, significant inter-laboratory variations occur with respect to data among laboratories. This raises concerns on the reliability and reproducibility of preclinical data that drives further development and translation. Lack of reproducibility may stem from a variety of factors such as poor scientist training, less than optimal experimental design, inadequate description of methodology, and impulse to publish only the positive data etc. Availability of technical manuals, standard operating procedures, accreditation avenues for laboratories performing such assays, inter-laboratory comparisons, and use of standardized protocols are necessary to enhance reliability and reproducibility. Thus, it is important that radiobiological assays are harmonized for laboratory protocols to ensure successful translation of pre-clinical research on radiation effect modulators to help design clinic trials with

MS transport assays for γ-aminobutyric acid transporters--an efficient alternative for radiometric assays.

Science.gov (United States)

Schmitt, Sebastian; Höfner, Georg; Wanner, Klaus T

2014-08-05

Transport assays for neurotransmitters based on radiolabeled substrates are widely spread and often indispensable in basic research and the drug development process, although the use of radioisotopes is inherently coupled to issues concerning radioactive waste and safety precautions. To overcome these disadvantages, we developed mass spectrometry (MS)-based transport assays for γ-aminobutyric acid (GABA), which is the major inhibitory neurotransmitter in the central nervous system (CNS). These "MS Transport Assays" provide all capabilities of [(3)H]GABA transport assays and therefore represent the first substitute for the latter. The performance of our approach is demonstrated for GAT1, the most important GABA transporter (GAT) subtype. As GABA is endogenously present in COS-7 cells employed as hGAT1 expression system, ((2)H6)GABA was used as a substrate to differentiate transported from endogenous GABA. To record transported ((2)H6)GABA, a highly sensitive, short, robust, and reliable HILIC-ESI-MS/MS quantification method using ((2)H2)GABA as an internal standard was developed and validated according to the Center for Drug Evaluation and Research (CDER) guidelines. Based on this LC-MS quantification, a setup to characterize hGAT1 mediated ((2)H6)GABA transport in a 96-well format was established, that enables automated processing and avoids any sample preparation. The K(m) value for ((2)H6)GABA determined for hGAT1 is in excellent agreement with results obtained from [(3)H]GABA uptake assays. In addition, the established assay format enables efficient determination of the inhibitory potency of GAT1 inhibitors, is capable of identifying those inhibitors transported as substrates, and furthermore allows characterization of efflux. The approach described here combines the strengths of LC-MS/MS with the high efficiency of transport assays based on radiolabeled substrates and is applicable to all GABA transporter subtypes.

Clonogenic assay: adherent cells.

Science.gov (United States)

Rafehi, Haloom; Orlowski, Christian; Georgiadis, George T; Ververis, Katherine; El-Osta, Assam; Karagiannis, Tom C

2011-03-13

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant

Genotoxicity testing: Comparison of the γH2AX focus assay with the alkaline and neutral comet assays.

Science.gov (United States)

Nikolova, Teodora; Marini, Federico; Kaina, Bernd

2017-10-01

Genotoxicity testing relies on the quantitative measurement of adverse effects, such as chromosome aberrations, micronuclei, and mutations, resulting from primary DNA damage. Ideally, assays will detect DNA damage and cellular responses with high sensitivity, reliability, and throughput. Several novel genotoxicity assays may fulfill these requirements, including the comet assay and the more recently developed γH2AX assay. Although they are thought to be specific for genotoxicants, a systematic comparison of the assays has not yet been undertaken. In the present study, we compare the γH2AX focus assay with the alkaline and neutral versions of the comet assay, as to their sensitivities and limitations for detection of genetic damage. We investigated the dose-response relationships of γH2AX foci and comet tail intensities at various times following treatment with four prototypical genotoxicants, methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), mitomycin C, and hydrogen peroxide (H 2 O 2 ) and we tested whether there is a correlation between the endpoints, i.e., alkali-labile sites and DNA strand breaks on the one hand and the cell's response to DNA double-strand breaks and blocked replication forks on the other. Induction of γH2AX foci gave a linear dose response and all agents tested were positive in the assay. The increase in comet tail intensity was also a function of dose; however, mitomycin C was almost completely ineffective in the comet assay, and the doses needed to achieve a significant effect were somewhat higher for some treatments in the comet assay than in the γH2AX foci assay, which was confirmed by threshold analysis. There was high correlation between tail intensity and γH2AX foci for MMS and H 2 O 2 , less for MNNG, and none for mitomycin C. From this we infer that the γH2AX foci assay is more reliable, sensitive, and robust than the comet assay for detecting genotoxicant-induced DNA damage. Copyright © 2017 Elsevier

Allogeneic effector/memory Th-1 cells impair FoxP3+ regulatory T lymphocytes and synergize with chaperone-rich cell lysate vaccine to treat leukemia.

Science.gov (United States)

Janikashvili, Nona; LaCasse, Collin J; Larmonier, Claire; Trad, Malika; Herrell, Amanda; Bustamante, Sara; Bonnotte, Bernard; Har-Noy, Michael; Larmonier, Nicolas; Katsanis, Emmanuel

2011-02-03

Therapeutic strategies combining the induction of effective antitumor immunity with the inhibition of the mechanisms of tumor-induced immunosuppression represent a key objective in cancer immunotherapy. Herein we demonstrate that effector/memory CD4(+) T helper-1 (Th-1) lymphocytes, in addition to polarizing type-1 antitumor immune responses, impair tumor-induced CD4(+)CD25(+)FoxP3(+) regulatory T lymphocyte (Treg) immunosuppressive function in vitro and in vivo. Th-1 cells also inhibit the generation of FoxP3(+) Tregs from naive CD4(+)CD25(-)FoxP3(-) T cells by an interferon-γ-dependent mechanism. In addition, in an aggressive mouse leukemia model (12B1), Th-1 lymphocytes act synergistically with a chaperone-rich cell lysate (CRCL) vaccine, leading to improved survival and long-lasting protection against leukemia. The combination of CRCL as a source of tumor-specific antigens and Th-1 lymphocytes as an adjuvant has the potential to stimulate efficient specific antitumor immunity while restraining Treg-induced suppression.

Multicentre comparison of a diagnostic assay

DEFF Research Database (Denmark)

Waters, Patrick; Reindl, Markus; Saiz, Albert

2016-01-01

) assays in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Coded samples from patients with neuromyelitis optica (NMO) or NMOSD (101) and controls (92) were tested at 15 European diagnostic centres using 21 assays including live (n=3) or fixed cell-based assays (n=10), flow cytometry (n=4...

Lipopolysaccharides of the cyanobacterium Microcystis aeruginosa

Energy Technology Data Exchange (ETDEWEB)

Raziuddin, S.; Siegelman, H.W.; Tornabene, T.G.

1983-01-01

Lipopolysaccharides (LPS) of two isolates of Microcystis aeruginosa were extracted with phenol/water and purified. Cesium chloride gradient ultracentrifugation of these preparations yielded only one fraction. The LPS contained significant amounts of 3-deoxy-D-manno-octulosonic acid, glucose, 3-deoxy sugars, glucosamine, fatty acids, fatty acid esters, hexoses, and phosphate. Heptose, a characteristic sugar component of the polysaccharide moiety of LPS of most gram-negative bacteria was absent. Lipopolysaccharides and lipid A hydrolysate of LPS preparations were active in mouse lethality and Limulus lysate gelation. The lipid A moiety was slightly less active in toxicity and Limulus lysate gelation assay than the intact LPS. The LPS and lipid A moiety of the two isolates of M. aeruginosa were less active in toxicity in mice and Limulus test than LPS of Salmonella abortus equi. 37 references, 1 figure, 3 tables.

An ultrafiltration assay for lysyl oxidase

International Nuclear Information System (INIS)

Shackleton, D.R.; Hulmes, D.J.

1990-01-01

A modification of the original microdistillation assay for lysyl oxidase is described in which Amicon C-10 microconcentrators are used to separate, by ultrafiltration, the 3H-labeled products released from a [4,5-3H]-lysine-labeled elastin substrate. Enzyme activity is determined by scintillation counting of the ultrafiltrate, after subtraction of radioactivity released in the presence of beta-aminopropionitrile, a specific inhibitor of the enzyme. Conditions are described which optimize both the sensitivity and the efficient use of substrate. The assay shows linear inhibition of activity in up to 1 M urea; hence, as the enzyme is normally diluted in the assay, samples in 6 M urea can be assayed directly, without prior dialysis, and corrected for partial inhibition. Comparable results are obtained when enzyme activity is assayed by ultrafiltration or microdistillation. The assay is simple and convenient and, by using disposable containers throughout, it eliminates the need for time-consuming decontamination of radioactive glassware

Human platelet lysate supplementation of mesenchymal stromal cell delivery: issues of xenogenicity and species variability.

Science.gov (United States)

Allen, Ashley B; Butts, Emily B; Copland, Ian B; Stevens, Hazel Y; Guldberg, Robert E

2017-10-01

Immunogenicity of fetal bovine serum (FBS) poses a problem for its use in the propagation of autologous mesenchymal stromal cells (MSCs) for cell therapy. Human platelet lysate (hPL), an enriched growth factor solution containing mitogenic and angiogenic cues, has potential utility in replacing FBS for human MSC (hMSC) delivery strategies. Despite its potentiation of hMSC number in vitro, little is known concerning its capacity to supplement implanted hMSC-seeded constructs and promote tissue regeneration in vivo. In this study, we tested the effects of incorporating hPL in cell-seeded constructs implanted subcutaneously into immunocompromised rats, investigated in vitro interactions between hPL and rat MSCs (rMSCs) and determined interspecies variability in the PL product [hPL vs rat PL (rPL)] and its effect on cultured MSCs (hPL/hMSCs vs rPL/rMSCs). The overarching aim was to determine the utility of hPL to foster MSC survival in preclinical rodent models. Exposure to hPL-supplemented media resulted in rMSC death, by a process attributable to heat-labile proteins, but not membrane attack complex formation. In the in vitro syngeneic model, the rodent product proved fundamentally distinct from the human product, with rPL having substantially lower growth factor content than hPL. Moreover, contrary to the positive effects of hPL on hMSC expansion, rPL did not reduce rMSC doubling time for the serum concentrations examined. When tested in vivo, hPL did not improve cell survival within hydrogel constructs through 2 weeks postimplantation. In summary, this study highlights the many facets of xenogenicity and interspecies variability that must be considered in the preclinical evaluation of hPL. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

LPS levels in root canals after the use of ozone gas and high frequency electrical pulses

Directory of Open Access Journals (Sweden)

Tiago André Fontoura de MELO

2016-01-01

Full Text Available Abstract The present study aims to verify the effect of ozone gas (OZY® System and high frequency electric pulse (Endox® System systems on human root canals previously contaminated with Escherichia colilipopolysaccharide (LPS. Fifty single-rooted teeth had their dental crowns removed and root lengths standardized to 16 mm. The root canals were prepared up to #60 hand K-files and sterilized using gamma radiation with cobalt 60. The specimens were divided into the following five groups (n = 10 based on the disinfection protocol used: OZY® System, one 120-second-pulse (OZY 1p; OZY® System, four 24-second-pulses (OZY 4p; and Endox® System (ENDOX. Contaminated and non-contaminated canals were exposed only to apyrogenic water and used as positive (C+ and negative (C- controls, respectively. LPS (O55:B55 was administered in all root canals except those belonging to group C-. After performing disinfection, LPS samples were collected from the canals using apyrogenic paper tips. Limulus Amoebocyte Lysate (LAL was used to quantify the LPS levels, and the data obtained was analyzed using one-way ANOVA. The disinfection protocols used were unable to reduce the LPS levels significantly (p = 0.019. The use of ozone gas and high frequency electric pulses was not effective in eliminating LPS from the root canals.

A rapid kinetic chromogenic method for quantification of bacterial endotoxins in lyophilized reagents for labeling with 99mTc radiopharmaceuticals

International Nuclear Information System (INIS)

Fukumori, Neuza T.O.; Campos, Domingos G.; Silva, Laercio; Fernandes, Adriana V.; Mengatti, Jair; Silva, Constancia P.G.; Matsuda, Margareth M.N.

2009-01-01

A rapid quantitative kinetic chromogenic test in an automated Portable Test System (PTS) has been developed for determination of bacterial endotoxins in water, in-process and end-products using the Limulus amebocyte lysate (LAL). The aim of this work was to validate the method for lyophilized reagents for labeling with 99m Tc radiopharmaceuticals with no interfering factors. Experiments were performed in three consecutive batches of the lyophilized reagents Methylenediphosphonic Acid (MDP) and Pyrophosphate (PYRO) produced at IPEN-CNEN/ SP using the PTS from Endosafe, Inc. TM , Charleston, SC. The Maximum Valid Dilution (MVD) was calculated to establish the extent of dilution to avoid interfering test conditions (MVD=500). Better results were obtained above 1:20 dilution factor for MDP and 1:100 for PYRO. The parameters of coefficient correlation (R) -0.980, RPPC between 50 - 200% and coefficient variation (CV) of the samples less than 25% were satisfied and the endotoxin concentration was lower than the lowest concentration of the standard curve (0.05 EU mL -1 ), therefore less than the established limit in pharmacopoeias. The PTS is a rapid, simple and accurate technique using the quantitative kinetic chromogenic method for bacterial endotoxin determination. For this reason, it is very practical in the radiopharmaceutical area and it trends to be the method of choice for the pyrogen test. For MDP and PYRO, the validation was successfully performed. (author)

Particulate systems based on pectin/chitosan association for the delivery of manuka honey components and platelet lysate in chronic skin ulcers.

Science.gov (United States)

Tenci, Marika; Rossi, Silvia; Bonferoni, Maria Cristina; Sandri, Giuseppina; Boselli, Cinzia; Di Lorenzo, Arianna; Daglia, Maria; Icaro Cornaglia, Antonia; Gioglio, Luciana; Perotti, Cesare; Caramella, Carla; Ferrari, Franca

2016-07-25

The aim of the present work was the development of a powder formulation for the delivery of manuka honey (MH) bioactive components and platelet lysate (PL) in chronic skin ulcers. In particular pectin (PEC)/chitosan (CS) particles were prepared by ionotropic gelation in the presence of calcium chloride and subsequently characterized for particle size, hydration properties and mechanical resistance. Different experimental conditions (calcium chloride and CS concentrations; rest time in the cationic solution) were considered in order to obtain particles characterized by optimal size, hydration properties and mechanical resistance. Two different fractions of MH were examined: one (Fr1), rich in methylglyoxal and the other (Fr2), rich in polyphenols. Particles were loaded with Fr1, fraction able to enhance in vitro proliferation of human fibroblasts, and with PL. The presence of CS in Fr1-loaded particles produced an improvement in cell proliferation. Moreover, PL loading into particles did not affect the biological activity of the hemoderivative. In vivo efficacy of PL- and Fr1-loaded particles was evaluated on a rat wound model. Both treatments markedly increased wound healing to the same extent. Copyright © 2016 Elsevier B.V. All rights reserved.

Radioreceptor assay for insulin

Energy Technology Data Exchange (ETDEWEB)

Suzuki, Kazuo [Tokyo Univ. (Japan). Faculty of Medicine

1975-04-01

Radioreceptor assay of insulin was discussed from the aspects of the measuring method, its merits and problems to be solved, and its clinical application. Rat liver 10 x g pellet was used as receptor site, and enzymatic degradation of insulin by the system contained in this fraction was inhibited by adding 1 mM p-CMB. /sup 125/I-labelled porcine insulin was made by lactoperoxidase method under overnight incubation at 4/sup 0/C and later purification by Sephadex G-25 column and Whatman CF-11 cellulose powder. Dog pancreatic vein serum insulin during and after the glucose load was determined by radioreceptor assay and radioimmunoassay resulting that both measurements accorded considerably. Radioreceptor assay would clarify the pathology of disorders of glucose metabolism including diabetes.

Nano-immunosafety: issues in assay validation

International Nuclear Information System (INIS)

Boraschi, Diana; Italiani, Paola; Oostingh, Gertie J; Duschl, Albert; Casals, Eudald; Puntes, Victor F; Nelissen, Inge

2011-01-01

Assessing the safety of engineered nanomaterials for human health must include a thorough evaluation of their effects on the immune system, which is responsible for defending the integrity of our body from damage and disease. An array of robust and representative assays should be set up and validated, which could be predictive of the effects of nanomaterials on immune responses. In a trans-European collaborative work, in vitro assays have been developed to this end. In vitro tests have been preferred for their suitability to standardisation and easier applicability. Adapting classical assays to testing the immunotoxicological effects of nanoparticulate materials has raised a series of issues that needed to be appropriately addressed in order to ensure reliability of results. Besides the exquisitely immunological problem of selecting representative endpoints predictive of the risk of developing disease, assay results turned out to be significantly biased by artefactual interference of the nanomaterials or contaminating agents with the assay protocol. Having addressed such problems, a series of robust and representative assays have been developed that describe the effects of engineered nanoparticles on professional and non-professional human defence cells. Two of such assays are described here, one based on primary human monocytes and the other employing human lung epithelial cells transfected with a reporter gene.

Data transformation methods for multiplexed assays

Science.gov (United States)

Tammero, Lance F. Bentley; Dzenitis, John M; Hindson, Benjamin J

2013-07-23

Methods to improve the performance of an array assay are described. A correlation between fluorescence intensity-related parameters and negative control values of the assay is determined. The parameters are then adjusted as a function of the correlation. As a result, sensitivity of the assay is improved without changes in its specificity.

Observations on the expression of human papillomavirus major capsid protein in HeLa cells.

Science.gov (United States)

Xiao, Chang-Yi; Fu, Bing-Bing; Li, Zhi-Ying; Mushtaq, Gohar; Kamal, Mohammad Amjad; Li, Jia-Hua; Tang, Gui-Cheng; Xiao, Shuo-Shuang

2015-01-01

The goal of this study was to identify the nature of the inclusion bodies that have been found in HeLa cells (cervical cancer immortal cell line) by electron microscope and to determine whether the major capsid protein (L1) of human papillomavirus (HPV) can be expressed in HPV-positive uterine cervix cancer cells. HPV L1 protein expression in HeLa cells was detected with anti-HPV L1 multivalent mice monoclonal antibody and rabbit polyclonal anti-HPV L1 antibody by ELISA, light microscope immunohistochemistry, electron microscope immunocytochemistry and Western blotting assays. Reverse transcriptional PCR (RT-PCR) was performed to detect the transcription of L1 mRNA in HeLa cells. The immortalized human keratinocyte HeCat was used as the negative control. HPV L1 proteins reacted positively in the lysate of HeLa cells by ELISA assays. HRP labeled light microscope immunohistochemistry assay showed that there was a strong HPV L1 positive reaction in HeLa cells. Under the electron microscope, irregular shaped inclusion bodies, assembled by many small and uniform granules, had been observed in the cytoplasm of some HeLa cells. These granules could be labeled by the colloidal gold carried by HPV L1 antibody. The Western blotting assay showed that there was a L1 reaction strap at 80-85 kDa in the HeLa cell lysates, hence demonstrating the existence of HPV18 L1 in HeLa cells. RT-PCR assay showed that the L1 mRNA was transcribed in HeLa cells. The inclusion bodies found in the cytoplasm of HeLa cells are composed of HPV18 L1 protein. Since HeLa cell line is a type of cervical cancer cells, this implies that HeLa cells have the ability to express HPV L1 proteins.

Spectrophotometric Enzyme Assays for High-Throughput Screening

Directory of Open Access Journals (Sweden)

Jean-Louis Reymond

2004-01-01

Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

Radioenzymatic assay of DOPA (3,4-dihydroxyphenylalanine)

International Nuclear Information System (INIS)

Johnson, G.A.; Gren, J.M.; Kupiecki, R.

1978-01-01

We modified the single-isotope radioenzymatic assay for catecholamines [Life Sci. 21, 625(1977)] to assay 3,4-dihydroxyphenylalanine (DOPA). DOPA decarboxylase is used to convert DOPA to dopamine, which concurrently is converted to [ 3 H]-3-O-methyldopamine in the presence of catechol-O-methyltransferase and [methyl- 3 H]-S-adenosylmethionine and assayed radioenzymatically. For assay of plasma DOPA, 50 μl of untreated plasma is added directly into the incubation mixture. A duplicate mixture containing an internal standard requires a second 50-μl aliquot of plasma. Because the assay measures both DOPA and endogenous dopamine, two additional aliquots of plasma must be assayed for dopamine in the absence of the decarboxylase by the differential assay; DOPA is estimated by difference. The assay is sensitive to 25 pg (500 ng/liter of plasma). Analysis of DOPA (DOPA plus dopamine) and the concurrent differential assay of catecholamines in at least 10 samples can be done in a single working day. Plasma DOPA concentrations for 42 normotensive adults were 1430 +- 19 ng/liter (mean +- SEM). In contrast, dopamine concentrations for these same subjects averaged 23 +- 20 ng/liter. Values for the 24 women subjects (1510 +- 62 ng/liter) significantly (P = 0.04) exceeded those for the men

RAS - Screens & Assays - Drug Discovery

Science.gov (United States)

The RAS Drug Discovery group aims to develop assays that will reveal aspects of RAS biology upon which cancer cells depend. Successful assay formats are made available for high-throughput screening programs to yield potentially effective drug compounds.

RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

OpenAIRE

Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

2008-01-01

Abstract Background Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of inter...

Systemic and Pulmonary Hypertension After Resuscitation with Cell-Free Hemoglobin

Science.gov (United States)

1992-07-01

Endotoxin was measured using a kinetic turbidimetric modification of the Limulus amoebocyte lysate assay (9). The rabbit pyrogen test was performed...renal failure. This injury occurred in half of the animals that were resuscitated with HPLC purified, pyrogen -negative, low-residual- phosphate HbAo...oxide: an endogenous modulator of leukocyte adhesion. Proc. Nat. Acad. Sci. USA 88:4651-4655, 1991. 17. Moncada, S., K. M. J. Palmer, E. A. Higgs. Nitric

The fluorometric microculture cytotoxicity assay.

Science.gov (United States)

Lindhagen, Elin; Nygren, Peter; Larsson, Rolf

2008-01-01

The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

Solution assay instrument operations manual

International Nuclear Information System (INIS)

Li, T.K.; Marks, T.; Parker, J.L.

1983-09-01

An at-line solution assay instrument (SAI) has been developed and installed in a plutonium purification and americium recovery process area in the Los Alamos Plutonium Processing Facility. The instrument was designed for accurate, timely, and simultaneous nondestructive analysis of plutonium and americium in process solutions that have a wide range of concentrations and americium/plutonium ratios and for routine operation by process technicians who lack instrumentation background. The SAI, based on transmission-corrected, high-resolution gamma-ray spectroscopy, has two measurement stations attached to a single multichannel analyzer/computer system. To ensure the quality of assay results, the SAI has an internal measurement control program, which requires daily and weekly check runs and monitors key aspects of all assay runs. For a 25-ml sample, the assay precision is 5 g/l within a 2000-s count time

Making transuranic assay measurements using modern controllers

International Nuclear Information System (INIS)

Kuckertz, T.H.; Caldwell, J.T.; Medvick, P.A.; Kunz, W.E.; Hastings, R.D.

1987-01-01

This paper describes methodology and computer-controlled instrumentation developed at the Los Alamos National Laboratory that accurately performs nondestructive assays of large containers bearing transuranic wastes and nonradioactive matrix materials. These assay systems can measure fissile isotopes with 1-mg sensitivity and spontaneous neutron-emitting isotopes at a 10-mg sensitivity. The assays are performed by neutron interrogation, detection, and counting in a custom assay chamber. An International Business Machines Personal Computer (IBM-PC) is used to control the CAMAC-based instrumentation system that acquires the assay data. 6 refs., 7 figs

Time-resolved immunofluorometric assay of serum ferritin

Energy Technology Data Exchange (ETDEWEB)

Yan, Yao [China Inst. of Atomic Energy, Beijing (China)

2007-06-15

This assay is a solid phase, two-site fluoroimmunometric assay based on the direct sandwish technique. Standards or samples containing ferritin are first reacted with immobilized anti-ferritin antibodies. Then the europium-lablled antibodies are reacted with the bound antigen. The range of this assay is 2-1000 ng/mL. The analytical sentivity is better than 0.05 ng/mL. The intra-assay variation and inter-assay variation are both below 5%; This kit was compared with Wallac DELFIA kit. The correlation is r=0.96. (authors)

Scintillation proximity assay

International Nuclear Information System (INIS)

Hart, H.

1980-01-01

In a method of immunological assay two different classes of particles which interact at short distances to produce characteristic detectable signals are employed in a modification of the usual latex fixation test. In one embodiment an aqueous suspension of antigen coated tritiated latex particles (LH) and antigen coated polystyrene scintillant particles (L*) is employed to assay antibody in the aqueous medium. The amount of (LH) (L*) dimer formation and higher order aggregation induced and therefore the concentration of antibody (or antigen) present which caused the aggregation can be determined by using standard liquid scintillation counting equipment. (author)

Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study

Directory of Open Access Journals (Sweden)

Franka Klatte-Schulz

2018-01-01

Full Text Available The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP; RegenLab (PRP-BCT, platelet concentrate (apheresis, PC, and PL (freezing-thawing destruction of PC. Additionally, ten commercial allogenic PLs (AlloPL from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1β, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

Directory of Open Access Journals (Sweden)

Lena Thoring

Full Text Available Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

Evaluation of a platelet lysate bilayered system for periodontal regeneration in a rat intrabony three-wall periodontal defect.

Science.gov (United States)

Babo, Pedro S; Cai, Xinjie; Plachokova, Adelina S; Reis, Rui L; Jansen, John; Gomes, Manuela E; Walboomers, X Frank

2018-02-01

With currently available therapies, full regeneration of lost periodontal tissues after periodontitis cannot be achieved. In this study, a combined compartmentalized system was tested, composed of (a) a platelet lysate (PL)-based construct, which was placed along the root aiming to regenerate the root cementum and periodontal ligament, and (b) a calcium phosphate cement composite incorporated with hyaluronic acid microspheres loaded with PL, aiming to promote the regeneration of alveolar bone. This bilayered system was assessed in a 3-wall periodontal defect in Wistar rats. The periodontal healing and the inflammatory response of the materials were scored for a period up to 6 weeks after implantation. Furthermore, histomorphometrical measurements were performed to assess the epithelial downgrowth, the formation of alveolar bone, and the formation of new connective tissue attachment. Our data showed that the stabilization of platelet-origin proteins on the root surface increased the overall periodontal healing score and restricted the formation of long epithelial junctions. Nevertheless, the faster degradation of the cement component with incorporated hyaluronic acid microspheres compromised the stability of the system, which hampered the periodontal regeneration. Overall, in this work, we proved the positive therapeutic effect of the immobilization of a PL-based construct over the root surface in a combined compartmentalized system to assist predictable healing of functional periodontium. Therefore, after optimization of the hard tissue analogue, the system should be further elaborated in (pre)clinical validation studies. Copyright © 2017 John Wiley & Sons, Ltd.

Assessing sediment contamination using six toxicity assays

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Allen G. BURTON Jr.

2001-08-01

Full Text Available An evaluation of sediment toxicity at Lake Orta, Italy was conducted to compare a toxicity test battery of 6 assays and to evaluate the extent of sediment contamination at various sediment depths. Lake Orta received excessive loadings of copper and ammonia during the 1900’s until a large remediation effort was conducted in 1989-90 using lime addition. Since that time, the lake has shown signs of a steady recovery of biological communities. The study results showed acute toxicity still exists in sediments at a depth of 5 cm and greater. Assays that detected the highest levels of toxicity were two whole sediment exposures (7 d using Hyalella azteca and Ceriodaphnia dubia. The MicrotoxR assay using pore water was the third most sensitive assay. The Thamnotox, Rototox, Microtox solid phase, and Seed Germination-Root Elongation (pore and solid phase assays showed occasional to no toxicity. Based on similarity of responses and assay sensitivity, the two most useful assays were the C. dubia (or H. azteca and Microtox pore water. These assays were effective at describing sediment toxicity in a weight-of-evidence approach.

Orsay: High-gradient experiment

International Nuclear Information System (INIS)

Anon.

1990-01-01

Maintaining the tradition of its contribution to the LEP Injector Linac (LIL), Orsay's Linear Accelerator Laboratory (LAL) is carrying out an R&D programme entitled 'New accelerator physics experiments at LAL' (NEPAL). The aim is to contribute to the long-term development of high energy electron-positron linear colliders, where progress can be of short-term benefit both to conventional accelerators and to injectors in rings or free-electron lasers

Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics

OpenAIRE

Aguisanda, Francis; Yeh, Charles D.; Chen, Catherine Z.; Li, Rong; Beers, Jeanette; Zou, Jizhong; Thorne, Natasha; Zheng, Wei

2017-01-01

Background Wolman disease (WD) is a rare lysosomal storage disorder that is caused by mutations in the LIPA gene encoding lysosomal acid lipase (LAL). Deficiency in LAL function causes accumulation of cholesteryl esters and triglycerides in lysosomes. Fatality usually occurs within the first year of life. While an enzyme replacement therapy has recently become available, there is currently no small-molecule drug treatment for WD. Results We have generated induced pluripotent stem cells (iPSCs...

Removal of endotoxins from bacteriophage preparations by extraction with organic solvents.

Directory of Open Access Journals (Sweden)

Bożena Szermer-Olearnik

Full Text Available Lipopolysaccharide (LPS, endotoxin, pyrogen constitutes a very troubling contaminant of crude phage lysates produced in Gram-negative bacteria. Toxicity of LPS depends on the strong innate immunity response including the cytokines. Therefore, its removal is important for bacteriophage applications. In this paper, we present a procedure for extractive removal of endotoxin from bacteriophage preparations with water immiscible solvents (1-octanol or 1-butanol. During extraction most of the phage lytic activity is retained in the aqueous phase, while endotoxin accumulates in the organic solvent. The levels of endotoxin (expressed as endotoxin units, EU in the aqueous bacteriophage-containing fraction determined by limulus amebocyte lysate or EndoLISA assay were exceptionally low. While the initial endotoxin levels in the crude phage lysates ranged between 10(3 and 10(5 EU/ml the average level after organic extraction remaining in the aqueous fraction was 5.3 EU/ml. These values when related to phage titers decreased from 10(3-10(5 EU/10(9 PFU (plaque forming units down to an average of 2.8 EU/10(9 PFU. The purification procedure is scalable, efficient and applicable to all the bacteriophages tested: T4, HAP1 (E. coli and F8 (P. aeruginosa.

A multiwell format assay for heparanase.

Science.gov (United States)

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

A quantitative comet infection assay for influenza virus

Science.gov (United States)

Lindsay, Stephen M.; Timm, Andrea; Yin, John

2011-01-01

Summary The virus comet assay is a cell-based virulence assay used to evaluate an antiviral drug or antibody against a target virus. The comet assay differs from the plaque assay in allowing spontaneous flows in 6-well plates to spread virus. When implemented quantitatively the comet assay has been shown to have an order-of-magnitude greater sensitivity to antivirals than the plaque assay. In this study, a quantitative comet assay for influenza virus is demonstrated, and is shown to have a 13-fold increase in sensitivity to ribavirin. AX4 cells (MDCK cells with increased surface concentration of α2–6 sialic acid, the influenza virus receptor) have reduced the comet size variability relative to MDCK cells, making them a better host cell for use in this assay. Because of enhanced antiviral sensitivity in flow-based assays, less drug is required, which could lead to lower reagent costs, reduced cytotoxicity, and fewer false-negative drug screen results. The comet assay also serves as a readout of flow conditions in the well. Observations from comets formed at varying humidity levels indicate a role for evaporation in the mechanism of spontaneous fluid flow in wells. PMID:22155578

Src inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity in Ramos Burkitt's lymphoma B cell line

International Nuclear Information System (INIS)

Hristov, K.; Mitev, V.; Knox, K.

2006-01-01

Reversible tyrosine phosphorylation, regulation of expression and proteolytic cleavage control tyrosine phosphatase contribution for the signalling pathways of B-cell antigen receptor (BCR), and CD40 during B cell selection. We used Ramos-BL B cell line to determine whether BCR and CD40 stimulation, or inhibition of the Src - tyrosine kinase, tyrosine phosphatase and caspase activity have an effect on the tyrosine phosphatase activities determined on in-gel phosphatase assay. The tyrosine phosphatase activities present in whole cell lysates of Ramos-BL B cells following treatment with 20 μg/ml anti-IgM, 1 μg/ml anti-CD40, 10 μM herbimycin A, 178 μM vanadate,100 μM phenylarsine oxide and 10 μM zVAD-fmk were detected with an in-gel phosphatase assay. Seven major tyrosine phosphatase activities with approximate molecular weight of 132.7, 63.9, 60.3, 54.2, 49.7, 44.6, and 39 kDa are present in whole cell lysates of Ramos-BL B cells. Treatment with Src-PTK inhibitor herbimycin A prevents 132.7 kDa tyrosine phosphatase activity. We conclude that the catalytic activity of Src-PTK in Ramos-BL B cells is critical for the presence of this 132.7 kDa tyrosine phosphatase activity. (authors)

Assay development status report for total cyanide

International Nuclear Information System (INIS)

Simpson, B.C.; Jones, T.E.; Pool, K.H.

1993-02-01

A validated cyanide assay that is applicable to a variety of tank waste matrices is necessary to resolve certain waste tank safety issues and for purposes of overall waste characterization. The target for this effort is an assay with an applicable range of greater than 1,000 ppM (0.10 wt%) total cyanide and a confidence level greater than 80%. Figure 1 illustrates the operating regime of the proposed cyanide assay method. The Assay Development Status Report for Total Cyanide will summarize the past experience with cyanide analyses on-tank waste matrices and will rate the status of the analytical methods used to assay total cyanide (CN - ion) in the tank waste matrices as acceptable or unacceptable. This paper will also briefly describe the current efforts for improving analytical resolution of the assays and the attempts at speciation

Prospects for cellular mutational assays in human populations

International Nuclear Information System (INIS)

Mendelsohn, M.L.

1984-01-01

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references

Prospects for cellular mutational assays in human populations

Energy Technology Data Exchange (ETDEWEB)

Mendelsohn, M.L.

1984-06-29

Practical, sensitive, and effective human cellular assays for detecting somatic and germinal mutations would have great value in environmental mutagenesis and carcinogenesis studies. Such assays would fill the void between human mutagenicity and the data that exist from short-term tests and from mutagenicity in other species. This paper discusses the following possible human cellular assays: (1) HPRT (hypoxanthine phosphoribosyltransferase) somatic cell mutation based on 6-thioguanine resistance; (2) hemoglobin somatic cell mutation assay; (3) glycophorin somatic cell mutation assay; and (4) LDH-X sperm cell mutation assay. 18 references.

A radiochemical assay for biotin in biological materials

International Nuclear Information System (INIS)

Hood, R.L.

1975-01-01

A radiochemical assay for biotin is described. The assay was sensitive to one nanogram and simple enough for routine biotin analyses. The assay yielded results which were comparable to those obtained from a microbiological assay using Lactobacillus plantarum. (author)

Donor T cells primed on leukemia lysate-pulsed recipient APCs mediate strong graft-versus-leukemia effects across MHC barriers in full chimeras.

Science.gov (United States)

Ghosh, Arnab; Koestner, Wolfgang; Hapke, Martin; Schlaphoff, Verena; Länger, Florian; Baumann, Rolf; Koenecke, Christian; Cornberg, Markus; Welte, Karl; Blazar, Bruce R; Sauer, Martin G

2009-04-30

Antigen-presenting cells (APCs) of host origin drive graft-versus-leukemia (GVL) effects but can also trigger life-threatening graft-versus-host disease (GVHD) after hematopoietic cell transplantation (HCT) across major histocompatibility complex (MHC) barriers. We show that in vitro priming of donor lymphocytes can circumvent the need of recipient-derived APCs in vivo for mediating robust GVL effects and significantly diminishes the risk of severe GVHD. In vitro, generated and expanded T cells (ETCs) mediate anti-leukemia effects only when primed on recipient-derived APCs. Loading of APCs in vitro with leukemia cell lysate, chimerism status of the recipient, and timing of adoptive transfer after HCT are important factors determining the outcome. Delayed transfer of ETCs resulted in strong GVL effects in leukemia-bearing full chimera (FC) and mixed chimera (MC) recipients, which were comparable with the GVL/GVHD rates observed after the transfer of naive donor lymphocyte infusion (DLI). Upon early transfer, GVL effects were more pronounced with ETCs but at the expense of significant GVHD. The degree of GVHD was most severe in MCs after transfer of ETCs that had been in vitro primed either on nonpulsed recipient-derived APCs or with donor-derived APCs.

New automated pellet/powder assay system

International Nuclear Information System (INIS)

Olsen, R.N.

1975-01-01

This paper discusses an automated, high precision, pellet/ powder assay system. The system is an active assay system using a small isotopic neutron source and a coincidence detection system. The handling of the pellet powder samples has been automated and a programmable calculator has been integrated into the system to provide control and data analysis. The versatile system can assay uranium or plutonium in either active or passive modes

Passive nondestructive assay of nuclear materials

International Nuclear Information System (INIS)

Reilly, D.; Ensslin, N.; Smith, H. Jr.; Kreiner, S.

1991-03-01

The term nondestructive assay (NDA) is applied to a series of measurement techniques for nuclear fuel materials. The techniques measure radiation induced or emitted spontaneously from the nuclear material; the measurements are nondestructive in that they do not alter the physical or chemical state of the nuclear material. NDA techniques are characterized as passive or active depending on whether they measure radiation from the spontaneous decay of the nuclear material or radiation induced by an external source. This book emphasizes passive NDA techniques, although certain active techniques like gamma-ray absorption densitometry and x-ray fluorescence are discussed here because of their intimate relation to passive assay techniques. The principal NDA techniques are classified as gamma-ray assay, neutron assay, and calorimetry. Gamma-ray assay techniques are treated in Chapters 1--10. Neutron assay techniques are the subject of Chapters 11--17. Chapters 11--13 cover the origin of neutrons, neutron interactions, and neutron detectors. Chapters 14--17 cover the theory and applications of total and coincidence neutron counting. Chapter 18 deals with the assay of irradiated nuclear fuel, which uses both gamma-ray and neutron assay techniques. Chapter 19 covers perimeter monitoring, which uses gamma-ray and neutron detectors of high sensitivity to check that no unauthorized nuclear material crosses a facility boundary. The subject of Chapter 20 is attribute and semiquantitative measurements. The goal of these measurements is a rapid verification of the contents of nuclear material containers to assist physical inventory verifications. Waste and holdup measurements are also treated in this chapter. Chapters 21 and 22 cover calorimetry theory and application, and Chapter 23 is a brief application guide to illustrate which techniques can be used to solve certain measurement problems

Real-time PCR analysis of genes encoding tumor antigens in esophageal tumors and a cancer vaccine

DEFF Research Database (Denmark)

Weinert, Brian T; Krishnadath, Kausilia K; Milano, Francesca

2009-01-01

Tumor antigens are the primary target of therapeutic cancer vaccines. We set out to define and compare the expression pattern of tumor antigen genes in esophagus carcinoma biopsies and in an allogeneic tumor lysate-based cancer vaccine, MelCancerVac. Cells used for vaccine production were treated...... in the production of the vaccine. Quantitative PCR was used to assay 74 tumor antigen genes in patients with squamous cell carcinoma of the esophagus. 81% (13/16) of tumors expressed more than five cancer/testis (CT) antigens. A total of 96 genes were assayed in the tumor cell clone (DDM1.7) used to make tumor cell...

Rover waste assay system

International Nuclear Information System (INIS)

Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J.

1997-01-01

The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched 235 U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for 137 Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs

Human platelet lysate is a successful alternative serum supplement for propagation of monocyte-derived dendritic cells.

Science.gov (United States)

Švajger, Urban

2017-04-01

Clinical protocols for dendritic cell (DC) generation from monocytes require the use of animal serum-free supplements. Serum-free media can also require up to 1% of serum supplementation. In addition, recommendations based on the 3Rs (Refinement, Reduction, Replacement) principle also recommend the use of non-animal sera in in vitro studies. The aim of this study was to explore the potential use of platelet lysate (PL) for generation of optimally differentiated DCs from monocytes. Cells were isolated from buffy coats from healthy volunteers using immunomagnetic selection. DCs were differentiated in RPMI1640 supplemented with either 10% fetal bovine serum (FBS), 10% AB serum or 10% PL with the addition of granulocyte monocyte colony stimulating factor and interleukin-4. Generated DCs were assessed for their morphology, viability, endocytotic capacity, surface phenotype (immature, mature and tolerogenic DCs) and activation of important signaling pathways. DC function was evaluated on the basis of their allostimulatory capacity, cytokine profile and ability to induce different T-helper subsets. DCs generated with PL displayed normal viability, morphology and endocytotic capacity. Their differentiation and maturation phenotype was comparable to FBS-cultured DCs. They showed functional plasticity and up-regulated tolerogenic markers in response to their environment. PL-cultured mature DCs displayed unhindered allostimulatory potential and the capacity to induce Th1 responses. The use of PL allowed for activation of crucial signaling proteins associated with DC differentiation and maturation. This study demonstrates for the first time that human PL represents a successful alternative to FBS in differentiation of DCs from monocytes. DCs display the major phenotypic and functional characteristics compared with existing culture protocols. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Potency testing of mesenchymal stromal cell growth expanded in human platelet lysate from different human tissues.

Science.gov (United States)

Fazzina, R; Iudicone, P; Fioravanti, D; Bonanno, G; Totta, P; Zizzari, I G; Pierelli, L

2016-08-25

Mesenchymal stromal cells (MSCs) have been largely investigated, in the past decade, as potential therapeutic strategies for various acute and chronic pathological conditions. MSCs isolated from different sources, such as bone marrow (BM), umbilical cord tissue (UCT) and adipose tissue (AT), share many biological features, although they may show some differences on cumulative yield, proliferative ability and differentiation potential. The standardization of MSCs growth and their functional amplification is a mandatory objective of cell therapies. The aim of this study was to evaluate the cumulative yield and the ex vivo amplification potential of MSCs obtained from various sources and different subjects, using defined culture conditions with a standardized platelet lysate (PL) as growth stimulus. MSCs isolated from BM, UCT and AT and expanded in human PL were compared in terms of cumulative yield and growth potential per gram of starting tissue. MSCs morphology, phenotype, differentiation potential, and immunomodulatory properties were also investigated to evaluate their biological characteristics. The use of standardized PL-based culture conditions resulted in a very low variability of MSC growth. Our data showed that AT has the greater capacity to generate MSC per gram of initial tissue, compared to BM and UCT. However, UCT-MSCs replicated faster than AT-MSCs and BM-MSCs, revealing a greater proliferation capacity of this source irrespective of its lower MSC yield. All MSCs exhibited the typical MSC phenotype and the ability to differentiate into all mesodermal lineages, while BM-MSCs showed the most prominent immunosuppressive effect in vitro. The adoption of standardized culture conditions may help researchers and clinicians to reveal particular characteristics and inter-individual variability of MSCs sourced from different tissues. These data will be beneficial to set the standards for tissue collection and MSCs clinical-scale expansion both for cell banking

Radioreceptor assay: theory and applications to pharmacology

International Nuclear Information System (INIS)

Perret, G.; Simon, P.

1984-01-01

The aim of the first part of this work is to present the theory of the radioreceptor assay and to compare it to the other techniques of radioanalysis (radioimmunoassay, competitive protein binding assays). The technology of the radioreceptor assay is then presented and its components (preparation of the receptors, radioligand, incubation medium) are described. The analytical characteristics of the radioreceptor assay (specificity, sensitivity, reproductibility, accuracy) and the pharmacological significance of the results are discussed. The second part is devoted to the description of the radioreceptor assays of some pharmacological classes (neuroleptics, tricyclic antidepressants, benzodiazepines, β-blockers, anticholinergic drugs) and to their use in therapeutic drug monitoring. In conclusion, by their nature, radioreceptor assays are highly sensitive, reliable, precise, accurate and simple to perform. Their chief disadvantage relates to specificity, since any substance having an appreciable affinity to the receptor site will displace the specifically bound radioligand. Paradoxically in some cases, this lack of specificity may be advantageous in that it allows for the detection of not only the apparent compound but of active metabolites and endogenous receptor agonists as well and in that radioreceptors assays can be devised for a whole pharmacological class and not only for one drug as it is the case for classical physico-chemical techniques. For all these reasons future of radioreceptor assay in pharmacology appears promising [fr

Assay optimization for molecular detection of Zika virus

NARCIS (Netherlands)

Corman, Victor M.; Rasche, Andrea; Baronti, Cecile; Aldabbagh, Souhaib; Cadar, Daniel; Reusken, Chantal Bem; Pas, Suzan D.; Goorhuis, Abraham; Schinkel, Janke; Molenkamp, Richard; Kümmerer, Beate M.; Bleicker, Tobias; Brünink, Sebastian; Eschbach-Bludau, Monika; Eis-Hübinger, Anna M.; Koopmans, Marion P.; Schmidt-Chanasit, Jonas; Grobusch, Martin P.; de Lamballerie, Xavier; Drosten, Christian; Drexler, Jan Felix

2016-01-01

To examine the diagnostic performance of real-time reverse transcription (RT)-polymerase chain reaction (PCR) assays for Zika virus detection. We compared seven published real-time RT-PCR assays and two new assays that we have developed. To determine the analytical sensitivity of each assay, we

The great tsunami of 26 December 2004: A description based on tide gauge data from Indian subcontinent and surrounding areas

Digital Repository Service at National Institute of Oceanography (India)

Nagarajan, B.; Suresh, I; Sundar, D.; Sharma, R; Lal, A.K.; Neetu, S.; Shenoi, S.S.C.; Shetye, S.R; Shankar, D.

-1 Earth Planets Space, 58, 211?215, 2006 The Great Tsunami of 26 December 2004: A description based on tide-gauge data from the Indian subcontinent and surrounding areas B. Nagarajan1, I. Suresh2, D. Sundar2, R. Sharma1,A.K.Lal1, S. Neetu2, S. S. C. Shenoi..., I. Suresh, D. Sundar, R. Sharma, A. K. Lal, S. Neetu, S. S. C. Shenoi, S. R. Shetye, and D. Shankar (e-mail: shankar@nio.org) ...

Research and Development on Inhalation Toxicologic Evaluation of Red Phosphorus/Butyl Rubber Combustion Products. Phase 4

Science.gov (United States)

1986-11-15

with Alizarin Red S or other suitable mordant dyes . References 1. Pucritier H and Meloar. SN. Demonstration o; phosphates in calcium deposits: A...mixed with Bio-Rad Protein Assay Dye Reagent and the optical density read at 595 nm in a spectrophotometer. Protein concentrations of AM lysates were...of concentration or concentration by recovery interaction, post hoc comparisons were made using Dunnett’s test. Per cent BC data were natural log

Thyroglobulin (Tg) Testing Revisited: Tg Assays, TgAb Assays, and Correlation of Results With Clinical Outcomes.

Science.gov (United States)

Netzel, Brian C; Grebe, Stefan K G; Carranza Leon, B Gisella; Castro, M Regina; Clark, Penelope M; Hoofnagle, Andrew N; Spencer, Carole A; Turcu, Adina F; Algeciras-Schimnich, Alicia

2015-08-01

Measurement of thyroglobulin (Tg) by mass spectrometry (Tg-MS) is emerging as a tool for accurate Tg quantification in patients with anti-Tg autoantibodies (TgAbs). The objective of the study was to perform analytical and clinical evaluations of two Tg-MS assays in comparison with immunometric Tg assays (Tg-IAs) and Tg RIAs (Tg-RIAs) in a cohort of thyroid cancer patients. A total of 589 samples from 495 patients, 243 TgAb-/252 TgAb+, were tested by Beckman, Roche, Siemens-Immulite, and Thermo-Brahms Tg and TgAb assays, two Tg-RIAs, and two Tg-MS assays. The frequency of TgAb+ was 58%, 41%, 27%, and 39% for Roche, Beckman, Siemens-Immulite, and Thermo-Brahms, respectively. In TgAb- samples, clinical sensitivities and specificities of 100% and 74%-100%, respectively, were observed across all assays. In TgAb+ samples, all Tg-IAs demonstrated assay-dependent Tg underestimation, ranging from 41% to 86%. In TgAb+ samples, the use of a common cutoff (0.5 ng/mL) for the Tg-MS, three Tg-IAs, and the USC-RIA improved the sensitivity for the Tg-MSs and Tg-RIAs when compared with the Tg-IAs. In up to 20% of TgAb+ cases, Tg-IAs failed to detect Tg that was detectable by Tg-MS. In Tg-RIAs false-high biases were observed in TgAb+ samples containing low Tg concentrations. Tg-IAs remain the method of choice for Tg quantitation in TgAb- patients. In TgAb+ patients with undetectable Tg by immunometric assay, the Tg-MS will detect Tg in up to 20% additional cases. The Tg-RIA will detect Tg in approximately 35% cases, but a significant proportion of these will be clinical false-positive results. The undetectable Tg-MS seen in approximately 40% of TgAb+ cases in patients with disease need further evaluation.

Development of chitosan oleate ionic micelles loaded with silver sulfadiazine to be associated with platelet lysate for application in wound healing.

Science.gov (United States)

Dellera, Eleonora; Bonferoni, Maria Cristina; Sandri, Giuseppina; Rossi, Silvia; Ferrari, Franca; Del Fante, Claudia; Perotti, Cesare; Grisoli, Pietro; Caramella, Carla

2014-11-01

In the treatment of chronic wounds, topical application of anti-infective drugs such as silver sulfadiazine (AgSD) is of primary importance to avoid infections and accelerate wound repair. AgSD is used in burns and chronic wounds for its wide antibacterial spectrum, but presents limitations due to poor solubility and cytotoxicity. In the present work polymeric micelles obtained by self-assembling of chitosan ionically modified by interaction with oleic acid were developed as carriers for AgSD to overcome the drawbacks of the drug. The AgSD loaded micelles were intended to be associated in wound healing with platelet lysate (PL), a hemoderivative rich in growth factors. Unloaded micelles demonstrated good compatibility with both fibroblasts and PL. The relevance of chitosan concentration and of the ratio between chitosan and oleic acid to the drug loading and the particle size of nanoparticles was studied. A marked increase (up to 100 times with respect to saturated solution) of AgSD concentration in micelle dispersion was obtained. Moreover, the encapsulation reduced the cytotoxic effect of the drug towards fibroblasts and the drug incompatibility with PDGF-AB (platelet derived growth factor), chosen as representative of platelet growth factors. Copyright © 2014. Published by Elsevier B.V.

Assessment and reduction of comet assay variation in relation to DNA damage: studies from the European Comet Assay Validation Group

DEFF Research Database (Denmark)

Møller, Peter; Möller, Lennart; Godschalk, Roger W L

2010-01-01

The alkaline single cell gel electrophoresis (comet) assay has become a widely used method for the detection of DNA damage and repair in cells and tissues. Still, it has been difficult to compare results from different investigators because of differences in assay conditions and because the data...... are reported in different units. The European Comet Assay Validation Group (ECVAG) was established for the purpose of validation of the comet assay with respect to measures of DNA damage formation and its repair. The results from this inter-laboratory validation trail showed a large variation in measured level...... reliability for the measurement of DNA damage by the comet assay but there is still a need for further validation to reduce both assay and inter-laboratory variation....

A European multicientre study on the comparison of HIV-1 viral loads between VERIS HIV-1 Assay and Roche COBAS® TAQMAN® HIV-1 test, Abbott RealTime HIV-1 Assay, and Siemens VERSANT HIV-1 Assay.

Science.gov (United States)

Braun, Patrick; Delgado, Rafael; Drago, Monica; Fanti, Diana; Fleury, Hervé; Hofmann, Jörg; Izopet, Jacques; Kühn, Sebastian; Lombardi, Alessandra; Mancon, Alessandro; Marcos, Mª Angeles; Mileto, Davide; Sauné, Karine; O'Shea, Siobhan; Pérez-Rivilla, Alfredo; Ramble, John; Trimoulet, Pascale; Vila, Jordi; Whittaker, Duncan; Artus, Alain; Rhodes, Daniel

2017-07-01

Viral load monitoring is essential for patients under treatment for HIV. Beckman Coulter has developed the VERIS HIV-1 Assay for use on the novel, automated DxN VERIS Molecular Diagnostics System. ¥ OBJECTIVES: Evaluation of the clinical performance of the new quantitative VERIS HIV-1 Assay at multiple EU laboratories. Method comparison with the VERIS HIV-1 Assay was performed with 415 specimens at 5 sites tested with COBAS ® AmpliPrep/COBAS ® TaqMan ® HIV-1 Test, v2.0, 169 specimens at 3 sites tested with RealTime HIV-1 Assay, and 202 specimens from 2 sites tested with VERSANT HIV-1 Assay. Patient monitoring sample results from 4 sites were also compared. Bland-Altman analysis showed the average bias between VERIS HIV-1 Assay and COBAS HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay to be 0.28, 0.39, and 0.61 log 10 cp/mL, respectively. Bias at low end levels below 1000cp/mL showed predicted bias to be <0.3 log 10 cp/mL for VERIS HIV-1 Assay versus COBAS HIV-1 Test and RealTime HIV-1 Assay, and <0.5 log 10 cp/mL versus VERSANT HIV-1 Assay. Analysis on 174 specimens tested with the 0.175mL volume VERIS HIV-1 Assay and COBAS HIV-1 Test showed average bias of 0.39 log 10 cp/mL. Patient monitoring results using VERIS HIV-1 Assay demonstrated similar viral load trends over time to all comparators. The VERIS HIV-1 Assay for use on the DxN VERIS System demonstrated comparable clinical performance to COBAS ® HIV-1 Test, RealTime HIV-1 Assay, and VERSANT HIV-1 Assay. Copyright © 2017 Elsevier B.V. All rights reserved.

Immune chromatography: a quantitative radioimmunological assay

International Nuclear Information System (INIS)

Davis, J.W.; Demetriades, M.; Bowen, J.M.

1984-01-01

Immune chromatography, a radioimmunological binding assay, employs paper chromatography to separate immune complexes from free antigen and antibodies. During chromatography free antigen and antibodies become distributed throughout the paper, while immune complexes remain near the bottoms of the strips. The chromatographic differences can be made quantitative by using either iodinated antigens or antibodies. Under these conditions nanogram quantities of antigen can be detected or antibodies in sera diluted several 1000-fold. The immune chromatography assay can also be performed as an indirect assay, since the paper strips are cut from nitrocellulose paper. In this case the immune components are absorbed by the paper during chromatography. Antigen is then detected with an iodinated second antibody. The indirect immune chromatography assay is particularly useful for identifying different sera that react with the same antigen. Reaction with the first serum before chromatography reduces the amount of antigen available to the second serum following chromatography. In addition to characterizing the immune chromatography procedure, we discuss the possible applications of chromatography assays for the quantitation of other types of molecular binding interactions. (Auth.)

Introducing MINA--The Molecularly Imprinted Nanoparticle Assay.

Science.gov (United States)

Shutov, Roman V; Guerreiro, Antonio; Moczko, Ewa; de Vargas-Sansalvador, Isabel Perez; Chianella, Iva; Whitcombe, Michael J; Piletsky, Sergey A

2014-03-26

A new ELISA- (enzyme-linked immunosorbent assay)-like assay is demonstrated in which no elements of biological origin are used for molecular recognition or signaling. Composite imprinted nanoparticles that contain a catalytic core and which are synthesized by using a solid-phase approach can simultaneously act as recognition/signaling elements, and be used with minimal modifications to standard assay protocols. This assay provides a new route towards replacement of unstable biomolecules in immunoassays. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rover waste assay system

Energy Technology Data Exchange (ETDEWEB)

Akers, D.W.; Stoots, C.M.; Kraft, N.C.; Marts, D.J. [Idaho National Engineering Lab., Idaho Falls, ID (United States)

1997-11-01

The Rover Waste Assay System (RWAS) is a nondestructive assay system designed for the rapid assay of highly-enriched {sup 235}U contaminated piping, tank sections, and debris from the Rover nuclear rocket fuel processing facility at the Idaho Chemical Processing Plant. A scanning system translates a NaI(Tl) detector/collimator system over the structural components where both relative and calibrated measurements for {sup 137}Cs are made. Uranium-235 concentrations are in operation and is sufficiently automated that most functions are performed by the computer system. These functions include system calibration, problem identification, collimator control, data analysis, and reporting. Calibration of the system was done through a combination of measurements on calibration standards and benchmarked modeling. A description of the system is presented along with the methods and uncertainties associated with the calibration and analysis of the system for components from the Rover facility. 4 refs., 2 figs., 4 tabs.

Assay system

International Nuclear Information System (INIS)

Patzke, J.B.; Rosenberg, B.J.

1984-01-01

The accuracy of assays for monitoring concentrations of basic drugs in biological fluids containing a 1 -acid glycoproteins, such as blood (serum or plasma), is improved by the addition of certain organic phosphate compounds to minimize the ''protein effect.'' Kits containing the elements of the invention are also disclosed

Medical Devices; Immunology and Microbiology Devices; Classification of the Assayed Quality Control Material for Clinical Microbiology Assays. Final order.

Science.gov (United States)

2017-07-27

The Food and Drug Administration (FDA, Agency, or we) is classifying the assayed quality control material for clinical microbiology assays into class II (special controls). The special controls that will apply to the device are identified in this order and will be part of the codified language for the assayed quality control material for clinical microbiology assays' classification. The Agency is classifying the device into class II (special controls) to provide a reasonable assurance of safety and effectiveness of the device.

Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress.

Science.gov (United States)

Homayouni Moghadam, Farshad; Tayebi, Tahereh; Moradi, Alireza; Nadri, Hamid; Barzegar, Kazem; Eslami, Gilda

2014-01-01

By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm(2)) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm(2)) for a long time (24 hours) or high tension (10 dyn/cm(2)) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel.

Capillary Electrophoresis Analysis of Conventional Splicing Assays

DEFF Research Database (Denmark)

de Garibay, Gorka Ruiz; Acedo, Alberto; García-Casado, Zaida

2014-01-01

of these assays is often challenging. Here, we explore this issue by conducting splicing assays in 31 BRCA2 genetic variants. All variants were assessed by RT-PCR followed by capillary electrophoresis and direct sequencing. If assays did not produce clear-cut outputs (Class-2 or Class-5 according to analytical...

Controlling variation in the comet assay

Directory of Open Access Journals (Sweden)

Andrew Richard Collins

2014-10-01

Full Text Available Variability of the comet assay is a serious issue, whether it occurs from experiment to experiment in the same laboratory, or between different laboratories analysing identical samples. Do we have to live with high variability, just because the comet assay is a biological assay rather than analytical chemistry? Numerous attempts have been made to limit variability by standardising the assay protocol, and the critical steps in the assay have been identified; agarose concentration, duration of alkaline incubation, and electrophoresis conditions (time, temperature and voltage gradient are particularly important. Even when these are controlled, variation seems to be inevitable. It is helpful to include in experiments reference standards, i.e. cells with a known amount of specific damage to the DNA. They can be aliquots frozen from a single large batch of cells, either untreated (negative controls or treated with, for example, H2O2 or X-rays to induce strand breaks (positive control for the basic assay, or photosensitiser plus light to oxidise guanine (positive control for Fpg- or OGG1-sensitive sites. Reference standards are especially valuable when performing a series of experiments over a long period - for example, analysing samples of white blood cells from a large human biomonitoring trial - to check that the assay is performing consistently, and to identify anomalous results necessitating a repeat experiment. The reference values of tail intensity can also be used to iron out small variations occurring from day to day. We present examples of the use of reference standards in human trials, both within one laboratory and between different laboratories, and describe procedures that can be used to control variation.

The effect of Platelet Lysate on osteoblast proliferation associated with a transient increase of the inflammatory response in bone regeneration.

Science.gov (United States)

Ruggiu, Alessandra; Ulivi, Valentina; Sanguineti, Francesca; Cancedda, Ranieri; Descalzi, Fiorella

2013-12-01

Platelet Lysate (PL) contains a cocktail of growth factors and cytokines, which actively participates in tissue repair and its clinical application has been broadly described. The aim of this study was to assess the regenerative potential of PL for bone repair. We demonstrated that PL stimulation induces a transient increase of the inflammatory response in quiescent human osteoblasts, via NF-kB activation, COX-2 induction, PGE2 production and secretion of pro-inflammatory cytokines. Furthermore, we showed that long-term PL stimulation enhances proliferation of actively replicating osteoblasts, without affecting their differentiation potential, along with changes of cell morphology, resulting in increased cell density at confluence. In confluent resting osteoblasts, PL treatment induced resumption of proliferation, change in cell morphology and increase of cell density at confluence. A burst of PL treatment (24-h) was sufficient to trigger such processes in both conditions. These results correlated with up-regulation of the proliferative and survival pathways ERKs and Akt and with cell cycle re-activation via induction of CyclinD1 and phosphorylation of Rb, following PL stimulation. Our findings demonstrate that PL treatment results in activation and expansion of resting osteoblasts, without affecting their differentiation potential. Therefore PL represents a good therapeutic candidate in regenerative medicine for bone repair. Copyright © 2013 Elsevier Ltd. All rights reserved.

Investigation of the bacteriophage community in induced lysates of undefined mesophilic mixed-strain DL-cultures using classical and metagenomic approaches.

Science.gov (United States)

Muhammed, Musemma K; Olsen, Mette L; Kot, Witold; Neve, Horst; Castro-Mejía, Josué L; Janzen, Thomas; Hansen, Lars H; Nielsen, Dennis S; Sørensen, Søren J; Heller, Knut J; Vogensen, Finn K

2018-05-02

To investigate the notion that starter cultures can be a reservoir of bacteriophages (phages) in the dairy environment, strains of three DL-starters (undefined mesophilic mixed-strain starters containing Lactococcus lactis subsp. lactis biovar. diacetylactis and Leuconostoc species) were selected and induced by mitomycin C, and the whole starters were induced spontaneously as well as by mitomycin C. Frequency of induction of 17%, 26% and 12% was estimated among the isolates of the three starters, with majority of the induced phages mostly showing morphological similarity to known P335 phages, and with a fraction of them showing atypical features. Sequences of P335 quasi-species phages were found to be the most frequent entities in almost all metaviromes derived from the induced lysates. However, sequences of Sk1virus phages (previously 936 phages) were emerged as the predominant entities following spontaneous induction of one of the starters, suggesting a phage-carrier state. Sequences of other phages such as 949, 1706, C2virus (previously c2 phages) and Leuconostoc species could also be observed but with a lower relative frequency. Taken together, the majority of the P335 quasi-species phages could represent the induced viral community of the starters and the remaining phage groups mainly represent the background ambient viral community. Copyright © 2018 Elsevier B.V. All rights reserved.

Evidence that the primary effect of phosphorylation of eukaryotic initiation factor 2(alpha) in rabbit reticulocyte lysate is inhibition of the release of eukaryotic initiation factor-2.GDP from 60 S ribosomal subunits

International Nuclear Information System (INIS)

Gross, M.; Redman, R.; Kaplansky, D.A.

1985-01-01

The phosphorylation of eukaryotic initiation factor (eIF) 2 alpha that occurs when rabbit reticulocyte lysate is incubated in the absence of hemin or with poly(I.C) causes inhibition of polypeptide chain initiation by preventing a separate factor (termed RF) from promoting the exchange of GTP for GDP on eIF-2. When lysate was incubated in the presence of hemin and [ 14 C] eIF-2 or [alpha- 32 P]GTP, the authors observed binding of eIF-2 and GDP or GTP to 60 S ribosomal subunits that was slightly greater than that bound to 40 S subunits and little binding to 80 S ribosomes. When incubation was in the absence of hemin or in the presence of hemin plus 0.1 microgram/ml poly(I.C), eIF-2 and GDP binding to 60 S subunits was increased 1.5- to 2-fold, that bound to 80 S ribosomes was almost as great as that bound to 60 S subunits, and that bound to 40 S subunits was unchanged. The data indicate that about 40% of the eIF-2 that becomes bound to 60 S subunits and 80 S ribosomes in the absence of hemin or with poly(I.C) is eIF-2(alpha-P) and suggest that the eIF-2 and GDP bound is probably in the form of a binary complex. The rate of turnover of GDP (presumably eIF-2.GDP) on 60 S subunits and 80 S ribosomes in the absence of hemin is reduced to less than 10% the control rate, because the dissociation of eIF-2.GDP is inhibited. Our findings suggest that eIF-2.GTP binding to and eIF-2.GDP release from 60 S subunits may normally occur and serve to promote subunit joining

233U Assay A Neutron NDA System

International Nuclear Information System (INIS)

Hensley, D.C.; Lucero, A.J.; Pierce, L.

1998-01-01

The assay of highly enriched 233 U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched 235 U do not convert easily over to the assay of 233 U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with γ ray isotopics information should give a good overall determination of 233 U material now stored in bldg. 3019 at the Oak Ridge National Laboratory

MCNP efficiency calculations of INEEL passive active neutron assay system for simulated TRU waste assays

International Nuclear Information System (INIS)

Yoon, W.Y.; Meachum, T.R.; Blackwood, L.G.; Harker, Y.D.

2000-01-01

The Idaho National Engineering and Environmental Laboratory Stored Waste Examination Pilot Plant (SWEPP) passive active neutron (PAN) radioassay system is used to certify transuranic (TRU) waste drums in terms of quantifying plutonium and other TRU element activities. Depending on the waste form involved, significant systematic and random errors need quantification in addition to the counting statistics. To determine the total uncertainty of the radioassay results, a statistical sampling and verification approach has been developed. In this approach, the total performance of the PAN nondestructive assay system is simulated using the computer models of the assay system, and the resultant output is compared with the known input to assess the total uncertainty. The supporting steps in performing the uncertainty analysis for the passive assay measurements in particular are as follows: (1) Create simulated waste drums and associated conditions; (2) Simulate measurements to determine the basic counting data that would be produced by the PAN assay system under the conditions specified; and (3) Apply the PAN assay system analysis algorithm to the set of counting data produced by simulating measurements to determine the measured plutonium mass. The validity of this simulation approach was verified by comparing simulated output against results from actual measurements using known plutonium sources and surrogate waste drums. The computer simulation of the PAN system performance uses the Monte Carlo N-Particle (MCNP) Code System to produce a neutron transport calculation for a simulated waste drum. Specifically, the passive system uses the neutron coincidence counting technique, utilizing the spontaneous fission of 240 Pu. MCNP application to the SWEPP PAN assay system uncertainty analysis has been very useful for a variety of waste types contained in 208-ell drums measured by a passive radioassay system. The application of MCNP to the active radioassay system is also feasible

Computer-determined assay time based on preset precision

International Nuclear Information System (INIS)

Foster, L.A.; Hagan, R.; Martin, E.R.; Wachter, J.R.; Bonner, C.A.; Malcom, J.E.

1994-01-01

Most current assay systems for special nuclear materials (SNM) operate on the principle of a fixed assay time which provides acceptable measurement precision without sacrificing the required throughput of the instrument. Waste items to be assayed for SNM content can contain a wide range of nuclear material. Counting all items for the same preset assay time results in a wide range of measurement precision and wastes time at the upper end of the calibration range. A short time sample taken at the beginning of the assay could optimize the analysis time on the basis of the required measurement precision. To illustrate the technique of automatically determining the assay time, measurements were made with a segmented gamma scanner at the Plutonium Facility of Los Alamos National Laboratory with the assay time for each segment determined by counting statistics in that segment. Segments with very little SNM were quickly determined to be below the lower limit of the measurement range and the measurement was stopped. Segments with significant SNM were optimally assays to the preset precision. With this method the total assay time for each item is determined by the desired preset precision. This report describes the precision-based algorithm and presents the results of measurements made to test its validity

Serotype determination of Salmonella by xTAG assay.

Science.gov (United States)

Zheng, Zhibei; Zheng, Wei; Wang, Haoqiu; Pan, Jincao; Pu, Xiaoying

2017-10-01

Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens. Compared with the serotyping results of traditional serum agglutination test, the sensitivity and specificity of the xTAG assay were 95.1% and 100%, respectively. The agreement rate of these two assays was 95.2%. Compared with Luminex xMAP® Salmonella Serotyping Assay (SSA) kit, the advantages of this xTAG assay are: First, the magnetic beads make it applicable to both the Luminex®100/200™ and MAGPIX® systems. Second, only primers rather than both primers and probes are needed in the xTAG assay, and the process of coupling antigen-specific oligonucleotide probes to beads is circumvented, which make the xTAG assay convenient to be utilized by other laboratories. The xTAG assay may serve as a rapid alternative or complementary method for traditional Salmonella serotyping tests, especially for laboratories that utilize the MAGPIX® systems. Copyright © 2017 Elsevier B.V. All rights reserved.

The comet assay: ready for 30 more years.

Science.gov (United States)

Møller, Peter

2018-02-24

During the last 30 years, the comet assay has become widely used for the measurement of DNA damage and repair in cells and tissues. A landmark achievement was reached in 2016 when the Organization for Economic Co-operation and Development adopted a comet assay guideline for in vivo testing of DNA strand breaks in animals. However, the comet assay has much more to offer than being an assay for testing DNA strand breaks in animal organs. The use of repair enzymes increases the range of DNA lesions that can be detected with the assay. It can also be modified to measure DNA repair activity. Still, despite the long-term use of the assay, there is a need for studies that assess the impact of variation in specific steps of the procedure. This is particularly important for the on-going efforts to decrease the variation between experiments and laboratories. The articles in this Special Issue of Mutagenesis cover important technical issues of the comet assay procedure, nanogenotoxicity and ionising radiation sensitivity on plant cells. The included biomonitoring studies have assessed seasonal variation and certain predictors for the basal level of DNA damage in white blood cells. Lastly, the comet assay has been used in studies on genotoxicity of environmental and occupational exposures in human biomonitoring studies and animal models. Overall, the articles in this Special Issue demonstrate the versatility of the comet assay and they hold promise that the assay is ready for the next 30 years.

Assays for calcitonin receptors

International Nuclear Information System (INIS)

Teitelbaum, A.P.; Nissenson, R.A.; Arnaud, C.D.

1985-01-01

The assays for calcitonin receptors described focus on their use in the study of the well-established target organs for calcitonin, bone and kidney. The radioligand used in virtually all calcitonin binding studies is 125 I-labelled salmon calcitonin. The lack of methionine residues in this peptide permits the use of chloramine-T for the iodination reaction. Binding assays are described for intact bone, skeletal plasma membranes, renal plasma membranes, and primary kidney cell cultures of rats. Studies on calcitonin metabolism in laboratory animals and regulation of calcitonin receptors are reviewed

Identification of oxidized protein hydrolase as a potential prodrug target in prostate cancer

International Nuclear Information System (INIS)

McGoldrick, Christopher A; Jiang, Yu-Lin; Paromov, Victor; Brannon, Marianne; Krishnan, Koyamangalath; Stone, William L

2014-01-01

Esterases are often overexpressed in cancer cells and can have chiral specificities different from that of the corresponding normal tissues. For this reason, ester prodrugs could be a promising approach in chemotherapy. In this study, we focused on the identification and characterization of differentially expressed esterases between non-tumorigenic and tumorigenic prostate epithelial cells. Cellular lysates from LNCaP, DU 145, and PC3 prostate cancer cell lines, tumorigenic RWPE-2 prostate epithelial cells, and non-tumorigenic RWPE-1 prostate epithelial cells were separated by native polyacrylamide gel electrophoresis (n-PAGE) and the esterase activity bands visualized using α-naphthyl acetate or α-naphthyl-N-acetylalaninate (ANAA) chiral esters and Fast Blue RR salt. The esterases were identified using nanospray LC/MS-MS tandem mass spectrometry and confirmed by Western blotting, native electroblotting, inhibition assays, and activity towards a known specific substrate. The serine protease/esterase oxidized protein hydrolase (OPH) was overexpressed in COS-7 cells to verify our results. The major esterase observed with the ANAA substrates within the n-PAGE activity bands was identified as OPH. OPH (EC 3.4.19.1) is a serine protease/esterase and a member of the prolyl oligopeptidase family. We found that LNCaP lysates contained approximately 40% more OPH compared to RWPE-1 lysates. RWPE-2, DU145 and PC3 cell lysates had similar levels of OPH activity. OPH within all of the cell lysates tested had a chiral preference for the S-isomer of ANAA. LNCaP cells were stained more intensely with ANAA substrates than RWPE-1 cells and COS-7 cells overexpressing OPH were found to have a higher activity towards the ANAA and AcApNA than parent COS-7 cells. These data suggest that prodrug derivatives of ANAA and AcApNA could have potential as chemotherapeutic agents for the treatment of prostate cancer tumors that overexpress OPH

Silver nanoparticle biosynthesis from newly isolated streptomyces genus from soil

Science.gov (United States)

Osman Adiguzel, Ali; Könen Adiguzel, Serpil; Mazmanci, Birgül; Tunçer, Münir; Mazmanci, Mehmet Ali

2018-04-01

This study aimed to green synthesis of silver nanoparticles (AgNPs) by cell lysates of actinobacterial strains isolated from Mersin soils. Also, free-radical scavenging potential, the inhibitory effect, and genotoxicity of synthesized AgNPs were investigated. As a result of the screening study, it was detected that cell lysate from isolate AOA21, which were classified in Streptomyces genus according to 16S rRNA gene sequences comparison, showed higher potential for AgNPs synthesis. The optimum pH, AgNO3 and cell lysate concentration for AgNPs synthesis were found to be pH 9.0, 1 mM AgNO3 and 2-fold diluted cell lysate, respectively. The FESEM analysis revealed that the size and shape of AgNPs were 35–60 nm and spherical. The x-ray diffraction patterns displayed typical peaks of crystalline AgNPs at 34.07°, 44.04°, 64.45°, 77.40° and 81.36°. The size of cubic crystalline AgNPs was found to be 9.35 nm. The FTIR analysis showed that the especially protein, peptide and amino acid component in the cell lysates of Streptomyces sp. AOA21 may be responsible in reduction of AgNO3 and stabilization of synthesized AgNPs. The MIC values of synthesized AgNPs for Bacillus cereus, Klebsiella pneumoniae, Escherichia coli and Staphylococcus aureus were found to be 8 μg ml‑1, 16 μg ml‑1, 16 μg ml‑1 and 32 μg ml‑1, respectively. Free-radical scavenging activity of synthesized AgNPs was 8.54%–55.58% at a concentration range of 800–2000 μg ml‑1. Furthermore, comet assay showed that synthesized AgNPs did not cause significant DNA damage in Saccharomyces cerevisiae at a concentration of 12.5 μg ml‑1 and 25 μg ml‑1.

Integrated bioassays in microfluidic devices: botulinum toxin assays.

Science.gov (United States)

Mangru, Shakuntala; Bentz, Bryan L; Davis, Timothy J; Desai, Nitin; Stabile, Paul J; Schmidt, James J; Millard, Charles B; Bavari, Sina; Kodukula, Krishna

2005-12-01

A microfluidic assay was developed for screening botulinum neurotoxin serotype A (BoNT-A) by using a fluorescent resonance energy transfer (FRET) assay. Molded silicone microdevices with integral valves, pumps, and reagent reservoirs were designed and fabricated. Electrical and pneumatic control hardware were constructed, and software was written to automate the assay protocol and data acquisition. Detection was accomplished by fluorescence microscopy. The system was validated with a peptide inhibitor, running 2 parallel assays, as a feasibility demonstration. The small footprint of each bioreactor cell (0.5 cm2) and scalable fluidic architecture enabled many parallel assays on a single chip. The chip is programmable to run a dilution series in each lane, generating concentration-response data for multiple inhibitors. The assay results showed good agreement with the corresponding experiments done at a macroscale level. Although the system has been developed for BoNT-A screening, a wide variety of assays can be performed on the microfluidic chip with little or no modification.

Comet assay on mice testicular cells

Directory of Open Access Journals (Sweden)

Anoop Kumar Sharma

2015-05-01

Full Text Available Heritable mutations may result in a variety of adverse outcomes including genetic disease in the offspring. In recent years the focus on germ cell mutagenicity has increased and the “Globally Harmonized System of Classification and Labelling of Chemicals (GHS” has published classification criteria for germ cell mutagens (Speit et al., 2009. The in vivo Comet assay is considered a useful tool for investigating germ cell genotoxicity. In the present study DNA strand breaks in testicular cells of mice were investigated. Different classes of chemicals were tested in order to evaluate the sensitivity of the comet assay in testicular cells. The chemicals included environmentally relevant substances such as Bisphenol A, PFOS and Tetrabrombisphenol A. Statistical power calculations will be presented to aid in the design of future Comet assay studies on testicular cells. Power curves were provided with different fold changes in % tail DNA, different number of cells scored and different number of gels (Hansen et al., 2014. An example is shown in Figure 1. A high throughput version of the Comet assay was used. Samples were scored with a fully automatic comet assay scoring system that provided faster scoring of randomly selected cells.

Immunoradiometric assay for cytomegalovirus-specific IgG antibodies; Assay development and evaluation in blood transfusion practice

Energy Technology Data Exchange (ETDEWEB)

Klapper, P.E.; Cleator, G.M.; Prinja-Wolks, D.; Morris, D.J. (Medical School, Manchester (United Kingdom). Department of Medical microbiology, Virology Unit); Morell, G. (Regional Blood Transfusion Centre, manchester (United Kingdom))

1990-03-01

An immunoradiometric assay (radio-immunosorbent test; RIST) for the detection of IgG antibodies to human herpesvirus 4 (human cytomegalovirus (CMV)) has been developed. The technique utilizes CMV antigen passively adsorbed to a polyvinyl microtitration plate and a radiolabelled murine monoclonal anti-human IgG antibody to detect binding of human antibody to the 'solid phase' reagent. The assay was optimized, and its specifity confirmed by testing paired acute and convalescent sera from patients with acute CMV or other human herpesvirus infections. To determine the assay's sensitivity 1433 blood donor sera were examined. The RIST was more sensitive than a standard complement fixation (CFT). Use of a monoclonal anti-human IgG antibody in the RIST reduced non-specific binding to the control uninfected cell antigen such that blood donor sera could be tested in the assay using only a CMV antigen without generating an unacceptable false positive rate. (author). 23 refs.; 1 tab.

233U Assay A Neutron NDA System

Energy Technology Data Exchange (ETDEWEB)

Hensley, D.C.; Lucero, A.J.; Pierce, L.

1998-11-17

The assay of highly enriched {sup 233}U material presents some unique challenges. Techniques which apply to the assay of materials of Pu or enriched {sup 235}U do not convert easily over to the assay of {sup 233}U. A specialized neutron assay device is being fabricated to exploit the singles neutron signal, the weak correlated neutron signal, and an active correlated signal. These pieces of information when combined with {gamma} ray isotopics information should give a good overall determination of {sup 233}U material now stored in bldg. 3019 at the Oak Ridge National Laboratory.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

International Nuclear Information System (INIS)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

2015-01-01

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

Energy Technology Data Exchange (ETDEWEB)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei, E-mail: kwng@ntu.edu.sg; Loo, Say Chye Joachim, E-mail: joachimloo@ntu.edu.sg [Nanyang Technological University, School of Materials Science and Engineering (Singapore)

2015-01-15

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein–particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

Nanoparticle-assay marker interaction: effects on nanotoxicity assessment

Science.gov (United States)

Zhao, Xinxin; Xiong, Sijing; Huang, Liwen Charlotte; Ng, Kee Woei; Loo, Say Chye Joachim

2015-01-01

Protein-based cytotoxicity assays such as lactate dehydrogenase (LDH) and tumor necrosis factor-alpha (TNF-α) are commonly used in cytotoxic evaluation of nanoparticles (NPs) despite numerous reports on possible interactions with protein markers in these assays that can confound the results obtained. In this study, conventional cytotoxicity assays where assay markers may (LDH and TNF- α) or may not (PicoGreen and WST-8) come into contact with NPs were used to evaluate the cytotoxicity of NPs. The findings revealed selective interactions between negatively charged protein assay markers (LDH and TNF- α) and positively charged ZnO NPs under abiotic conditions. The adsorption and interaction with these protein assay markers were strongly influenced by surface charge, concentration, and specific surface area of the NPs, thereby resulting in less than accurate cytotoxic measurements, as observed from actual cell viability measurements. An improved protocol for LDH assay was, therefore, proposed and validated by eliminating any effects associated with protein-particle interactions. In view of this, additional measures and precautions should be taken when evaluating cytotoxicity of NPs with standard protein-based assays, particularly when they are of opposite charges.

Qualification of standard membrane-feeding assay with Plasmodium falciparum malaria and potential improvements for future assays.

Directory of Open Access Journals (Sweden)

Kazutoyo Miura

Full Text Available Vaccines that interrupt malaria transmission are of increasing interest and a robust functional assay to measure this activity would promote their development by providing a biologically relevant means of evaluating potential vaccine candidates. Therefore, we aimed to qualify the standard membrane-feeding assay (SMFA. The assay measures the transmission-blocking activity of antibodies by feeding cultured P. falciparum gametocytes to Anopheles mosquitoes in the presence of the test antibodies and measuring subsequent mosquito infection. The International Conference on Harmonisation (ICH Harmonised Tripartite Guideline Q2(R1 details characteristics considered in assay validation. Of these characteristics, we decided to qualify the SMFA for Precision, Linearity, Range and Specificity. The transmission-blocking 4B7 monoclonal antibody was tested over 6 feeding experiments at several concentrations to determine four suitable concentrations that were tested in triplicate in the qualification experiments (3 additional feeds to evaluate Precision, Linearity and Range. For Specificity, 4B7 was tested in the presence of normal mouse IgG. We determined intra- and inter-assay variability of % inhibition of mean oocyst intensity at each concentration of 4B7 (lower concentrations showed higher variability. We also showed that % inhibition was dependent on 4B7 concentration and the activity is specific to 4B7. Since obtaining empirical data is time-consuming, we generated a model using data from all 9 feeds and simulated the effects of different parameters on final readouts to improve the assay procedure and analytical methods for future studies. For example, we estimated the effect of number of mosquitoes dissected on variability of % inhibition, and simulated the relationship between % inhibition in oocyst intensity and % inhibition of prevalence of infected mosquitos at different mean oocysts in the control. SMFA is one of the few biological assays used in

Metal-amplified Density Assays, (MADAs), including a Density-Linked Immunosorbent Assay (DeLISA).

Science.gov (United States)

Subramaniam, Anand Bala; Gonidec, Mathieu; Shapiro, Nathan D; Kresse, Kayleigh M; Whitesides, George M

2015-02-21

This paper reports the development of Metal-amplified Density Assays, or MADAs - a method of conducting quantitative or multiplexed assays, including immunoassays, by using Magnetic Levitation (MagLev) to measure metal-amplified changes in the density of beads labeled with biomolecules. The binding of target analytes (i.e. proteins, antibodies, antigens) to complementary ligands immobilized on the surface of the beads, followed by a chemical amplification of the binding in a form that results in a change in the density of the beads (achieved by using gold nanoparticle-labeled biomolecules, and electroless deposition of gold or silver), translates analyte binding events into changes in density measureable using MagLev. A minimal model based on diffusion-limited growth of hemispherical nuclei on a surface reproduces the dynamics of the assay. A MADA - when performed with antigens and antibodies - is called a Density-Linked Immunosorbent Assay, or DeLISA. Two immunoassays provided a proof of principle: a competitive quantification of the concentration of neomycin in whole milk, and a multiplexed detection of antibodies against Hepatitis C virus NS3 protein and syphilis T. pallidum p47 protein in serum. MADAs, including DeLISAs, require, besides the requisite biomolecules and amplification reagents, minimal specialized equipment (two permanent magnets, a ruler or a capillary with calibrated length markings) and no electrical power to obtain a quantitative readout of analyte concentration. With further development, the method may be useful in resource-limited or point-of-care settings.

Chromosome aberration assays in Allium

Energy Technology Data Exchange (ETDEWEB)

Grant, W.F.

1982-01-01

The common onion (Allium cepa) is an excellent plant for the assay of chromosome aberrations after chemical treatment. Other species of Allium (A. cepa var. proliferum, A. carinatum, A. fistulosum and A. sativum) have also been used but to a much lesser extent. Protocols have been given for using root tips from either bulbs or seeds of Allium cepa to study the cytological end-points, such as chromosome breaks and exchanges, which follow the testing of chemicals in somatic cells. It is considered that both mitotic and meiotic end-points should be used to a greater extent in assaying the cytogenetic effects of a chemical. From a literature survey, 148 chemicals are tabulated that have been assayed in 164 Allium tests for their clastogenic effect. Of the 164 assays which have been carried out, 75 are reported as giving a positive reaction, 49 positive and with a dose response, 1 positive and temperature-related, 9 borderline positive, and 30 negative; 76% of the chemicals gave a definite positive response. It is proposed that the Allium test be included among those tests routinely used for assessing chromosomal damage induced by chemicals.

Principles of validation of diagnostic assays for infectious diseases

International Nuclear Information System (INIS)

Jacobson, R.H.

1998-01-01

Assay validation requires a series of inter-related processes. Assay validation is an experimental process: reagents and protocols are optimized by experimentation to detect the analyte with accuracy and precision. Assay validation is a relative process: its diagnostic sensitivity and diagnostic specificity are calculated relative to test results obtained from reference animal populations of known infection/exposure status. Assay validation is a conditional process: classification of animals in the target population as infected or uninfected is conditional upon how well the reference animal population used to validate the assay represents the target population; accurate predictions of the infection status of animals from test results (PV+ and PV-) are conditional upon the estimated prevalence of disease/infection in the target population. Assay validation is an incremental process: confidence in the validity of an assay increases over time when use confirms that it is robust as demonstrated by accurate and precise results; the assay may also achieve increasing levels of validity as it is upgraded and extended by adding reference populations of known infection status. Assay validation is a continuous process: the assay remains valid only insofar as it continues to provide accurate and precise results as proven through statistical verification. Therefore, the work required for validation of diagnostic assays for infectious diseases does not end with a time-limited series of experiments based on a few reference samples rather, to assure valid test results from an assay requires constant vigilance and maintenance of the assay, along with reassessment of its performance characteristics for each unique population of animals to which it is applied. (author)

Analysis of the Effects of Normal Walking on Ankle Joint Contact Characteristics After Acute Inversion Ankle Sprain.

Science.gov (United States)

Bae, Ji Yong; Park, Kyung Soon; Seon, Jong Keun; Jeon, Insu

2015-12-01

To show the causal relationship between normal walking after various lateral ankle ligament (LAL) injuries caused by acute inversion ankle sprains and alterations in ankle joint contact characteristics, finite element simulations of normal walking were carried out using an intact ankle joint model and LAL injury models. A walking experiment using a volunteer with a normal ankle joint was performed to obtain the boundary conditions for the simulations and to support the appropriateness of the simulation results. Contact pressure and strain on the talus articular cartilage and anteroposterior and mediolateral translations of the talus were calculated. Ankles with ruptured anterior talofibular ligaments (ATFLs) had a higher likelihood of experiencing increased ankle joint contact pressures, strains and translations than ATFL-deficient ankles. In particular, ankles with ruptured ATFL + calcaneofibular ligaments and all ruptured ankles had a similar likelihood as the ATFL-ruptured ankles. The push off stance phase was the most likely situation for increased ankle joint contact pressures, strains and translations in LAL-injured ankles.

Factor IX assay

Science.gov (United States)

... this page: //medlineplus.gov/ency/article/003679.htm Factor IX assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

Factor VIII assay

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... this page: //medlineplus.gov/ency/article/003678.htm Factor VIII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

Factor II assay

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... this page: //medlineplus.gov/ency/article/003674.htm Factor II assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

Factor VII assay

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... this page: //medlineplus.gov/ency/article/003676.htm Factor VII assay To use the sharing features on ... M. is also a founding member of Hi-Ethics and subscribes to the principles of the Health ...

A high-throughput 2D-analytical technique to obtain single protein parameters from complex cell lysates for in silico process development of ion exchange chromatography.

Science.gov (United States)

Kröner, Frieder; Elsäßer, Dennis; Hubbuch, Jürgen

2013-11-29

The accelerating growth of the market for biopharmaceutical proteins, the market entry of biosimilars and the growing interest in new, more complex molecules constantly pose new challenges for bioseparation process development. In the presented work we demonstrate the application of a multidimensional, analytical separation approach to obtain the relevant physicochemical parameters of single proteins in a complex mixture for in silico chromatographic process development. A complete cell lysate containing a low titre target protein was first fractionated by multiple linear salt gradient anion exchange chromatography (AEC) with varying gradient length. The collected fractions were subsequently analysed by high-throughput capillary gel electrophoresis (HT-CGE) after being desalted and concentrated. From the obtained data of the 2D-separation the retention-volumes and the concentration of the single proteins were determined. The retention-volumes of the single proteins were used to calculate the related steric-mass action model parameters. In a final evaluation experiment the received parameters were successfully applied to predict the retention behaviour of the single proteins in salt gradient AEC. Copyright © 2013 Elsevier B.V. All rights reserved.

Multiplexing a high-throughput liability assay to leverage efficiencies.

Science.gov (United States)

Herbst, John; Anthony, Monique; Stewart, Jeremy; Connors, David; Chen, Taosheng; Banks, Martyn; Petrillo, Edward W; Agler, Michele

2009-06-01

In order to identify potential cytochrome P-450 3A4 (drug-metabolizing enzyme) inducers at an early stage of the drug discovery process, a cell-based transactivation high-throughput luciferase reporter assay for the human pregnane X receptor (PXR) in HepG2 cells has been implemented and multiplexed with a viability end point for data interpretation, as part of a Lead Profiling portfolio of assays. As a routine part of Lead Profiling operations, assays are periodically evaluated for utility as well as for potential improvements in technology or process. We used a recent evaluation of our PXR-transactivation assay as a model for the application of Lean Thinking-based process analysis to lab-bench assay optimization and automation. This resulted in the development of a 384-well multiplexed homogeneous assay simultaneously detecting PXR transactivation and HepG2 cell cytotoxicity. In order to multiplex fluorescent and luminescent read-outs, modifications to each assay were necessary, which included optimization of multiple assay parameters such as cell density, plate type, and reagent concentrations. Subsequently, a set of compounds including known cytotoxic compounds and PXR inducers were used to validate the multiplexed assay. Results from the multiplexed assay correlate well with those from the singleplexed assay formats measuring PXR transactivation and viability separately. Implementation of the multiplexed assay for routine compound profiling provides improved data quality, sample conservation, cost savings, and resource efficiencies.

Plasma concentrations of alpha-melanocyte-stimulating hormone are elevated in patients on chronic haemodialysis.

Science.gov (United States)

Airaghi, L; Garofalo, L; Cutuli, M G; Delgado, R; Carlin, A; Demitri, M T; Badalamenti, S; Graziani, G; Lipton, J M; Catania, A

2000-08-01

Clinical and/or laboratory signs of systemic inflammation occur frequently in patients undergoing long-term haemodialysis. It is likely, therefore, that a compensatory release of endogenous anti-inflammatory molecules occurs to limit host reactions. The aim of the present research was to determine if the potent anti-inflammatory peptide alpha-melanocyte-stimulating hormone (alpha-MSH), a pro-opiomelanocortin derivative, is increased in plasma of haemodialysis patients. Because endotoxin and cytokines induce alpha-MSH in vivo and in vitro, we also measured plasma concentrations of endotoxin, interleukin-6 (IL-6), and tumour necrosis factor alpha (TNF-alpha), and the two circulating products of activated monocytes, nitric oxide (NO) and neopterin. Thirty-five chronic haemodialysis patients, 20 patients with chronic renal failure not yet on dialysis, and 35 normal controls were included in the study. In the haemodialysis group, blood samples were obtained before and at the end of a dialysis session. Plasma alpha-MSH was measured using a double antibody radioimmunoassay, and IL-6, TNF-alpha, and neopterin using specific enzyme-linked immunosorbent assays. Plasma nitrites were determined by a colorimetric method, and endotoxin with the quantitative chromogenic LAL (limulus amoebocyte lysate) method. Mean plasma alpha-MSH was higher in haemodialysis patients than in control subjects, with the peptide concentrations being particularly elevated in dialysed patients with detectable endotoxin. High alpha-MSH concentrations were observed in the pre-dialysis samples, with no substantial change at the end of the dialysis session. Plasma concentrations of IL-6, TNF-alpha, neopterin, and NO were generally elevated in chronic haemodialysis patients and there was a negative correlation between circulating alpha-MSH and IL-6. In patients with renal failure not yet on dialysis, mean plasma alpha-MSH was similar to that of normal subjects. alpha-MSH is increased in the circulation of

Evaluation of Presto(plus) assay and LightMix kit Trichomonas vaginalis assay for detection of Trichomonas vaginalis in dry vaginal swabs.

Science.gov (United States)

de Waaij, Dewi J; Ouburg, Sander; Dubbink, Jan Henk; Peters, Remco P H; Morré, Servaas A

2016-08-01

This is an evaluation study of the Presto(plus) Assay for T. vaginalis by comparing to the TIB MOLBIOL LightMix Kit Trichomonas vaginalis Assay using 615 dry collected vaginal and rectal swabs. Discordant samples were analyzed by the Qiagen® Microbial DNA qPCR for TV Assay. Both assays showed comparable performances (McNemar p>0.05). Copyright © 2016 Elsevier B.V. All rights reserved.

Nondestructive assay methods for irradiated nuclear fuels

International Nuclear Information System (INIS)

Hsue, S.T.; Crane, T.W.; Talbert, W.L. Jr.; Lee, J.C.

1978-01-01

This report is a review of the status of nondestructive assay (NDA) methods used to determine burnup and fissile content of irradiated nuclear fuels. The gamma-spectroscopy method measures gamma activities of certain fission products that are proportional to the burnup. Problems associated with this method are migration of the fission products and gamma-ray attenuation through the relatively dense fuel material. The attenuation correction is complicated by generally unknown activity distributions within the assemblies. The neutron methods, which usually involve active interrogation and prompt or delayed signal counting, are designed to assay the fissile content of the spent-fuel elements. Systems to assay highly enriched spent-fuel assemblies have been tested extensively. Feasibility studies have been reported of systems to assay light-water reactor spent-fuel assemblies. The slowing-down spectrometer and neutron resonance absorption methods can distinguish between the uranium and plutonium fissile contents, but they are limited to the assay of individual rods. We have summarized the status of NDA techniques for spent-fuel assay and present some subjects in need of further investigation. Accuracy of the burnup calculations for power reactors is also reviewed

The microculture-kinetic (MiCK) assay: the role of a drug-induced apoptosis assay in drug development and clinical care.

Science.gov (United States)

Bosserman, Linda; Prendergast, Franklyn; Herbst, Roy; Fleisher, Martin; Salom, Emery; Strickland, Steven; Raptis, Anastasios; Hallquist, Allan; Perree, Mathieu; Rajurkar, Swapnil; Karimi, Misagh; Rogers, Karl; Davidson, Dirk; Willis, Carl; Penalver, Manuel; Homesley, Howard; Burrell, Matthew; Garrett, Audrey; Rutledge, James; Chernick, Michael; Presant, Cary A

2012-08-15

A drug-induced apoptosis assay, termed the microculture-kinetic (MiCK) assay, has been developed. Blinded clinical trials have shown higher response rates and longer survival in groups of patients with acute myelocytic leukemia and epithelial ovarian cancer who have been treated with drugs that show high apoptosis in the MiCK assay. Unblinded clinical trials in multiple tumor types have shown that the assay will be used frequently by clinicians to determine treatment, and when used, results in higher response rates, longer times to relapse, and longer survivals. Model economic analyses suggest possible cost savings in clinical use based on increased generic drug use and single-agent substitution for combination therapies. Two initial studies with drugs in development are promising. The assay may help reduce costs and speed time to drug approval. Correlative studies with molecular biomarkers are planned. This assay may have a role both in personalized clinical therapy and in more efficient drug development. ©2012 AACR.

Bone Formation with Deproteinized Bovine Bone Mineral or Biphasic Calcium Phosphate in the Presence of Autologous Platelet Lysate: Comparative Investigation in Rabbit

Directory of Open Access Journals (Sweden)

Carole Chakar

2014-01-01

Full Text Available Bone substitutes alone or supplemented with platelet-derived concentrates are widely used to promote bone regeneration but their potency remains controversial. The aim of this study was, therefore, to compare the regenerative potential of preparations containing autologous platelet lysate (APL and particles of either deproteinized bovine bone mineral (DBBM or biphasic calcium phosphate (BCP, two bone substitutes with different resorption patterns. Rabbit APL was prepared by freeze-thawing a platelet suspension. Critical-size defects in rabbit femoral condyle were filled with DBBM or DBBM+APL and BCP or BCP+APL. Rabbits were sacrificed after six weeks and newly formed bone and residual implanted material were evaluated using nondemineralized histology and histomorphometry. New bone was observed around particles of all fillers tested. In the defects filled with BCP, the newly formed bone area was greater (70%; P<0.001 while the residual material area was lower (60%; P<0.001 than that observed in those filled with DBBM. New bone and residual material area of defects filled with either APL+DBBM or APL+BCP were similar to those observed in those filled with the material alone. In summary, osteoconductivity and resorption of BCP were greater than those of DBBM, while APL associated with either DBBM or BCP did not have an additional benefit.

Radioligand assay in reproductive biology

International Nuclear Information System (INIS)

Korenman, S.G.; Sherman, B.M.

1975-01-01

Radioligand assays have been developed for the principal reproductive steroids and peptide hormones. Specific binding reagents have included antibodies, plasma binders, and intracellular receptors. In each assay, problems of specificity, sensitivity, and nonspecific inhibitors were encountered. Many features of the endocrine physiology in childhood, during puberty, and in adulthood have been characterized. Hormonal evaluations of endocrine disorders of reproduction are characterized on the basis of their characteristic pathophysiologic alterations. (U.S.)

Thermometric enzyme linked immunosorbent assay: TELISA.

Science.gov (United States)

Mattiasson, B; Borrebaeck, C; Sanfridson, B; Mosbach, K

1977-08-11

A new method, thermometric enzyme linked immunosorbent assay (TELISA), for the assay of endogenous and exogenous compounds in biological fluids is described. It is based on the previously described enzyme linked immunosorbent assay technique, ELISA, but utilizes enzymic heat formation which is measured in an enzyme thermistor unit. In the model system studied determination of human serum albumin down to a concentration of 10(-10) M (5 ng/ml) was achieved, with both normal and catalase labelled human serum albumin competing for the binding sites on the immunosorbent, which was rabbit antihuman serum albumin immobilized onto Sepharose CL-4B.

Leishmania braziliensis: aislamiento de lesiones por inoculación de hámsteres con o sin adición de lisado de glándulas salivares de Lutzomyia youngi Leishmania braziliensis: isolation of lesions by inoculation of hamsters with and without the addition of salivary gland lysates of Lutzomyia youngi

Directory of Open Access Journals (Sweden)

Elina Rojas

1995-02-01

Full Text Available Homogeneizados de biopsias de lesiones cutáneas de 50 casos de leishmaniasis tegumentaria de Trujillo, Venezuela, han sido inoculados en hámsteres machos. Se ha comparado la infectvidad de Leishamania braziliensis, de homogeneizados simples, con la de los mezclados con lisado de glándula salival de Lutzomyia youngi, registrandose un 58,5% de infecciones para una media de 12 semanas de prepatencia con los homogeneizados simples, contra 92% de infecciones con una media de 3 semanas de prepatencia, cuando cada uno de los inóculos de homogeneizado se mezcló con lisado equivalente al de una glándula salival de flebótomo.Homogenized biopsy tissue from the cutaneous leishmaniasis lesions of 50 patients from Trujillo, Venezuela, were inoculated subcutaneously into the tarsi of male hamsters. Homogenized tissue either alone or mixed with salivary gland lysates of Lutzomyia youngi were used for inoculation. Homogenized tissue alone yielded 58.5% of infections with a mean of twelve weeks for prepatency, while those mixed with sandfly lysate resulted in 92% of infections with a mean prepatency of three weeks.

Use of enzyme-linked immunosorbent assay and dipstick assay for detection of Strongyloides stercoralis infection in humans

NARCIS (Netherlands)

van Doorn, H. Rogier; Koelewijn, Rob; Hofwegen, Henk; Gilis, Henk; Wetsteyn, Jose C. F. M.; Wismans, Pieter J.; Sarfati, Claudine; Vervoort, Tony; van Gool, Tom

2007-01-01

A homemade enzyme-linked immunosorbent assay (ELISA) (Academic Medical Center ELISA [AMC-ELISA]) and a dipstick assay for the detection of anti-Strongyloides stercoralis antibodies in serum were developed and evaluated together with two commercially available ELISAs (IVD-ELISA [IVD Research, Inc.

Parallel force assay for protein-protein interactions.

Science.gov (United States)

Aschenbrenner, Daniela; Pippig, Diana A; Klamecka, Kamila; Limmer, Katja; Leonhardt, Heinrich; Gaub, Hermann E

2014-01-01

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

Nondestructive assay of HTGR fuel rods

International Nuclear Information System (INIS)

Menlove, H.O.

1974-01-01

Performance characteristics of three different radioactive source NDA systems are compared for the assay of HTGR fuel rods and stacks of rods. These systems include the fast neutron Sb-Be assay system, the 252 Cf ''Shuffler,'' and the thermal neutron PAPAS assay system. Studies have been made to determinethe perturbation on the measurements from particle size, kernel Th/U ratio, thorium content, and hydrogen content. In addition to the total 235 U determination, the pellet-to-pellet or rod-to-rod uniformity of HTGR fuel rod stacks has been measured by counting the delayed gamma rays with a NaI through-hole in the PAPAS system. These measurements showed that rod substitutions can be detected easily in a fuel stack, and that detailed information is available on the loading variations in a uniform stack. Using a 1.0 mg 252 Cf source, assay rates of 2 to 4 rods/s are possible, thus facilitating measurement of 100 percent of a plant's throughput. (U.S.)

Barcoded microchips for biomolecular assays.

Science.gov (United States)

Zhang, Yi; Sun, Jiashu; Zou, Yu; Chen, Wenwen; Zhang, Wei; Xi, Jianzhong Jeff; Jiang, Xingyu

2015-01-20

Multiplexed assay of analytes is of great importance for clinical diagnostics and other analytical applications. Barcode-based bioassays with the ability to encode and decode may realize this goal in a straightforward and consistent manner. We present here a microfluidic barcoded chip containing several sets of microchannels with different widths, imitating the commonly used barcode. A single barcoded microchip can carry out tens of individual protein/nucleic acid assays (encode) and immediately yield all assay results by a portable barcode reader or a smartphone (decode). The applicability of a barcoded microchip is demonstrated by human immunodeficiency virus (HIV) immunoassays for simultaneous detection of three targets (anti-gp41 antibody, anti-gp120 antibody, and anti-gp36 antibody) from six human serum samples. We can also determine seven pathogen-specific oligonucleotides by a single chip containing both positive and negative controls.

Performance characteristics of the ARCHITECT anti-HCV assay.

Science.gov (United States)

Jonas, Gesa; Pelzer, Claudia; Beckert, Christian; Hausmann, Michael; Kapprell, Hans-Peter

2005-10-01

The ARCHITECT Anti-HCV assay is a fully automated high throughput chemiluminescent microparticle immunoassay (CMIA) for the detection of antibodies to structural and nonstructural proteins of the hepatitis C virus (HCV). To further enhance the performance of this test, the assay was modified to improve the specificity for blood donor specimens. The specificity of the enhanced ARCHITECT Anti-HCV assay was evaluated by screening blood donor samples randomly collected from various German blood banks, as well as hospitalized patient samples derived from Germany and the US. Additionally, antibody sensitivity was determined on commercially available anti-HCV seroconversion panels and on a commercially available worldwide anti-HCV genotype performance panel. Apparent specificity of the modified ARCHITECT Anti-HCV assay in a blood donor population consisting of 3811 specimens was 99.92%, compared to 99.76% for the current on-market assay. Additionally, antibody sensitivity was determined on commercially available anti-HCV seroconversion panels. Seroconversion sensitivity equivalent to or better than the current on-market product was observed by testing 33 seroconversion panels. This study demonstrates that the modified version of the ARCHITECT Anti-HCV assay shows improved specificity for blood donor specimens compared to the current assay on market without compromising sensitivity. With the availability of the improved ARCHITECT Anti-HCV assay and the recent launch of the ARCHITECT HIV Ag/Ab Combo assay, the ARCHITECT system now offers a full hepatitis/retrovirus menu with excellent performance on a high throughput, random access, automated analyzer, ideally suited for blood screening and diagnostic applications.

Capture of lipopolysaccharide (endotoxin) by the blood clot: a comparative study.

Science.gov (United States)

Armstrong, Margaret T; Rickles, Frederick R; Armstrong, Peter B

2013-01-01

In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot) and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse) operate to capture lipopolysaccharide (LPS, endotoxin). The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL) test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.

Capture of lipopolysaccharide (endotoxin by the blood clot: a comparative study.

Directory of Open Access Journals (Sweden)

Margaret T Armstrong

Full Text Available In vertebrates and arthropods, blood clotting involves the establishment of a plug of aggregated thrombocytes (the cellular clot and an extracellular fibrillar clot formed by the polymerization of the structural protein of the clot, which is fibrin in mammals, plasma lipoprotein in crustaceans, and coagulin in the horseshoe crab, Limulus polyphemus. Both elements of the clot function to staunch bleeding. Additionally, the extracellular clot functions as an agent of the innate immune system by providing a passive anti-microbial barrier and microbial entrapment device, which functions directly at the site of wounds to the integument. Here we show that, in addition to these passive functions in immunity, the plasma lipoprotein clot of lobster, the coagulin clot of Limulus, and both the platelet thrombus and the fibrin clot of mammals (human, mouse operate to capture lipopolysaccharide (LPS, endotoxin. The lipid A core of LPS is the principal agent of gram-negative septicemia, which is responsible for more than 100,000 human deaths annually in the United States and is similarly toxic to arthropods. Quantification using the Limulus Amebocyte Lysate (LAL test shows that clots capture significant quantities of LPS and fluorescent-labeled LPS can be seen by microscopy to decorate the clot fibrils. Thrombi generated in the living mouse accumulate LPS in vivo. It is suggested that capture of LPS released from gram-negative bacteria entrapped by the blood clot operates to protect against the disease that might be caused by its systemic dispersal.

Short communication. Microculture syncytia assay for bovine leukemia virus

Energy Technology Data Exchange (ETDEWEB)

Paul, P.S.; Castro, A.E.; Pomeroy, K.A.; Johnson, D.W.; Muscoplat, C.C.

1978-01-01

A microculture syncytia assay for the detection of bovine leukemia virus (BLV) has been described and compared with the conventional macroculture assay. The microculture assay required fewer indicator cells, was as sensitive as the macroculture assay and provided a reproducible test for the detection and titration of BLV.

Determination of Immunogenic Relevant Antigens in the Excretory-Secretory (ES Products and the Lysates of Ascaridia galli Larvae

Directory of Open Access Journals (Sweden)

S Saffi

2008-04-01

Full Text Available Background: Ascaridia galli, the largest nematode of small intestine of birds, especially the native poultry, may give rise to serious illness, pathological defects and economical losses even in modern poultry production systems. Although various measures have been undertaken to vaccinate poultry against A.galli, no satisfactory results were obtained so far. However, there is no report on the efficacy of excretory-secretory (ES proteins of A.galli larvae in immunization of poultry. Thus, the aim of the present research project was based on the use of the ES products of the larvae, in order to find the protective anti­gens.Methods: Five hundred native poultry were autopsied and adult A.galli was removed form their intestines. The eggs were harvested form the uterus of female worms and cultured at 25 ˚C in water containing 0.1 N sulphuric acid for almost a fort­night. The larvae were then freed mechanically and kept in Earl's salt solution for a few days. The supernatant solution of alive larvae containing the ES products of the larvae, as well as the sonicated alive and dead larvae, was analyzed by SDS-PAGE.Result: Many protein fractions of 15 kDa up to 200 kDa were demonstrated in lysate of these larvae. Using the serum of a hen, infected with a high numbers of A.galli, an immunogenic antigen was identified between 55 kDa to 72 kDa by Western blotting procedure.Conclusion: Finding the protein band between 55 and 72 kDa can be promising for preparation of vaccine, though more investigations are needed to prove the protective ability of this antigen.

Senescence and quiescence in adipose-derived stromal cells: Effects of human platelet lysate, fetal bovine serum and hypoxia.

Science.gov (United States)

Søndergaard, Rebekka Harary; Follin, Bjarke; Lund, Lisbeth Drozd; Juhl, Morten; Ekblond, Annette; Kastrup, Jens; Haack-Sørensen, Mandana

2017-01-01

Adipose-derived stromal cells (ASCs) are attractive sources for cell-based therapies. The hypoxic niche of ASCs in vivo implies that cells will benefit from hypoxia during in vitro expansion. Human platelet lysate (hPL) enhances ASC proliferation rates, compared with fetal bovine serum (FBS) at normoxia. However, the low proliferation rates of FBS-expanded ASCs could be signs of senescence or quiescence. We aimed to determine the effects of hypoxia and hPL on the expansion of ASCs and whether FBS-expanded ASCs are senescent or quiescent. ASCs expanded in FBS or hPL at normoxia or hypoxia until passage 7 (P7), or in FBS until P5 followed by culture in hPL until P7, were evaluated by proliferation rates, cell cycle analyses, gene expression and β-galactosidase activity. hPL at normoxia and hypoxia enhanced proliferation rates and expression of cyclins, and decreased G0/G1 fractions and expression of p21 and p27, compared with FBS. The shift from FBS to hPL enhanced cyclin levels, decreased p21 and p27 levels and tended to decrease G0/G1 fractions. Hypoxia does not add to the effect of hPL during ASC expansion with regard to proliferation, cell cycle regulation and expression of cyclins, p21 and p27. hPL rejuvenates FBS-expanded ASCs with regard to cell cycle regulation and expression of cyclins, p21 and p27. This indicates a reversible arrest. Therefore, we conclude that ASCs expanded until P7 are not senescent regardless of culture conditions. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Platelet lysate activates quiescent cell proliferation and reprogramming in human articular cartilage: Involvement of hypoxia inducible factor 1.

Science.gov (United States)

Nguyen, Van Thi; Cancedda, Ranieri; Descalzi, Fiorella

2018-03-01

The idea of rescuing the body self-repair capability lost during evolution is progressively gaining ground in regenerative medicine. In particular, growth factors and bioactive molecules derived from activated platelets emerged as promising therapeutic agents acting as trigger for repair of tissue lesions and restoration of tissue functions. Aim of this study was to assess the potential of a platelet lysate (PL) for human articular cartilage repair considering its activity on progenitor cells and differentiated chondrocytes. PL induced the re-entry in the cell cycle of confluent, growth-arrested dedifferentiated/progenitor cartilage cells. In a cartilage permissive culture environment, differentiated cells also resumed proliferation after exposure to PL. These findings correlated with an up-regulation of the proliferation/survival pathways ERKs and Akt and with an induction of cyclin D1. In short- and long-term cultures of articular cartilage explants, we observed a release of proliferating chondroprogenitors able to differentiate and form an "in vitro" tissue with properties of healthy articular cartilage. Moreover, in cultured cartilage cells, PL induced a hypoxia-inducible factor (HIF-1) alpha increase, its nuclear relocation and the binding to HIF-1 responsive elements. These events were possibly related to the cell proliferation because the HIF-1 inhibitor acriflavine inhibited HIF-1 binding to HIF-1 responsive elements and cell proliferation. Our study demonstrates that PL induces quiescent cartilage cell activation and proliferation leading to new cartilage formation, identifies PL activated pathways playing a role in these processes, and provides a rationale to the application of PL for therapeutic treatment of damaged articular cartilage. Copyright © 2017 John Wiley & Sons, Ltd.

Evaluation of intra-articular injection of autologous platelet lysate (PL) in horses with osteoarthritis of the distal interphalangeal joint.

Science.gov (United States)

Tyrnenopoulou, Panagiota; Diakakis, Nikolaos; Karayannopoulou, Maria; Savvas, Ioannis; Koliakos, Georgios

2016-06-01

Regenerative medicine has become one of the most promising therapies of equine osteoarthritis. Platelet lysate (PL) is rich in bioactive proteins and growth factors that play a crucial role in tissue healing. To evaluate the efficacy of intra-articularly injected autologous PL in equine athletes with naturally occurring osteoarthritis. Fifteen warmblood geldings aged 8-19 years with osteoarthritis of the distal interphalangeal joint were included in this study. They were randomly divided into two groups; 10 horses received intra-articular injections of PL and 5 of normal saline (controls). Before treatment, platelet-derived growth factor (PDGF) levels in basal plasma and prepared PL were estimated. Each joint was injected twice within a three-week period. Lameness was evaluated using the American Association of Equine Practitioners grading system, before treatment and 10 days after each intra-articular injection. Horses were examined fortnightly for one year. Radiographic examination was performed six months post-treatment. The generalized estimating equation test was used for statistical analysis. Acceptable levels of PDGF were detected in PLs (mean ± SD: 258.0 ± 52.3 pg/ml). The majority of horses (9/10) responded positively to PL treatment presenting lower lameness grades (p < 0.0005) compared to controls 10 days after the second injection, and returned to normal athletic activity. Radiographs revealed no changes in osteoarthritis lesions six months after treatment. One year post-injections, however, all horses relapsed to their initial degree of lameness. Intra-articularly injected autologous PL is an efficient method for temporarily managing osteoarthritis of the distal interphalangeal joint in athletic horses.

Mesenchymal Stromal Cells Implantation in Combination with Platelet Lysate Product Is Safe for Reconstruction of Human Long Bone Nonunion.

Science.gov (United States)

Labibzadeh, Narges; Emadedin, Mohsen; Fazeli, Roghayeh; Mohseni, Fatemeh; Hosseini, Seyedeh Esmat; Moghadasali, Reza; Mardpour, Soura; Azimian, Vajiheh; Ghorbani Liastani, Maede; Mirazimi Bafghi, Ali; Baghaban Eslaminejad, Mohamadreza; Aghdami, Nasser

2016-01-01

Nonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion. In this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period. From a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control Xray evidence, bony union had occurred. Results from the present study suggest that the implantation of bone marrow-derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179).

Comparison of human platelet lysate alternatives using expired and freshly isolated platelet concentrates for adipose-derived stromal cell expansion.

Science.gov (United States)

Dessels, Carla; Durandt, Chrisna; Pepper, Michael S

2018-03-19

Pooled human platelet lysate (pHPL) has been used to expand adipose-derived stromal cells (ASCs) and can be formulated using fresh or expired buffy coats (BCs) which are then resuspended in either plasma or an additive solution. Not much is known about the effects that expired products and additive solutions have on ASC expansion, and the need for quality control and release criteria has been expressed. This pilot study compared proliferation, cell size, morphology and immunophenotype of ASCs expanded in the different pHPL alternatives versus foetal bovine serum (FBS). Quality control criteria were assessed prior to and during the manufacture of the pHPL alternatives. ASCs were then expanded in 1%, 2.5%, 5% or 10% of the different pHPL alternatives or in 10% FBS. Cell size, morphology, cell number and immunophenotype were measured using microscopy and flow cytometry. The majority of the pHPL alternatives were within the recommended ranges for the quality control criteria. ASCs expanded in the pHPL alternatives were smaller in size, displayed a tighter spindle-shaped morphology, increased cell growth and had a similar immunophenotype (with the exception of CD34 and CD36) when compared to ASCs expanded in FBS. Here we report on the effects that expired BC products and additive solutions have on ASC expansion. When taken together, our findings indicate that all of the pHPL alternatives can be considered to be suitable replacements for FBS for ASC expansion, and that expired BC products can be used as an alternative to fresh BC products.

21 CFR 864.7525 - Heparin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Heparin assay. 864.7525 Section 864.7525 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7525 Heparin assay. (a) Identification. A...

Suitability of a liquid chromatography assay of neomycin sulfate to replace the microbiological assay for neomycin in USP Monographs.

Science.gov (United States)

Hanko, Valoran P; Rohrer, Jeffrey S

2010-01-05

The current USP National Formulary contains 65 Monographs for drug formulations containing neomycin. All 65 Monographs prescribe a bioassay for neomycin assay. This bioassay, based on cell culture, is labor intensive, has poor precision, and cannot be adapted for purity or identification. High-performance anion-exchange chromatography with integrated pulsed amperometric detection (HPAE-IPAD), a liquid chromatography technique, has been shown to be suitable for neomycin purity analysis and neomycin assay of an over-the-counter first aid cream (Hanko and Rohrer [17]). Here we propose that an HPAE-IPAD assay can replace the bioassay in the 65 neomycin-containing Monographs. We applied the HPAE-IPAD assay to four neomycin-containing drug products representing the four classes of formulations found in the 65 Monographs, liquid, solid, suspension, and cream. Each drug was analyzed with two chromatography systems, and on 3 separate days. For all products, HPAE-IPAD measurements were precise and accurate with respect to the label concentrations. There was also high accuracy for spike recovery of neomycin from the four drug products throughout 70-150% of the labeled concentration. These results suggest that an HPAE-IPAD assay would be an accurate assay for neomycin, and would be faster and more precise than the current bioassay.

Human cord blood-derived platelet lysate enhances the therapeutic activity of adipose-derived mesenchymal stromal cells isolated from Crohn's disease patients in a mouse model of colitis.

Science.gov (United States)

Forte, Dorian; Ciciarello, Marilena; Valerii, Maria Chiara; De Fazio, Luigia; Cavazza, Elena; Giordano, Rosaria; Parazzi, Valentina; Lazzari, Lorenza; Laureti, Silvio; Rizzello, Fernando; Cavo, Michele; Curti, Antonio; Lemoli, Roberto M; Spisni, Enzo; Catani, Lucia

2015-09-09

Due to their immunomodulatory properties, mesenchymal stromal cells (MSCs) have been used for auto-immune disease treatment. Crohn disease (CD) and ulcerative colitis are two major inflammatory bowel diseases (IBDs), resulting from pathological immune responses to environmental or microbial antigens. Preclinical and clinical studies have suggested that MSC-based cellular therapy hold promising potential for IBD treatment. However, open issues include the selection of the proper cell dose, the source and the optimal route of administration of MSCs for more effective results. Platelet lysate has gained clinical interest due to its efficacy in accelerating wound healing. Thus, we propose to combine the administration of MSCs with a human umbilical cord blood-derived platelet lysate (hCBPL) as a novel strategy to improve MSC-based therapy for IBD resolution. Colitis was induced in 8-week-old C57BL/6J mice by daily oral administration of dextran sulphate sodium (DSS) (1.5 % w/v in tap water) for 9 days. MSCs were isolated from adipose tissue of CD patients (adCD-MSCs), expanded in proliferation medium, resuspended in hCBPL or PBS and administrated via enema for three times (1 × 10(6) cells/mouse/time) every other day starting on day +7 from DSS induction. The colitis evolution was evaluated by daily monitoring of body weight, stool consistency and bleeding. Histopathological analysis was performed. Inflammatory cytokine plasma levels were determined. adCD-MSCs stained with lipophilic membrane dye Nile Red, were injected in DSS mice as described above. Colon section of mice sacrificed 24 hours after last cell administration, were analyzed by confocal microscopy. We found that adCD-MSCs could be easily isolated and expanded from CD patients. Upon injection, adCD-MSCs exerted a therapeutic effect on DSS-induced colitis. Moreover, hCBPL increased adCD-MSCs efficacy by significantly reducing colitis scores, extension of the colon inflamed area and plasma levels of

Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

Science.gov (United States)

Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

2006-03-01

Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

The narrow therapeutic window of glycated hemoglobin and assay variability.

Science.gov (United States)

Hosseini, S S; Bibler, I; Charles, M A

1999-12-01

Glycated hemoglobin is measured by a variety of assays, each of which has a unique normal level. Our purpose is to show that among the different assays available in the United States, using the same patient's blood sample, assay results may vary widely and may more or less easily achieve a glycated hemoglobin value within the normal range. The following assays were compared using the same patient's blood sample for each pair of assays: glycohemoglobin affinity assay (GHB Reader; Isolab, Akron, OH) versus gel electrophoresis assay (n = 76); Isolab versus ion capture assay (IMX; Abbott Laboratories, Irving, TX) (n = 57); monoclonal antibody assay (DCA2000; Bayer Diagnostics, Pittsburgh, PA) versus IMX (n = 100); and high-performance liquid chromatography (HPLC) assay (Bio-Rad Variant A1c; Bio-Rad Laboratories, Richmond, CA) versus IMX assay (n = 55). Our analyses indicate that a relative ranking can be established for the ease of achieving a normal glycated hemoglobin level. The ranking indicates that the most stringent or difficult assays for achieving a normal level are the Isolab and DCA2000 assays. The intermediate assays are the IMX and Bio-Rad Variant, and the easiest method for achieving a normal value is the gel electrophoresis assay. Our results indicate that various glycated hemoglobin assays vary widely and are associated with more or less difficulty for an individual patient to achieve a glycated hemoglobin level within the normal range. These results are especially significant with respect to (1) the clinically narrow therapeutic window of glycated hemoglobin values in type 1 diabetes to avoid rapidly advancing severe hypoglycemia rates and chronic microvascular complication rates, and (2) the glycated hemoglobin threshold for rapidly advancing macrovascular disease in both type 1 and type 2 patients.

Parallel force assay for protein-protein interactions.

Directory of Open Access Journals (Sweden)

Daniela Aschenbrenner

Full Text Available Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.

Have you stress tested your assay?

Directory of Open Access Journals (Sweden)

Zheng Cao

2016-08-01

Full Text Available Objectives: When a clinical assay is stressed with extraordinarily high volume of specimens over a short period of time, extra caution may be needed to avoid systematic errors and biases. Here we report our experience with a HgbA1c assay used for high volume wellness screening purpose, to illustrate the importance of stress testing during assay validation. Design and Methods: Over 15,000 whole blood specimens were tested for HgbA1c in a period of 2 months. HgbA1c was tested by an immunoturbidimetric method on a high through-put automation line. The HgbA1c population distribution in our study was compared to that from the NHANES database. Daily distributions of HgbA1c values ≥6%, means and medians were plotted. Correlation studies were performed between the high through-put immunoturbidimetric assay and a medium through-put HPLC method. Results: We observed a shift of HgbA1c distribution to the higher values compared to the NHANES. A bias of 15–20% was noted from further stress testing where large number of samples were batched and tested using the immunoturbidimetric assay. A 5–7% higher bias remained after implementing a cuvette washing program after each HgbA1c sample. We hypothesized this bias was caused by build-up of blood cell fragments in the cuvettes when continuous whole blood samples are run through the system. Our experience suggests stress testing needs to be incorporated early in the test validation process for high volume batched screening applications. This seemingly extra validation step may save significant troubleshooting and retesting efforts down the road. Keywords: Hemoglobin A1c, Immunoturbidimetric assay, HPLC, Quality assurance, Systematic bias, High volume, Automation

Rapid colorimetric assay for gentamicin injection.

Science.gov (United States)

Tarbutton, P

1987-01-01

A rapid colorimetric method for determining gentamicin concentration in commercial preparations of gentamicin sulfate injection was developed. Methods currently available for measuring gentamicin concentration via its colored complex with cupric ions in alkaline solution were modified to reduce the time required for a single analysis. The alkaline copper tartrate (ACT) reagent solution was prepared such that each milliliter contained 100 mumol cupric sulfate, 210 mumol potassium sodium tartrate, and 1.25 mmol sodium hydroxide. The assay involves mixing 0.3 mL gentamicin sulfate injection 40 mg/mL (of gentamicin), 1.0 mL ACT reagent, and 0.7 mL water; the absorbance of the resulting solution at 560 nm was used to calculate the gentamicin concentration in the sample. For injections containing 10 mg/mL of gentamicin, the amount of the injection was increased to 0.5 mL and water decreased to 0.5 mL. The concentration of gentamicin in samples representing 11 lots of gentamicin sulfate injection 40 mg/mL and 8 lots of gentamicin sulfate injection 10 mg/mL was determined. The specificity, reproducibility, and accuracy of the assay were assessed. The colored complex was stable for at least two hours. Gentamicin concentration ranged from 93.7 to 108% and from 95 to 109% of the stated label value of the 40 mg/mL and the 10 mg/mL injections, respectively. No components of the preservative system present in the injections interfered with the assay. Since other aminoglycosides produced a colored complex, the assay is not specific for gentamicin. The assay was accurate and reproducible over the range of 4-20 mg of gentamicin. This rapid and accurate assay can be easily applied in the hospital pharmacy setting.

Comparison of Parasite Burden Using Real-Time Polymerase Chain Reaction Assay and Limiting Dilution Assay in Leishma-nia major Infected Mouse

Directory of Open Access Journals (Sweden)

Somayeh GHOTLOO

2015-12-01

Full Text Available Background:Limiting dilution assay is considered as the gold standard method for quantifying the number of parasites in the animal model of Leishmania infection. Nowadays, real-time PCR is being increasingly applied to quantify infectious agents. In the present study, a real-time PCR assay was developed to estimate para­site burdens in lymph nodes of Leishmania major infected BALB/C mice. Enumera­tion of parasites was also performed by limiting dilution assay and compared with the results of real-time PCR based quantification.Methods:The SYBR Green based real- time PCR assay was performed to amplify a 75 bp fragment of superoxide dismutase B1 gene in the lymph nodes of L. major infected BALB/C mice 8 weeks post infection. Mice were infected subcutaneously at the base of their tail with 2 × 105L. major promastigotes in the stationary phase of growth. To compare parasite burdens obtained by real-time PCR assay with those of limiting dilution assay, twelve 8-fold serial dilutions of the lymph node homoge­nates were prepared in the Schneider medium and incubated at 26°C.After 7 days, wells containing motile parasites were identified by direct observation under an inverted light microscope and the total number of parasites was estimated using the ELIDA software.Results:Spearman's correlation coefficient of the parasite burdens between real-time PCR and limiting dilution assay was 0.72 (Pvalue = 0.008.Conclusion:Real-time PCR assay is an appropriate replacement to existing limit­ing dilution assay in quantifying parasite burden in the experimental model of Leishma­nia infection.

Regulation of laminin beta2 chain gene expression in human cancer cell lines

DEFF Research Database (Denmark)

Durkin, M E; Nielsen, F C; Loechel, F

2001-01-01

of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain m......RNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates....

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver.

Science.gov (United States)

Ding, Wei; Bishop, Michelle E; Lyn-Cook, Lascelles E; Davis, Kelly J; Manjanatha, Mugimane G

2016-05-04

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

Ecotoxicological Assessment of Aquatic Genotoxicity Using the Comet Assay

Directory of Open Access Journals (Sweden)

KHUSNUL YAQIN

2006-09-01

Full Text Available Comet assay is a novel biological analysis, which is a sensitive, flexible, simple, rapid, and inexpensive method to assess aquatic genotoxicant. Since Singh and co-workers developed the method in 1988, its use has increased exponentially in various fields. This review discourses on the application of this assay in aquatic ecosystems. Various types of cells from various aquatic organisms have been tested by various genotoxicant both direct- and indirect-acting using the comet assay. The applications of this assay suggest that it is a useful assay to assess aquatic genotoxicants. However, there are some factors, which should be taken into account when using this assay as aquatic ecotoxicological assessment device such as inter-animal and cell variability.

Minimum pricing of alcohol versus volumetric taxation: which policy will reduce heavy consumption without adversely affecting light and moderate consumers?

Science.gov (United States)

Sharma, Anurag; Vandenberg, Brian; Hollingsworth, Bruce

2014-01-01

We estimate the effect on light, moderate and heavy consumers of alcohol from implementing a minimum unit price for alcohol (MUP) compared with a uniform volumetric tax. We analyse scanner data from a panel survey of demographically representative households (n = 885) collected over a one-year period (24 Jan 2010-22 Jan 2011) in the state of Victoria, Australia, which includes detailed records of each household's off-trade alcohol purchasing. The heaviest consumers (3% of the sample) currently purchase 20% of the total litres of alcohol (LALs), are more likely to purchase cask wine and full strength beer, and pay significantly less on average per standard drink compared to the lightest consumers (A$1.31 [95% CI 1.20-1.41] compared to $2.21 [95% CI 2.10-2.31]). Applying a MUP of A$1 per standard drink has a greater effect on reducing the mean annual volume of alcohol purchased by the heaviest consumers of wine (15.78 LALs [95% CI 14.86-16.69]) and beer (1.85 LALs [95% CI 1.64-2.05]) compared to a uniform volumetric tax (9.56 LALs [95% CI 9.10-10.01] and 0.49 LALs [95% CI 0.46-0.41], respectively). A MUP results in smaller increases in the annual cost for the heaviest consumers of wine ($393.60 [95% CI 374.19-413.00]) and beer ($108.26 [95% CI 94.76-121.75]), compared to a uniform volumetric tax ($552.46 [95% CI 530.55-574.36] and $163.92 [95% CI 152.79-175.03], respectively). Both a MUP and uniform volumetric tax have little effect on changing the annual cost of wine and beer for light and moderate consumers, and likewise little effect upon their purchasing. While both a MUP and a uniform volumetric tax have potential to reduce heavy consumption of wine and beer without adversely affecting light and moderate consumers, a MUP offers the potential to achieve greater reductions in heavy consumption at a lower overall annual cost to consumers.

[Comparison of the clinical performance of the ECLusys HBsAg II assay with the Lumipulse f and HISCL 2000-i HBsAg screening assays].

Science.gov (United States)

Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

2012-02-01

We compared the ECLusys HBsAgII (ECL HBsAg) assay to the Lumipulse Forte (LPf HBsAg) and HISCL (HIS HBsAg) assays. Measurement of dilution panels for which the WHO HBsAg international reference panel was the parent specimen revealed that the ECL and HIS assays enabled detection to a theoretical level of 0.04 IU/mL, whereas the LPf assay enabled detection to a level of 0.08 IU/mL. In a specificity test using high RF positive specimens (n = 33), pregnancy specimens (n = 35), cytomegalovirus antibody positive specimens (n = 36), and high M protein positive specimens (n = 21) that were confirmed negative for HBsAg by the LPf assay, negative results were obtained for all specimens on the HIS assay, but the ECL assay yielded a positive result for one of the high RF positive specimens. This individual was suggested on further testing to be an HBV carrier who was strongly positive for HBc antibody. In HBsAg mutants detection test, the detection rate was 92.3% with the ECL assay and 69.2% with the HIS assay. In a correlation test using routinely collected clinical specimens (n = 155), including positive stock specimens, aside from the one case where the LPf assay gave a negative result but both the ECL and HIS assays gave positive results, all of the results were consistent for all specimens. The above results confirmed that the ECL assay is both highly sensitive and specific, and also enables a high rate of HBsAg mutant detection.

Evaluation of the Abbott ARCHITECT Toxo IgM assay.

Science.gov (United States)

Sickinger, Eva; Braun, Hans-Bertram; Praast, Gerald; Stieler, Myriam; Gundlach, Cordelia; Birkenbach, Claudia; Prostko, John; Palafox, Mary Ann; Frias, Edwin; Hsu, Stephen; Matias, Matthew; Pucci, Dominick; Hausmann, Michael; Sagel, Ulrich; Smith, Darwin

2009-07-01

Development of the ARCHITECT Toxo IgM assay has been done to assist the clinician in acute Toxoplasma gondii infection detection, especially in pregnant women. Its use, in conjunction with ARCHITECT Toxo IgG and Toxo Avidity assays, will provide an array of assays particularly useful in the monitoring of pregnant females to determine the risk of maternal transmission of the parasite. Specificity results from 2 testing sites, using populations of pregnant females, hospital patients, and blood donors, demonstrated that the assay has an overall resolved relative specificity of 99.89% (confidence interval, 99.68-99.98%). Relative specificity for pregnant female specimens was 99.95% (n = 2031). Excellent seroconversion sensitivity was observed for the ARCHITECT Toxo IgM assay, which was similar to the Abbott AxSYM Toxo IgM assay (Abbott Laboratories, Abbott Park, IL). In more than 90% of the panels tested, the 1st bleed detected in the serial bleeds was the same for both assays.

A novel dressing for the combined delivery of platelet lysate and vancomycin hydrochloride to chronic skin ulcers: Hyaluronic acid particles in alginate matrices.

Science.gov (United States)

Rossi, S; Mori, M; Vigani, B; Bonferoni, M C; Sandri, G; Riva, F; Caramella, C; Ferrari, F

2018-06-15

The aim of the present work was to develop a medication allowing for the combined delivery of platelet lysate (PL) and an anti-infective model drug, vancomycin hydrochloride (VCM), to chronic skin ulcers. A simple method was set up for the preparation of hyaluronic acid (HA) core-shell particles, loaded with PL and coated with calcium alginate, embedded in a VCM containing alginate matrix. Two different CaCl 2 concentrations were investigated to allow for HA/PL core-shell particle formation. The resulting dressings were characterized for mechanical and hydration properties and tested in vitro (on fibroblasts) and ex-vivo (on skin biopsies) for biological activity. They were found of sufficient mechanical strength to withstand packaging and handling stress and able to absorb a high amount of wound exudate and to form a protective gel on the lesion area. The CaCl 2 concentration used for shell formation did not affect VCM release from the alginate matrix, but strongly modified the release of PGFAB (chosen as representative of growth factors present in PL) from HA particles. In vitro and ex vivo tests provided sufficient proof of concept of the ability of dressings to improve skin ulcers healing. Copyright © 2018 Elsevier B.V. All rights reserved.

Production and assay of forskolin antibodies

International Nuclear Information System (INIS)

Ho, L.T.; Ho, R.J.

1986-01-01

Forskolin (Fo), a cardiovascular active diterpene of plant origin, has been widely used as a research tool in regulation of the catalytic activity of adenylate cyclase (AC). A linear relationship of Fo binding to plasma membrane with activation of AC has been reported. The present abstract describes the production and assay of Fo antibodies (AB). 7-0-Hemisuccinyl-7-deacetyl Fo, coupled to either human serum albumin or goat IgG, was injected into goats to elicit AB to Fo haptan. AB to Fo in antiserum or an isolated IgG fraction was tested by two assay methods, a radioimmunoassay using 3 H-Fo as a tracer and a colorimetric enzyme-linked immunosorbent assay (ELISA) using horse radish peroxidase-rabbit anti goat IgG as indicator. The titers for Fo antiserum were 4000-10,000. In the defined assay condition, approximately 20-25% of the added 3 H-Fo was found to bind to AB. The bound radioactivity was displaced by Fo-HSA or Fo-goat IgG or free unlabelled Fo ranging from 0.5-50 pmol/tube, or 5-500 nM. The IC 50 was approximately 8-10 pmol/tube or 80-100 nM. The binding of HRP-rabbit anti goat IgG in the ELISA was inhibited by proper Fo conjugate. The development of methods for production and assay for Fo AB may be useful in the study of mechanism of activation of AC by Fo and Fo-like compound

Hyaluronic Acid Assays

DEFF Research Database (Denmark)

Itenov, Theis S; Kirkby, Nikolai S; Bestle, Morten H

2015-01-01

BACKGROUD: Hyaluronic acid (HA) is proposed as a marker of functional liver capacity. The aim of the present study was to compare a new turbidimetric assay for measuring HA with the current standard method. METHODS: HA was measured by a particle-enhanced turbidimetric immunoassay (PETIA) and enzyme...

A novel microculture kinetic assay (MiCK assay) for malignant cell growth and chemosensitivity.

Science.gov (United States)

Kravtsov, V D

1994-01-01

The THERMOmax microplate reader was adapted for monitoring the growth kinetics of human leukaemic OCI/AML-2 and mouse tumour J-774.1 cell lines in continuous culture. Fluid evaporation from wells, CO2 escape and contamination were prevented by hermetic sealing of the microcultures in wells of a 96-well microplate, thus enabling the cells to grow exponentially for 72 h under the conditions of the incubated microplate reader. For both OCI/AML-2 cells, which grow in suspension, and adherent J-774.1 cells, a linear correlation was demonstrated between the number of unstained cells seeded in a given microplate well and the optical density (OD) of that well. Therefore, the OD/time curve of the culture could be deemed to be its growth curve. By the use of the linear fit equation, the actual number of the cells in the wells was computable at any time point of the assay. In the chemosensitivity test, an inhibitory effect of ARA-C on the growth of the cells could be estimated by viewing of the growth curves plotted on the screen. The maximum kinetic rates (Vmax) of the curves in the control and the ARA-C-treated wells were compared, yielding a growth inhibition index (GII). Comparison of results of the kinetic chemosensitivity assay with those of a [3H]thymidine incorporation assay revealed that the novel assay is suitable for precise quantitation of the cell chemosensitivity, is more informative and has the added technical advantage of performance without recourse to radioactive or chemically hazardous substances.

Diagnostic sensitivity of two radio receptor assays (TRAK Assay and TRAK Dyno human) for the detection of TSH receptor antibodies

International Nuclear Information System (INIS)

Paunkovic, N.; Paunkovic, J.

2003-01-01

Radio receptor assays for the detection of TSH receptor antibodies in serum are typically based on binding the competition of TSH-R antibodies and 125I -labelled-TSH for membrane preparation of thyrocytes (TBII tests). The sensitivity of the available tests utilizing porcine cell membranes was found to be around 80%. A new test (TRAK Dyno human, BRAHMS) utilizes human recombinant TSH receptor and human standard material that is supposed to improve the performance of the test. We have compared the results of these two assays. The sensitivity of the TRAK Assay tested in 356 patients with untreated Grave's disease was found to be 85%, and 97.5% for TRAK Dyno human in 111 newly diagnosed patients. Both tests were performed from the same serum specimen for 60 of the investigated patients. The TRAK Assay was positive in 50 patients (83.2%) and TRAK Dyno human in 59 patients (98.3%). The specificity of the new radio receptor assay was also improved. (author)

Cell Culture Assay for Human Noroviruses [response

Energy Technology Data Exchange (ETDEWEB)

Straub, Tim M.; Honer Zu Bentrup, Kerstin; Orosz Coghlan, Patricia; Dohnalkova, Alice; Mayer, Brooke K.; Bartholomew, Rachel A.; Valdez, Catherine O.; Bruckner-Lea, Cindy J.; Gerba, Charles P.; Abbaszadegan, Morteza A.; Nickerson, Cheryl A.

2007-07-01

We appreciate the comments provided by Leung et al., in response to our recently published article “In Vitro Cell Culture Infectivity Assay for Human Noroviruses” by Straub et al. (1). The specific aim of our project was to develop an in vitro cell culture infectivity assay for human noroviruses (hNoV) to enhance risk assessments when they are detected in water supplies. Reverse transcription (RT) qualitative or quantitative PCR are the primary assays for waterborne NoV monitoring. However, these assays cannot distinguish between infectious vs. non-infectious virions. When hNoV is detected in water supplies, information provided by our infectivity assay will significantly improve risk assessment models and protect human health, regardless of whether we are propagating NoV. Indeed, in vitro cell culture infectivity assays for the waterborne pathogen Cryptosporidium parvum that supplement approved fluorescent microscopy assays, do not result in amplification of the environmentally resistant hard-walled oocysts (2). However, identification of life cycle stages in cell culture provides evidence of infectious oocysts in a water supply. Nonetheless, Leung et al.’s assertion regarding the suitability of our method for the in vitro propagation of high titers of NoV is valid for the medical research community. In this case, well-characterized challenge pools of virus would be useful for developing and testing diagnostics, therapeutics, and vaccines. As further validation of our published findings, we have now optimized RT quantitative PCR to assess the level of viral production in cell culture, where we are indeed finding significant increases in viral titer. The magnitude and time course of these increases is dependent on both virus strain and multiplicity of infection. We are currently preparing a manuscript that will discuss these findings in greater detail, and the implications this may have for creating viral challenge pools

Radioimmune assay of human platelet prostaglandin synthetase

International Nuclear Information System (INIS)

Roth, G.J.; Machuga, E.T.

1982-01-01

Normal platelet function depends, in part, on platelet PG synthesis. PG synthetase (cyclo-oxygenase) catalyzes the first step in PG synthesis, the formation of PGH 2 from arachidonic acid. Inhibition of the enzyme by ASA results in an abnormality in the platelet release reaction. Patients with pparent congenital abnormalities in the enzyme have been described, and the effects have been referred to as ''aspirin-like'' defects of the platelet function. These patients lack platelet PG synthetase activity, but the actual content of PG synthetase protein in these individuals' platelets is unknown. Therefore an RIA for human platelet PG synthetase would provide new information, useful in assessing the aspirin-like defects of platelet function. An RIA for human platelet PG synthetase is described. The assay utilizes a rabbit antibody directed against the enzyme and [ 125 I]-labelled sheep PG synthetase as antigen. The human platelet enzyme is assayed by its ability to inhibit precipitation of the [ 125 I]antigen. The assay is sensitive to 1 ng of enzyme. By the immune assay, human platelets contain approximately 1200 ng of PG synethetase protein per 1.5 mg of platelet protein (approximately 10 9 platelets). This content corresponds to 10,000 enzyme molecules per platelet. The assay provides a rapid and convenient assay for the human platelet enzyme, and it can be applied to the assessment of patients with apparent platelet PG synthetase (cyclo-oxygenase) deficiency

Complementing in vitro screening assays with in silico ...

Science.gov (United States)

High-throughput in vitro assays offer a rapid, cost-efficient means to screen thousands of chemicals across hundreds of pathway-based toxicity endpoints. However, one main concern involved with the use of in vitro assays is the erroneous omission of chemicals that are inactive under assay conditions but that can generate active metabolites under in vivo conditions. To address this potential issue, a case study will be presented to demonstrate the use of in silico tools to identify inactive parents with the ability to generate active metabolites. This case study used the results from an orthogonal assay designed to improve confidence in the identification of active chemicals tested across eighteen estrogen receptor (ER)-related in vitro assays by accounting for technological limitations inherent within each individual assay. From the 1,812 chemicals tested within the orthogonal assay, 1,398 were considered inactive. These inactive chemicals were analyzed using Chemaxon Metabolizer software to predict the first and second generation metabolites. From the nearly 1,400 inactive chemicals, over 2,200 first-generation (i.e., primary) metabolites and over 5,500 second-generation (i.e., secondary) metabolites were predicted. Nearly 70% of primary metabolites were immediately detoxified or converted to other metabolites, while over 70% of secondary metabolites remained stable. Among these predicted metabolites, those that are most likely to be produced and remain

Global optimization in the adaptive assay of subterranean uranium nodules

International Nuclear Information System (INIS)

Vulkan, U.; Ben-Haim, Y.

1989-01-01

An adaptive assay is one in which the design of the assay system is modified during operation in response to measurements obtained on-line. The present work has two aims: to design an adaptive system for borehole assay of isolated subterranean uranium nodules, and to investigate globality of optimal design in adaptive assay. It is shown experimentally that reasonably accurate estimates of uranium mass are obtained for a wide range of nodule shapes, on the basis of an adaptive assay system based on a simple geomorphological model. Furthermore, two concepts are identified which underlie the optimal design of the assay system. The adaptive assay approach shows promise for successful measurement of spatially random material in many geophysical applications. (author)

[Comparison of the clinical performance of the ECLusys HIV combi assay with the Lumipulse f and HISCL 2000-i HIV-1/2 ab screening assays].

Science.gov (United States)

Sugiura, Aya; Iwahara, Kunihiro; Suga, Yasuyuki; Uchiyama, Sachinori; Maekawa, Masato

2012-04-01

We compared the ECLusys HIV combi assay (ECL HIV Ag/Ab) to the Lumipulse Forte (LPf HIV 1/2 Ab) and HISCL (HIS HIV 1/2 Ab) assays. In a dilution sensitivity test using dilution panels of WHO HIV antibody international reference panel (HIV-1 Subtype A, B, C, E, HIV-1 Group O, HIV-2) and HIV-1/2 Ab CE marked material(HIV-1, HIV-2) parent specimens, the ECL assay enabled detection at a higher level of sensitivity than either the LPf assay or the HIS assay for all dilution panels. In an early detection test in the early phase of infection in which a BBI HIV seroconversion panel was used, the ECL assay enabled detection 7 days after initial blood sample collection, whereas the LPf and HIS assays enabled detection after 27 days. In a specificity test using high RF positive specimens (n=33), pregnancy specimens (n=35), cytomegalovirus antibody positive specimens (n=36), and high M protein positive specimens (n=21) that were confirmed negative for HIV-1/2 antibodies by the LPf assay, negative results were obtained for all specimens on both the ECL assay and the HIS assay. In a correlation test using routinely collected clinical specimens (n=121), including positive stock specimens, the ECL and HIS assays demonstrated the highest agreement rate 98.3%. The above results confirmed that the fourth-generation reagent ECL assay, which simultaneously detects both HIV-1/2 antibodies and p24 antigens, is both highly sensitive and specific, and is a suitable assay for use in routine testing.

Monitoring environmental exposures with semen assays

International Nuclear Information System (INIS)

Anon.

1979-01-01

Semen studies in humans and animals have yielded extensive and compelling evidence that sperm can be used to assess reproductive potential and diagnose pathology. More recent studies on mutagens and carcinogens both at this and other laboratories suggest that a combination of mouse and human assays can be an efficient, effective approach to monitoring for reproductive hazards in the environment. We are investigating the potential of using variability in sperm morphology and DNA content to quantify and monitor the effects of environmental agents on the human testes. Here we review the status of human and mouse assays for environmental surveillance, discuss the genetic and fertility implications of chemically induced semen changes, and describe the high-speed flow methods being developed to automate sperm assays

Assay for mutagenesis in heterozygous diploid human lymphoblasts

Science.gov (United States)

Skopek, Thomas R.; Liber, Howard L.; Penman, Bruce W.; Thilly, William G.; Hoppe, IV, Henry

1981-01-01

An assay is disclosed for determining mutagenic damage caused by the administration of a known or suspected mutagen to diploid human lymphoblastoid cell lines. The gene locus employed for this assay is the gene for thymidine kinase, uridine kinase, or cytidine deaminase. Since human lymphoblastoid cells contain two genes for these enzymes, heterozygotes of human lymphoblastoid cells are used in this assay.

Clinical validation of the Tempus xO assay

Science.gov (United States)

Beaubier, Nike; Tell, Robert; Huether, Robert; Bontrager, Martin; Bush, Stephen; Parsons, Jerod; Shah, Kaanan; Baker, Tim; Selkov, Gene; Taxter, Tim; Thomas, Amber; Bettis, Sam; Khan, Aly; Lau, Denise; Lee, Christina; Barber, Matthew; Cieslik, Marcin; Frankenberger, Casey; Franzen, Amy; Weiner, Ali; Palmer, Gary; Lonigro, Robert; Robinson, Dan; Wu, Yi-Mi; Cao, Xuhong; Lefkofsky, Eric; Chinnaiyan, Arul; White, Kevin P.

2018-01-01

We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification. PMID:29899824

Nondestructive assay measurements applied to reprocessing plants

International Nuclear Information System (INIS)

Ruhter, Wayne D.; Lee, R. Stephen; Ottmar, Herbert; Guardini, Sergio

1999-01-01

Nondestructive assay for reprocessing plants relies on passive gamma-ray spectrometry for plutonium isotopic and plutonium mass values of medium-to-low-density samples and holdup deposits; on active x-ray fluorescence and densitometry techniques for uranium and plutonium concentrations in solutions; on calorimetry for plutonium mass in product; and passive neutron techniques for plutonium mass in spent fuel, product, and waste. This paper will describe the radiation-based nondestructive assay techniques used to perform materials accounting measurements. The paper will also discuss nondestructive assay measurements used in inspections of reprocessing plants [ru

Comparison of Five Assays for Detection of Clostridium difficile Toxin

Science.gov (United States)

Chapin, Kimberle C.; Dickenson, Roberta A.; Wu, Fongman; Andrea, Sarah B.

2011-01-01

Performance characteristics of five assays for detection of Clostridium difficile toxin were compared using fresh stool samples from patients with C. difficile infection (CDI). Assays were performed simultaneously and according to the manufacturers' instructions. Patients were included in the study if they exhibited clinical symptoms consistent with CDI. Nonmolecular assays included glutamate dehydrogenase antigen tests, with positive findings followed by the Premier Toxin A and B Enzyme Immunoassay (GDH/EIA), and the C. Diff Quik Chek Complete test. Molecular assays (PCR) included the BD GeneOhm Cdiff Assay, the Xpert C. difficile test, and the ProGastro Cd assay. Specimens were considered true positive if results were positive in two or more assays. For each method, the Youden index was calculated and cost-effectiveness was analyzed. Of 81 patients evaluated, 26 (32.1%) were positive for CDI. Sensitivity of the BD GeneOhm Cdiff assay, the Xpert C. difficile test, the ProGastro Cd assay, C. Diff Quik Chek Complete test, and two-step GDH/EIA was 96.2%, 96.2%, 88.5%, 61.5%, and 42.3%, respectively. Specificity of the Xpert C. difficile test was 96.4%, and for the other four assays was 100%. Compared with nonmolecular methods, molecular methods detected 34.7% more positive specimens. Assessment of performance characteristics and cost-effectiveness demonstrated that the BD GeneOhm Cdiff assay yielded the best results. While costly, the Xpert C. difficile test required limited processing and yielded rapid results. Because of discordant results, specimen processing, and extraction equipment requirements, the ProGastro Cd assay was the least favored molecular assay. The GDH/EIA method lacked sufficient sensitivity to be recommended. PMID:21704273

A molecular gram stain using broad range PCR and pyrosequencing technology: a potentially useful tool for diagnosing orthopaedic infections.

Science.gov (United States)

Kobayashi, Naomi; Bauer, Thomas W; Togawa, Daisuke; Lieberman, Isador H; Sakai, Hiroshige; Fujishiro, Takaaki; Tuohy, Marion J; Procop, Gary W

2005-06-01

The bacteria associated with orthopaedic infections are usually common gram-positive and gram-negative bacteria. This fundamental grouping of bacteria is a necessary first step in the selection of appropriate antibiotics. Since polymerase chain reaction (PCR) is more rapid and may be more sensitive than culture, we developed a postamplification pyrosequencing method to subcategorize bacteria based on a few nucleotide polymorphisms in the 16S rRNA gene. We validated this method using well-characterized strains of bacteria and applied it to specimens from spinal surgery cases with suspected infections. Lysates of 114 bacteria including 75 species were created following standard cultivation to obtain DNA. The DNA was amplified by a broad-range real-time PCR. The amplicons were evaluated by pyrosequencing and were classified as gram-positive, gram-negative, or acid-fast bacilli based on the first three to five nucleotides sequenced. In addition, clinical cases of suspected infection were obtained from spinal surgery. The results of the "molecular Gram stain" were compared with the results of traditional Gram stain and culture. The lysates of 107 (93.9%) of the bacteria extracts tested were appropriately categorized as gram-positive and gram-negative or as acid-fast bacilli on the basis of this assay. The sensitivity and specificity of this assay were 100% and 97.4% for gram-positive and 88.3% and 100% for gram-negative isolates. All of the five clinical samples were appropriately categorized as containing gram-positive or gram-negative bacteria with this assay. This study demonstrates that high sensitivity and specificity of a molecular gram stain may be achieved using broad-range real-time PCR and pyrosequencing.

Momordica charantia Inhibits Inflammatory Responses in Murine Macrophages via Suppression of TAK1.

Science.gov (United States)

Yang, Woo Seok; Yang, Eunju; Kim, Min-Jeong; Jeong, Deok; Yoon, Deok Hyo; Sung, Gi-Ho; Lee, Seungihm; Yoo, Byong Chul; Yeo, Seung-Gu; Cho, Jae Youl

2018-01-01

Momordica charantia known as bitter melon is a representative medicinal plant reported to exhibit numerous pharmacological activities such as antibacterial, antidiabetic, anti-inflammatory, anti-oxidant, antitumor, and hypoglycemic actions. Although this plant has high ethnopharmacological value for treating inflammatory diseases, the molecular mechanisms by which it inhibits the inflammatory response are not fully understood. In this study, we aim to identify the anti-inflammatory mechanism of this plant. To this end, we studied the effects of its methanol extract (Mc-ME) on lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Specifically, we evaluated nitric oxide (NO) production, mRNA expression of inflammatory genes, luciferase reporter gene activity, and putative molecular targets. Mc-ME blocked NO production in a dose-dependent manner in RAW264.7 cells; importantly, no cytotoxicity was observed. Moreover, the mRNA expression levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 were decreased by Mc-ME treatment in a dose-dependent manner. Luciferase assays and nuclear lysate immunoblotting analyses strongly indicated that Mc-ME decreases the levels of p65 [a nuclear factor (NF)-[Formula: see text]B subunit] and c-Fos [an activator protein (AP)-1 subunit]. Whole lysate immunoblotting assays, luciferase assays, and overexpression experiments suggested that transforming growth factor [Formula: see text]-activated kinase 1 (TAK1) is targeted by Mc-ME, thereby suppressing NF-[Formula: see text]B and AP-1 activity via downregulation of extracellular signal-regulated kinases (ERKs) and AKT. These results strongly suggest that Mc-ME exerts its anti-inflammatory activity by reducing the action of TAK1, which also affects the activation of NF-[Formula: see text]B and AP-1.

Detection of radiation-induced apoptosis using the comet assay

International Nuclear Information System (INIS)

Wada, Seiichi; Kobayashi, Yasuhiko; Funayama, Tomoo; Yamamoto, Kazuo; Khoa, Tran Van; Natsuhori, Masahiro; Ito, Nobuhiko

2003-01-01

The electrophoresis pattern of apoptotic cells detected by the comet assay has a characteristic small head and spread tail. This image has been referred to as an apoptotic comet, but it has not been previously proven to be apoptotic cells by any direct method. In order to identify this image obtained by the comet assay as corresponding to an apoptotic cell, the frequency of appearance of apoptosis was examined using CHO-K1 and L5178Y cells which were exposed to gamma irradiation. As a method for detecting apoptosis, the terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) assay was used. When the frequency of appearance of apoptotic cells following gamma irradiation was observed over a period of time, there was a significant increase in appearance of apoptosis when using the TUNEL assay. However, there was only a slight increase when using the comet assay. In order to verify the low frequency of appearance of apoptosis when using the comet assay, we attempted to use the TUNEL assay to satin the apoptotic comets detected in the comet assay. The apoptotic comets were TUNEL positive and the normal comets were TUNEL negative. This indicates that the apoptotic comets were formed from DNA fragments with 3'-hydroxy ends that are generated as cells undergo apoptosis. Therefore, it was understood that the characteristic pattern of apoptotic comets detected by the comet assay corresponds to cells undergoing apoptosis. (author)

Neutron Resonance Transmission Analysis (NRTA): A Nondestructive Assay Technique for the Next Generation Safeguards Initiative’s Plutonium Assay Challenge

Energy Technology Data Exchange (ETDEWEB)

J. W. Sterbentz; D. L. Chichester

2010-12-01

This is an end-of-year report for a project funded by the National Nuclear Security Administration's Office of Nuclear Safeguards (NA-241). The goal of this project is to investigate the feasibility of using Neutron Resonance Transmission Analysis (NRTA) to assay plutonium in commercial light-water-reactor spent fuel. This project is part of a larger research effort within the Next-Generation Safeguards Initiative (NGSI) to evaluate methods for assaying plutonium in spent fuel, the Plutonium Assay Challenge. The first-year goals for this project were modest and included: 1) developing a zero-order MCNP model for the NRTA technique, simulating data results presented in the literature, 2) completing a preliminary set of studies investigating important design and performance characteristics for the NRTA measurement technique, and 3) documentation of this work in an end of the year report (this report). Research teams at Los Alamos National Laboratory (LANL), Lawrence Berkeley National Laboratory (LBNL), Pacific Northwest National Laboratory (PNNL), and at several universities are also working to investigate plutonium assay methods for spent-fuel safeguards. While the NRTA technique is well proven in the scientific literature for assaying individual spent fuel pins, it is a newcomer to the current NGSI efforts studying Pu assay method techniques having just started in March 2010; several analytical techniques have been under investigation within this program for two to three years or more. This report summarizes a nine month period of work.

Endoproteolytic activity assay in malting barley

Directory of Open Access Journals (Sweden)

Blanca Gómez Guerrero

2013-12-01

Full Text Available Hydrolysis of barley proteins into peptides and amino acids is one of the most important processes during barley germination.The degradation of the endosperm stored proteins facilitates water and enzyme movements, enhances modification, liberates starch granules and increases soluble amino nitrogen. Protease activity is the result of the activities of a mixture of exo- and endo-proteases. The barley proteins are initially solubilized by endo-proteases and the further by exo-proteases. Four classes of endo-proteases have been described: serine-proteases, cysteine-proteases, aspartic-proteases and metallo-proteases. The objective of this work was to develop a rapid and colorimetric enzymatic assay to determine the endo-proteolytic activity of the four endo-protease classes using two different substrates: azo-gelatin and azo-casein. Optimum conditions for the assays such as: pH,reaction time and temperature and absorbance scale were determined. Azo-gelatin presented several difficulties in standardizing an “in solution” assay. On the other hand, azo-casein allowed standardization of the assay for the four enzyme classes to produce consistent results. The endo-proteoteolytic method developed was applied to determine the endo-protease activity in barley, malt and wort.