Cloning, sequence analysis, and expression of the large subunit of the human lymphocyte activation antigen 4F2

Energy Technology Data Exchange (ETDEWEB)

Lumadue, J.A.; Glick, A.B.; Ruddle, F.H.

1987-12-01

Among the earliest expressed antigens on the surface of activated human lymphocytes is the surface antigen 4F2. The authors have used DNA-mediated gene transfer and fluorescence-activated cell sorting to obtain cell lines that contain the gene encoding the large subunit of the human 4F2 antigen in a mouse L-cell background. Human DNAs cloned from these cell lines were subsequently used as hybridization probes to isolate a full-length cDNA clone expressing 4F2. Sequence analysis of the coding region has revealed an amino acid sequence of 529 residues. Hydrophobicity plotting has predicted a probable structure for the protein that includes an external carboxyl terminus, an internal leader sequence, a single hydrophobic transmembrane domain, and two possible membrane-associated domains. The 4F2 cDNA detects a single 1.8-kilobase mRNA in T-cell and B-cell lines. RNA gel blot analysis of RNA derived from quiescent and serum-stimulated Swiss 3T3 fibroblasts reveals a cell-cycle modulation of 4F2 gene expression: the mRNA is present in quiescent fibroblasts but increases 8-fold 24-36 hr after stimulation, at the time of maximal DNA synthesis.

Keratinocyte growth factor mRNA expression in periodontal ligament fibroblasts

DEFF Research Database (Denmark)

Dabelsteen, S; Wandall, H H; Grøn, B

1997-01-01

Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF mRNA is expres......Keratinocyte growth factor (KGF) is a fibroblast growth factor which mediates epithelial growth and differentiation. KGF is expressed in subepithelial fibroblasts, but generally not in fibroblasts of deep connective tissue, such as fascia and ligaments. Here we demonstrate that KGF m......RNA is expressed in periodontal ligament fibroblasts, and that the expression is increased upon serum stimulation. Fibroblasts from human periodontal ligament, from buccal mucosa, from gingiva, and from skin were established from explants. Alkaline phosphatase activity was used as an indicator of the periodontal...

Human tissue factor: cDNA sequence and chromosome localization of the gene

International Nuclear Information System (INIS)

Scarpati, E.M.; Wen, D.; Broze, G.J. Jr.; Miletich, J.P.; Flandermeyer, R.R.; Siegel, N.R.; Sadler, J.E.

1987-01-01

A human placenta cDNA library in λgt11 was screened for the expression of tissue factor antigens with rabbit polyclonal anti-human tissue factor immunoglobulin G. Among 4 million recombinant clones screened, one positive, λHTF8, expressed a protein that shared epitopes with authentic human brain tissue factor. The 1.1-kilobase cDNA insert of λHTF8 encoded a peptide that contained the amino-terminal protein sequence of human brain tissue factor. Northern blotting identified a major mRNA species of 2.2 kilobases and a minor species of ∼ 3.2 kilobases in poly(A) + RNA of placenta. Only 2.2-kilobase mRNA was detected in human brain and in the human monocytic U937 cell line. In U937 cells, the quantity of tissue factor mRNA was increased several fold by exposure of the cells to phorbol 12-myristate 13-acetate. Additional cDNA clones were selected by hybridization with the cDNA insert of λHTF8. These overlapping isolates span 2177 base pairs of the tissue factor cDNA sequence that includes a 5'-noncoding region of 75 base pairs, an open reading frame of 885 base pairs, a stop codon, a 3'-noncoding region of 1141 base pairs, and a poly(a) tail. The open reading frame encodes a 33-kilodalton protein of 295 amino acids. The predicted sequence includes a signal peptide of 32 or 34 amino acids, a probable extracellular factor VII binding domain of 217 or 219 amino acids, a transmembrane segment of 23 acids, and a cytoplasmic tail of 21 amino acids. There are three potential glycosylation sites with the sequence Asn-X-Thr/Ser. The 3'-noncoding region contains an inverted Alu family repetitive sequence. The tissue factor gene was localized to chromosome 1 by hybridization of the cDNA insert of λHTF8 to flow-sorted human chromosomes

Gene expression of fibroblast growth factors in human gliomas and meningiomas: Demonstration of cellular source of basic fibroblast growth factor mRNA and peptide in tumor tissues

International Nuclear Information System (INIS)

Takahashi, J.A.; Mori, Hirotaka; Fukumoto, Manabu; Oda, Yoshifumi; Kikuchi, Haruhiko; Hatanaka, Masakazu; Igarashi, Koichi; Jaye, M.

1990-01-01

The growth autonomy of human tumor cells is considered due to the endogenous production of growth factors. Transcriptional expression of candidates for autocrine stimulatory factors such as basic fibroblast growth factor (FGF), acidic FGF, and transforming growth factor type β were determined in human brain tumors. Basic FGF was expressed abundantly in 17 of 18 gliomas, 20 of 22 meningiomas, and 0 of 5 metastatic brain tumors. The level of mRNA expression of acidic FGF in gliomas was significant. In contrast, transforming growth factor type β1 was expressed in all the samples investigated. The mRNA for basic FGF and its peptide were localized in tumor cells in vivo by in situ hybridization and immunohistochemistry, showing that basic FGF is actually produced in tumor cells. The results suggest that tumor-derived basic FGF is involved in the progression of gliomas and meningiomas in vivo, whereas acidic FGF is expressed in a tumor origin-specific manner, suggesting that acidic FGF works in tandem with basic FGF in glioma tumorigenesis

Radioimmunoassay of human Hageman factor (factor XII)

International Nuclear Information System (INIS)

Saito, H.; Ratnoff, O.D.; Pensky, J.

1976-01-01

A specific, sensitive, and reproducible radioimmunoassay for human Hageman factor (HF, factor XII) has been developed with purified human HF and monospecific rabbit antibody. Precise measurements of HF antigen were possible for concentrations as low as 0.1 percent of that in normal pooled plasma. A good correlation (correlation coefficient = 0.82) existed between the titers of HF measured by clot-promoting assays and radioimmunoassays among 42 normal adults. Confirming earlier studies, HF antigen was absent in Hageman trait plasma, but other congenital deficient plasmas, including those of individuals with Fletcher trait and Fitzgerald trait, contained normal amounts of HF antigen. HF antigen was reduced in the plasmas of patients with disseminated intravascular coagulation or advanced liver cirrhosis, but it was normal in those of patients with chronic renal failure or patients under treatment with warfarin. HF antigen was detected by this assay in plasmas of primates, but not detectable in plasmas of 11 nonprimate mammalian and one avian species

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

International Nuclear Information System (INIS)

Lazarus, Kyren A.; Zhao, Zhe; Knower, Kevin C.; To, Sarah Q.; Chand, Ashwini L.; Clyne, Colin D.

2013-01-01

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E 2 ), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E 2 , showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E 2 treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer

Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

Energy Technology Data Exchange (ETDEWEB)

Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

2013-08-30

Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER−ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER− and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor α (ERα) positive (ER+) breast cancer cells compared to ER− cells. However, the presence of LRH-1 protein in ER− cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER− breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER− compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER− versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ERα. Our data demonstrates that in ER− cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER− cells as well as ER− tumors suggests a possible role in the development of ER− tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER− and ER+ breast cancer.

Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate

Directory of Open Access Journals (Sweden)

Deutsch Eric W

2008-05-01

Full Text Available Abstract Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate – basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial – and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63. Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50 but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.

Cup regulates oskar mRNA stability during oogenesis.

Science.gov (United States)

Broyer, Risa M; Monfort, Elena; Wilhelm, James E

2017-01-01

The proper regulation of the localization, translation, and stability of maternally deposited transcripts is essential for embryonic development in many organisms. These different forms of regulation are mediated by the various protein subunits of the ribonucleoprotein (RNP) complexes that assemble on maternal mRNAs. However, while many of the subunits that regulate the localization and translation of maternal transcripts have been identified, relatively little is known about how maternal mRNAs are stockpiled and stored in a stable form to support early development. One of the best characterized regulators of maternal transcripts is Cup - a broadly conserved component of the maternal RNP complex that in Drosophila acts as a translational repressor of the localized message oskar. In this study, we have found that loss of cup disrupts the localization of both the oskar mRNA and its associated proteins to the posterior pole of the developing oocyte. This defect is not due to a failure to specify the oocyte or to disruption of RNP transport. Rather, the localization defects are due to a drop in oskar mRNA levels in cup mutant egg chambers. Thus, in addition to its role in regulating oskar mRNA translation, Cup also plays a critical role in controlling the stability of the oskar transcript. This suggests that Cup is ideally positioned to coordinate the translational control function of the maternal RNP complex with its role in storing maternal transcripts in a stable form. Published by Elsevier Inc.

Electroporation of mRNA as Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

Science.gov (United States)

Gerer, Kerstin F; Hoyer, Stefanie; Dörrie, Jan; Schaft, Niels

2017-01-01

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than a decade to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs), and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers, and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs for therapeutic vaccination in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types, (2) scalability from 10 6 to approximately 10 8 cells per shot, (3) high transfection efficiency (80-99 %), (4) homogenous protein expression, (5) GMP compliance if the EP is performed in a class A clean room, and (6) no transgene integration into the genome. The provided protocol involves: Opti-MEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time is altered. Next to the basic protocol, we also provide an extensive list of hints and tricks, which in our opinion are of great value for everyone who intends to use this transfection technique.

T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

Science.gov (United States)

Fauser, S; Kern, P

1997-04-01

In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response.

Effect of human vascular endothelial growth factor gene transfer on endogenous vascular endothelial growth factor mRNA expression in a rat fibroblast and osteoblast culture model.

Science.gov (United States)

Li, Ru; Li, Claire H; Nauth, Aaron; McKee, Michael D; Schemitsch, Emil H

2010-09-01

Vascular endothelial growth factor (VEGF) plays an important role in promoting angiogenesis and osteogenesis during fracture repair. Our previous studies have shown that cell-based VEGF gene therapy enhances bone healing of a rabbit tibia segmental bone defect in vivo. The aim of this project was to examine the effect of exogenous human VEGF on the endogenous rat VEGF messenger RNA (mRNA) expression in a cell-based gene transfer model. Rat fibroblasts and osteoblasts were harvested from the dermal tissue and periosteum, respectively, of Fisher 344 rats. The cells were then cultured and transfected with pcDNA-human VEGF using Superfect reagent (Qiagen). Four experimental groups were created: 1) fibroblast-VEGF; 2) osteoblast-VEGF; 3) nontransfected fibroblast controls; and 4) nontransfected osteoblast controls. The cultured cells were harvested at 1, 3, and 7 days after the gene transfection. The total mRNA was extracted (Trizol; Invitrogen); both human VEGF and rat VEGF mRNA were measured by reverse transcriptase-polymerase chain reaction and quantified by VisionWorksLS. The human VEGF165 mRNA was detected by reverse transcriptase-polymerase chain reaction from transfected fibroblasts and osteoblasts at 1, 3, and 7 days after gene transfection. The human VEGF165 levels peaked at Day 1 and then gradually reduced expression in both transfected fibroblasts and osteoblasts. Two endogenous rat VEGF isoforms were detected in this cell culture model: rat VEGF120 and rat VEGF164. We compared the rat VEGF120 and rat VEGF164 expression level of the fibroblasts or osteoblasts that were transfected with human VEGF165, with nontransfected control cells. Both the transfected fibroblasts and osteoblasts showed greater expression of rat VEGF164 than nontransfected controls at Day 1 (peak level) and Day 3, but not at Day 7. The expression of rat VEGF120 was lower in transfected fibroblasts, but higher in transfected osteoblasts, than the relevant control groups at any time point

Development of Anti-Human Mesothelin-Targeted Chimeric Antigen Receptor Messenger RNA-transfected Peripheral Blood Lymphocytes for Ovarian Cancer Therapy.

Science.gov (United States)

Hung, Chien-Fu; Xu, Xuequn; Li, Linhong; Ma, Ying; Jin, Qiu; Viley, Angelia; Allen, Cornell; Natarajan, Pachai; Shivakumar, Rama; Peshwa, Madhusudan V; Emens, Leisha A

2018-04-02

CD19-targeted chimeric antigen receptor (CAR) engineered T/natural killer (NK)-cell therapies can result in durable clinical responses in B-cell malignancies. However, CAR-based immunotherapies have been much less successful in solid cancers, in part due to "on-target off-tumor" toxicity related to expression of target tumor antigens on normal tissue. Based on preliminary observations of safety and clinical activity in proof-of-concept clinical trials, tumor antigen-specific messenger RNA (mRNA) CAR transfection into selected, activated, and expanded T/NK cells may permit prospective control of "on-target off-tumor" toxicity. To develop a commercial product for solid tumors, mesothelin was selected as an antigen target based on its association with poor prognosis and overexpression in multiple solid cancers. It was hypothesized that selecting, activating, and expanding cells ex vivo prior to mRNA CAR transfection would not be necessary, thus simplifying the complexity and cost of manufacturing. Now, the development of anti-human mesothelin mRNA CAR transfected peripheral blood lymphocytes (CARMA-hMeso) is reported, demonstrating the manufacture and cryopreservation of multiple cell aliquots for repeat administrations from a single human leukapheresis. A rapid, automated, closed system for cGMP-compliant transfection of mRNA CAR in up to 20 × 10 9 peripheral blood lymphocytes was developed. Here we show that CARMA-hMeso cells recognize and lyse tumor cells in a mesothelin-specific manner. Expression of CAR was detectable over approximately 7 days in vitro, with a progressive decline of CAR expression that appears to correlate with in vitro cell expansion. In a murine ovarian cancer model, a single intraperitoneal injection of CARMA-hMeso resulted in the dose-dependent inhibition of tumor growth and improved survival of mice. Furthermore, repeat weekly intraperitoneal administrations of the optimal CARMA-hMeso dose further prolonged disease control and survival

Studies on the isolation, structural analysis and tissue localization of fetal antigen 1 and its relation to a human adrenal-specific cDNA, pG2

DEFF Research Database (Denmark)

Jensen, Charlotte Harken; Teisner, Børge; Højrup, Peter

1993-01-01

Fetal antigen 1 was purified from second trimester human amniotic fluid by immunospecific affinity chromatography followed by reversed-phase chromatography. Fetal antigen 1 is a single chain glycoprotein with a M(r) of 32-38 kDa. The amino acid composition revealed a high content of cysteines......, prolines and amino acids (aa) with acidic side-chains indicating that fetal antigen 1 is a compactly folded, strongly hydrophilic molecule. The N-terminal amino acid sequence (37 aa) revealed no homology to other known protein sequences, implying that fetal antigen 1 is a 'novel' human protein. When the aa...... sequence was back-translated into the appropriate degenerate sequence of nucleic acids, fetal antigen 1 could be partially aligned to a 'human adrenal-specific mRNA, pG2'. The indirect immunoperoxidase technique demonstrated fetal antigen 1 in fetal hepatocytes, glandular cells of fetal pancreas...

A universal trend of reduced mRNA stability near the translation-initiation site in prokaryotes and eukaryotes.

Directory of Open Access Journals (Sweden)

Wanjun Gu

2010-02-01

Full Text Available Recent studies have suggested that the thermodynamic stability of mRNA secondary structure near the start codon can regulate translation efficiency in Escherichia coli, and that translation is more efficient the less stable the secondary structure. We survey the complete genomes of 340 species for signals of reduced mRNA secondary structure near the start codon. Our analysis includes bacteria, archaea, fungi, plants, insects, fishes, birds, and mammals. We find that nearly all species show evidence for reduced mRNA stability near the start codon. The reduction in stability generally increases with increasing genomic GC content. In prokaryotes, the reduction also increases with decreasing optimal growth temperature. Within genomes, there is variation in the stability among genes, and this variation correlates with gene GC content, codon bias, and gene expression level. For birds and mammals, however, we do not find a genome-wide trend of reduced mRNA stability near the start codon. Yet the most GC rich genes in these organisms do show such a signal. We conclude that reduced stability of the mRNA secondary structure near the start codon is a universal feature of all cellular life. We suggest that the origin of this reduction is selection for efficient recognition of the start codon by initiator-tRNA.

A small RNA activates CFA synthase by isoform-specific mRNA stabilization.

Science.gov (United States)

Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

2013-11-13

Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability.

Self-amplifying mRNA vaccines.

Science.gov (United States)

Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

2015-01-01

This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

Suberoylanilide hydroxamic acid (SAHA) inhibits EGF-induced cell transformation via reduction of cyclin D1 mRNA stability

International Nuclear Information System (INIS)

Zhang, Jingjie; Ouyang, Weiming; Li, Jingxia; Zhang, Dongyun; Yu, Yonghui; Wang, York; Li, Xuejun; Huang, Chuanshu

2012-01-01

Suberoylanilide hydroxamic acid (SAHA) inhibiting cancer cell growth has been associated with its downregulation of cyclin D1 protein expression at transcription level or translation level. Here, we have demonstrated that SAHA inhibited EGF-induced Cl41 cell transformation via the decrease of cyclin D1 mRNA stability and induction of G0/G1 growth arrest. We found that SAHA treatment resulted in the dramatic inhibition of EGF-induced cell transformation, cyclin D1 protein expression and induction of G0/G1 growth arrest. Further studies showed that SAHA downregulation of cyclin D1 was only observed with endogenous cyclin D1, but not with reconstitutionally expressed cyclin D1 in the same cells, excluding the possibility of SAHA regulating cyclin D1 at level of protein degradation. Moreover, SAHA inhibited EGF-induced cyclin d1 mRNA level, whereas it did not show any inhibitory effect on cyclin D1 promoter-driven luciferase reporter activity under the same experimental conditions, suggesting that SAHA may decrease cyclin D1 mRNA stability. This notion was supported by the results that treatment of cells with SAHA decreased the half-life of cyclin D1 mRNA from 6.95 h to 2.57 h. Consistent with downregulation of cyclin D1 mRNA stability, SAHA treatment also attenuated HuR expression, which has been well-characterized as a positive regulator of cyclin D1 mRNA stability. Thus, our study identifies a novel mechanism responsible for SAHA inhibiting cell transformation via decreasing cyclin D1 mRNA stability and induction of G0/G1 growth arrest in Cl41 cells. -- Highlights: ► SAHA inhibits cell transformation in Cl41 cells. ► SAHA suppresses Cyclin D1 protein expression. ► SAHA decreases cyclin D1 mRNA stability.

Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

International Nuclear Information System (INIS)

Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M.

1990-01-01

The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures

Lea blood group antigen on human platelets

International Nuclear Information System (INIS)

Dunstan, R.A.; Simpson, M.B.; Rosse, W.F.

1985-01-01

One- and two-stage radioligand assays were used to determine if human platelets possess the Lea antigen. Goat IgG anti-Lea antibody was purified by multiple adsorptions with Le(a-b-) human red blood cells, followed by affinity chromatography with synthetic Lea substance and labeling with 125 I. Human IgG anti-Lea antibody was used either in a two stage radioassay with 125 I-labeled mouse monoclonal IgG anti-human IgG as the second antibody or, alternatively, purified by Staph protein A chromatography, labeled with 125 I, and used in a one-stage radioassay. Platelets from donors of appropriate red blood cell phenotypes were incubated with the antisera, centrifuged through phthalate esters, and assayed in a gamma scintillation counter. Dose response and saturation curve analysis demonstrate the presence of Lewis a antigen on platelets from Lea+ donors. Furthermore, platelets from an Le(a-b-) donor incubated in Le (a+b-) plasma adsorb Lea antigen in a similar manner to red blood cells. The clinical significance of these antigens in platelet transfusion remains undefined

Precision cancer immunotherapy: optimizing dendritic cell-based strategies to induce tumor antigen-specific T-cell responses against individual patient tumors.

Science.gov (United States)

Osada, Takuya; Nagaoka, Koji; Takahara, Masashi; Yang, Xiao Yi; Liu, Cong-Xiao; Guo, Hongtao; Roy Choudhury, Kingshuk; Hobeika, Amy; Hartman, Zachary; Morse, Michael A; Lyerly, H Kim

2015-05-01

Most dendritic cell (DC)-based vaccines have loaded the DC with defined antigens, but loading with autologos tumor-derived antigens would generate DCs that activate personalized tumor-specific T-cell responses. We hypothesized that DC matured with an optimized combination of reagents and loaded with tumor-derived antigens using a clinically feasible electroporation strategy would induce potent antitumor immunity. We first studied the effects on DC maturation and antigen presentation of the addition of picibanil (OK432) to a combination of zoledronic acid, tumor necrosis factor-α, and prostaglandin E2. Using DC matured with the optimized combination, we tested 2 clinically feasible sources of autologous antigen for electroloading, total tumor mRNA or total tumor lysate, to determine which stimulated more potent antigen-specific T cells in vitro and activated more potent antitumor immunity in vivo. The combination of tumor necrosis factor-α/prostaglandin E2/zoledronic acid/OK432 generated DC with high expression of maturation markers and antigen-specific T-cell stimulatory function in vitro. Mature DC electroloaded with tumor-derived mRNA [mRNA electroporated dendritic cell (EPDC)] induced greater expansion of antigen-specific T cells in vitro than DC electroloaded with tumor lysate (lysate EPDC). In a therapeutic model of MC38-carcinoembryonic antigen colon cancer-bearing mice, vaccination with mRNA EPDC induced the most efficient anti-carcinoembryonic antigen cellular immune response, which significantly suppressed tumor growth. In conclusion, mature DC electroloaded with tumor-derived mRNA are a potent cancer vaccine, especially useful when specific tumor antigens for vaccination have not been identified, allowing autologous tumor, and if unavailable, allogeneic cell lines to be used as an unbiased source of antigen. Our data support clinical testing of this strategy.

Photobiomodulation effects on mRNA levels from genomic and chromosome stabilization genes in injured muscle.

Science.gov (United States)

da Silva Neto Trajano, Larissa Alexsandra; Trajano, Eduardo Tavares Lima; da Silva Sergio, Luiz Philippe; Teixeira, Adilson Fonseca; Mencalha, Andre Luiz; Stumbo, Ana Carolina; de Souza da Fonseca, Adenilson

2018-04-26

Muscle injuries are the most prevalent type of injury in sports. A great number of athletes have relapsed in muscle injuries not being treated properly. Photobiomodulation therapy is an inexpensive and safe technique with many benefits in muscle injury treatment. However, little has been explored about the infrared laser effects on DNA and telomeres in muscle injuries. Thus, the aim of this study was to evaluate photobiomodulation effects on mRNA relative levels from genes related to telomere and genomic stabilization in injured muscle. Wistar male rats were randomly divided into six groups: control, laser 25 mW, laser 75 mW, injury, injury laser 25 mW, and injury laser 75 mW. Photobiomodulation was performed with 904 nm, 3 J/cm 2 at 25 or 75 mW. Cryoinjury was induced by two applications of a metal probe cooled in liquid nitrogen directly on the tibialis anterior muscle. After euthanasia, skeletal muscle samples were withdrawn and total RNA extracted for evaluation of mRNA levels from genomic (ATM and p53) and chromosome stabilization (TRF1 and TRF2) genes by real-time quantitative polymerization chain reaction. Data show that photobiomodulation reduces the mRNA levels from ATM and p53, as well reduces mRNA levels from TRF1 and TRF2 at 25 and 75 mW in injured skeletal muscle. In conclusion, photobiomodulation alters mRNA relative levels from genes related to genomic and telomere stabilization in injured skeletal muscle.

Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines

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Barredo Julio C

2002-03-01

Full Text Available Abstract Background We describe an alternative method to determine mRNA half-life (t1/2 based on the Real-Time RT-PCR procedure. This approach was evaluated by using the β-actin gene as a reference molecule for measuring of mRNA stability. Results Human leukemia Nalm-6 and CCRF-CEM cells were treated with various concentrations of Actinomycin D to block transcription and aliquots were removed periodically. Total RNA was isolated and quantified using the RiboGreen® fluorescent dye with the VersaFluor Fluorometer System. One μg of total RNA was reverse transcribed and used as template for the amplification of a region of the β-actin gene (231 bp. To generate the standard curve, serial ten-fold dilutions of the pBactin-231 vector containing the cDNA amplified fragment were employed, β-actin mRNAs were quantified by Real-Time RT-PCR using the SYBR® Green I fluorogenic dye and data analyzed using the iCycle iQ system software. Using this method, the β-actin mRNA exhibited a half-life of 6.6 h and 13.5 h in Nalm-6 and CCRF-CEM cells, respectively. The t1/2 value obtained for Nalm-6 is comparable to those estimated from Northern blot studies, using normal human leukocytes (5.5 h. Conclusions We have developed a rapid, sensitive, and reliable method based on Real-Time RT-PCR for measuring mRNA half-life. Our results confirm that β-actin mRNA half-life can be affected by the cellular growth rate.

The physical stability of the recombinant tuberculosis fusion antigens h1 and h56

DEFF Research Database (Denmark)

Hamborg, Mette; Kramer, Ryan; Schanté, Carole E

2013-01-01

a gradual loss of secondary as well as tertiary structure as a function of temperature, with no cooperative transitions observed. Preformulation studies with the Th1-inducing cationic adjuvant CAF01 showed that the antigens were almost completely surface adsorbed. Upon adsorption, the liposome size...... and it is therefore important to characterize their conformational stability in solution as well as upon interaction with adjuvants. In this study, the physical stability of the two antigens was characterized using a number of biophysical techniques. Dynamic light scattering and sodium dodecyl sulfate...... increased; however, the physical stabilities of the bound and the unbound antigens were comparable. This study provides important information about the biophysical properties of H1 and H56 and highlights the analytical challenges of characterizing complex vaccine formulations....

Detection of melatonin receptor mRNA in human muscle

International Nuclear Information System (INIS)

Li Lei

2004-01-01

To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

A radioimmunoassay for human antibody specific for microbial antigens

International Nuclear Information System (INIS)

Tew, J.G.; Burmeister, J.; Greene, E.J.; Pflaumer, S.K.; Goldstein, J.

1977-01-01

A simple and sensitive method for detecting and quantitating antibody specific or microbial antigens is described. Bacterial, fungal, parasitic or viral antigens attached to bromoacetyl cellulose or the intact cells themselves were added to a series of two-fold dilutions of human serum. After a short incubation period, which allowed human antibody to attach to the antigens, the complex was thoroughly washed and carbon-14 labeled anti-human light chain antibody was added to each dilution. The resulting complex was washed, collected on a filter pad, placed in a scintillation vial and radioassayed. The relationship between radioactivity bound and -log 2 of the serum dilution was linear. The endpoint for each assay and a confidence interval was calculated by doing inverse prediction from simple linear regression. Results obtained using this assay indicated the presence of antibody in a pool of normal human sera specific for herpes virus and for both cell surface and intracellular antigens of Streptococcus mutans, Naegleria fowleri and Cryptococcus neoformans. In general the dominant response was against the intracellular antigens rather than cell surface antigens

Nucleolin and YB-1 are required for JNK-mediated interleukin-2 mRNA stabilization during T-cell activation

DEFF Research Database (Denmark)

Chen, C Y; Gherzi, R; Andersen, Jens S.

2000-01-01

Regulated mRNA turnover is a highly important process, but its mechanism is poorly understood. Using interleukin-2 (IL-2) mRNA as a model, we described a role for the JNK-signaling pathway in stabilization of IL-2 mRNA during T-cell activation, acting via a JNK response element (JRE) in the 5' un...

Nerve growth factor mRNA in brain: localization by in situ hybridization

International Nuclear Information System (INIS)

Rennert, P.D.; Heinrich, G.

1986-01-01

Nerve Growth Factor is a 118 amino acid polypeptide that plays an important role in the differentiation and survival of neurons. The recent discovery that a mRNA that encodes beta Nerve Growth Factor is present in brain suggests that the Nerve Growth Factor gene may not only regulate gene expression of peripheral but also of central neurons. To identify the site(s) of Nerve Growth Factor mRNA production in the brain and to determine which cells express the Nerve Growth Factor gene, the technique of in situ hybridization was employed. A 32P-labeled RNA probe complementary to Nerve Growth Factor mRNA hybridized to cells in the stratum granulosum of the dentate gyrus and the stratum pyramidale of the hippocampus. These observations identify for the first time cellular sites of Nerve Growth Factor gene expression in the central nervous system, and suggest that Nerve Growth Factor mRNA is produced by neurons

Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA.

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Lu, Song; Chow, Christie C; Zhou, Junwei; Leung, Po Sing; Tsui, Stephen K; Lui, Kathy O

2016-01-01

In this chapter, we describe a highly efficient genetic modification strategy for human pancreatic progenitor cells using modified mRNA-encoding GFP and Neurogenin-3. The properties of modified mRNA offer an invaluable platform to drive protein expression, which has broad applicability in pathway regulation, directed differentiation, and lineage specification. This approach can also be used to regulate expression of other pivotal transcription factors during pancreas development and might have potential therapeutic values in regenerative medicine.

PKA- and PKC-dependent regulation of angiopoietin 2 mRNA in human granulosa lutein cells.

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Witt, P S; Pietrowski, D; Keck, C

2004-02-01

New blood vessels develop from preexisting vessels in response to growth factors or hypoxic conditions. Recent studies have shown that angiopoietin 2 (ANGPT-2) plays an important role in the modulation of angiogenesis and vasculogenesis in humans and mice. The signaling pathways that lead to the regulation of ANGPT-2 are largely unclear. Here, we report that protein kinase C and protein kinase A activators (ADMB, 8-Cl-cAMP) increased the mRNA levels of ANGPT-2 in human Granulosa cells, whereas PKC and PKA Inhibitors (Rp-cAMP, GO 6983) decreased markedly the level of ANGPT-2 mRNA. Due to varying specificity of the modulators for certain protein kinases subunits, we conclude that the conventional PKCs, but not PKC alpha and beta1, the atypical PKCs and the PKA I, are involved in the regulation of ANGPT-2. These findings may help to explain the role of both PKA and PKC dependent signaling cascades in the regulation of ANGPT-2 mRNA.

Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies

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Britta Schneider

2012-01-01

Full Text Available Bispecific antibodies (bsAbs that bind to cell surface antigens and to digoxigenin (Dig were used for targeted small interfering RNA (siRNA delivery. They are derivatives of immunoglobulins G (IgGs that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs or into lipid-based nanoparticles (LNPs. The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

Science.gov (United States)

2016-09-07

sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

Factors associated with anti-human leukocyte antigen antibodies in patients supported with continuous-flow devices and effect on probability of transplant and post-transplant outcomes

DEFF Research Database (Denmark)

Alba, Ana C; Tinckam, Kathryn; Foroutan, Farid

2015-01-01

BACKGROUND: One major disadvantage of ventricular assist device (VAD) therapy is the development of human-leukocyte antigen (HLA) antibodies. We aimed to identify factors associated with HLA antibodies during continuous flow (CF)-VAD support and assess the effect on transplant probability...

Nonsense mutations in the human β-globin gene affect mRNA metabolism

International Nuclear Information System (INIS)

Baserga, S.J.; Benz, E.J. Jr.

1988-01-01

A number of premature translation termination mutations (nonsense mutations) have been described in the human α- and β-globin genes. Studies on mRNA isolated from patients with β 0 -thalassemia have shown that for both the β-17 and the β-39 mutations less than normal levels of β-globin mRNA accumulate in peripheral blood cells. (The codon at which the mutation occurs designates the name of the mutation; there are 146 codons in human β-globin mRNA). In vitro studies using the cloned β-39 gene have reproduced this effect in a heterologous transfection system and have suggested that the defect resides in intranuclear metabolism. The authors have asked if this phenomenon of decreased mRNA accumulation is a general property of nonsense mutations and if the effect depends on the location or the type of mutation. Toward this end, they have studied the effect of five nonsense mutations and two missense mutations on the expression of human β-globin mRNA in a heterologous transfection system. In all cases studied, the presence of a translation termination codon correlates with a decrease in the steady-state level of mRNA. The data suggest that the metabolism of a mammalian mRNA is affected by the presence of a mutation that affects translation

Monoclonal Antibody Production against Human Spermatozoal Surface Antigens

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M Jedi-Tehrani

2005-10-01

Full Text Available Introduction: As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. Material and Methods: To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund’s adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Results: Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. Conclusion: The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization.

Radioimmunoassay for a human prostate specific antigen

International Nuclear Information System (INIS)

Machida, T.; Miki, M.; Ohishi, Y.; Kido, A.; Morikawa, J.; Ogawa, Y.

1983-01-01

As a marker for prostatic cancer, a prostate-specific antigen was purified from human prostatic tissues. Double antibody radioimmunoassay utilizing immune reaction was developed on the basis of the purified prostatic antigen (PA). Measurement results have revealed that PA radioimmunoassay is much better than prostatic acid phosphatase (PAP) radioimmunoassay in the diagnosis of prostatic cancer

Radioimmunoassay for tumor antigen of human cervical squamous cell carcinoma

International Nuclear Information System (INIS)

Kato, H.; Torigoe, T.

1977-01-01

A heterologous antiserum for human cervical squamous cell carcinoma was prepared and specificity determined by Ouchterlony immunodiffusion and immunofluorescence studies. With this antiserum, a tumor antigen was purified from human cervical squamous cell carcinoma tissue. The specificities of the antigen and the antiserum were then re-examined by a radioimmunoassay method using 125 I-labeled purified antigen. Although normal cervical tissue extract showed a moderate cross-reactivity in the radioimmunoassay, the circulating antigen activity could not be detected in normal women or in several patients with other carcinomas, whereas 27 of 35 patients with cervical squamous cell carcinoma showed detectable serum antigen activity. All patients with advanced stages of cervical squamous cell carcinoma showed detectable antigen levels. These results indicate that there is a quantitative abnormality, at least, of this tumor antigen in patients with cervical squamous cell carcinoma and that the radioimmunoassay for the antigen is a potentially useful tool in clinical care

Antigen presentation and MHC class II expression by human esophageal epithelial cells: role in eosinophilic esophagitis.

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Mulder, Daniel J; Pooni, Aman; Mak, Nanette; Hurlbut, David J; Basta, Sameh; Justinich, Christopher J

2011-02-01

Professional antigen-presenting cells (APCs) play a crucial role in initiating immune responses. Under pathological conditions, epithelial cells at mucosal surfaces act as nonprofessional APCs, thereby regulating immune responses at the site of exposure. Epithelial cells in the esophagus may contribute to the pathogenesis of eosinophilic esophagitis (EoE) by presenting antigens on the major histocompatibility complex (MHC) class II. Our goal was to demonstrate the ability of esophageal epithelial cells to process and present antigens on the MHC class II system and to investigate the contribution of epithelial cell antigen presentation to EoE. Immunohistochemistry detected HLA-DR, CD80, and CD86 expression and enzyme-linked immunosorbent assay detected interferon-γ (IFNγ) in esophageal biopsies. Antigen presentation was studied using the human esophageal epithelial cell line HET-1A by reverse transcriptase-PCR, flow cytometry, and confocal microscopy. T helper cell lymphocyte proliferation was assessed by flow cytometry and IL-2 secretion. IFNγ and MHC class II were increased in mucosa of patients with EoE. IFNγ increased mRNA of HLA-DP, HLA-DQ, HLA-DR, and CIITA in HET-1A cells. HET-1A engulfed cell debris and processed ovalbumin. HET-1A cells expressed HLA-DR after IFNγ treatment. HET-1A stimulated T helper cell activation. In this study, we demonstrated the ability of esophageal epithelial cells to act as nonprofessional APCs in the presence of IFNγ. Esophageal epithelial cell antigen presentation may contribute to the pathophysiology of eosinophilic esophagitis. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

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Daniela Toro-Ascuy

2016-11-01

Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

Generation of human β-thalassemia induced pluripotent cell lines by reprogramming of bone marrow-derived mesenchymal stromal cells using modified mRNA.

Science.gov (United States)

Varela, Ioanna; Karagiannidou, Angeliki; Oikonomakis, Vasilis; Tzetis, Maria; Tzanoudaki, Marianna; Siapati, Elena-Konstantina; Vassilopoulos, George; Graphakos, Stelios; Kanavakis, Emmanuel; Goussetis, Evgenios

2014-12-01

Synthetic modified mRNA molecules encoding pluripotency transcription factors have been used successfully in reprogramming human fibroblasts to induced pluripotent stem cells (iPSCs). We have applied this method on bone marrow-derived mesenchymal stromal cells (BM-MSCs) obtained from a patient with β-thalassemia (β-thal) with the aim to generate trangene-free β-thal-iPSCs. Transfection of 10(4) BM-MSCs by lipofection with mRNA encoding the reprogramming factors Oct4, Klf4, Sox2, cMyc, and Lin28 resulted in formation of five iPSC colonies, from which three were picked up and expanded in β-thal-iPSC lines. After 10 serial passages in vitro, β-thal-iPSCs maintain genetic stability as shown by array comparative genomic hybridization (aCGH) and are capable of forming embryoid bodies in vitro and teratomas in vivo. Their gene expression profile compared to human embryonic stem cells (ESCs) and BM-MSCs seems to be similar to that of ESCs, whereas it differs from the profile of the parental BM-MSCs. Differentiation cultures toward a hematopoietic lineage showed the generation of CD34(+) progenitors up to 10%, but with a decreased hematopoietic colony-forming capability. In conclusion, we report herein the generation of transgene-free β-thal-iPSCs that could be widely used for disease modeling and gene therapy applications. Moreover, it was demonstrated that the mRNA-based reprogramming method, used mainly in fibroblasts, is also suitable for reprogramming of human BM-MSCs.

Molecular cloning of cDNA for the human tumor-associated antigen CO-029 and identification of related transmembrane antigens

International Nuclear Information System (INIS)

Szala, S.; Kasai, Yasushi; Steplewski, Z.; Rodeck, U.; Koprowski, H.; Linnenbach, A.J.

1990-01-01

The human tumor-associated antigen CO-029 is a monoclonal antibody-defined cell surface glycoprotein of 27-34 kDa. By using the high-efficiency COS cell expression system, a full-length cDNA clone for CO-029 was isolated. When transiently expressed in COS cells, the cDNA clone directed the synthesis of an antigen reactive to monoclonal antibody CO-029 in mixed hemadsorption and immunoblot assays. Sequence analysis revealed that CO-029 belongs to a family of cell surface antigens that includes the melanoma-associated antigen ME491, the leukocyte cell surface antigen CD37, and the Sm23 antigen of the parasitic helminth Schistosoma mansoni. CO-029 and ME491 antigen expression and the effect of their corresponding monoclonal antibodies on cell growth were compared in human tumor cell lines of various histologic origins

Effective antigen presentation to helper T cells by human eosinophils.

Science.gov (United States)

Farhan, Ruhaifah K; Vickers, Mark A; Ghaemmaghami, Amir M; Hall, Andrew M; Barker, Robert N; Walsh, Garry M

2016-12-01

Although eosinophils are inflammatory cells, there is increasing attention on their immunomodulatory roles. For example, murine eosinophils can present antigen to CD4 + T helper (Th) cells, but it remains unclear whether human eosinophils also have this ability. This study determined whether human eosinophils present a range of antigens, including allergens, to activate Th cells, and characterized their expression of MHC class II and co-stimulatory molecules required for effective presentation. Human peripheral blood eosinophils purified from non-allergic donors were pulsed with the antigens house dust mite extract (HDM), Timothy Grass extract (TG) or Mycobacterium tuberculosis purified protein derivative (PPD), before co-culture with autologous CD4 + Th cells. Proliferative and cytokine responses were measured, with eosinophil expression of HLA-DR/DP/DQ and the co-stimulatory molecules CD40, CD80 and CD86 determined by flow cytometry. Eosinophils pulsed with HDM, TG or PPD drove Th cell proliferation, with the response strength dependent on antigen concentration. The cytokine responses varied with donor and antigen, and were not biased towards any particular Th subset, often including combinations of pro- and anti-inflammatory cytokines. Eosinophils up-regulated surface expression of HLA-DR/DP/DQ, CD80, CD86 and CD40 in culture, increases that were sustained over 5 days when incubated with antigens, including HDM, or the major allergens it contains, Der p I or Der p II. Human eosinophils can, therefore, act as effective antigen-presenting cells to stimulate varied Th cell responses against a panel of antigens including HDM, TG or PPD, an ability that may help to determine the development of allergic disease. © 2016 John Wiley & Sons Ltd.

Regular endurance training reduces the exercise induced HIF-1alpha and HIF-2alpha mRNA expression in human skeletal muscle in normoxic conditions

DEFF Research Database (Denmark)

Lundby, Carsten; Gassmann, Max; Pilegaard, Henriette

2005-01-01

and 2 (HIFs) are clearly related heterodimeric transcription factors that consist of an oxygen-depended alpha-subunit and a constitutive beta-subunit. With hypoxic exposure, HIF-1alpha and HIF-2alpha protein are stabilized. Upon heterodimerization, HIFs induce the transcription of a variety of genes......Regular exercise induces a variety of adaptive responses that enhance the oxidative and metabolic capacity of human skeletal muscle. Although the physiological adjustments of regular exercise have been known for decades, the underlying mechanisms are still unclear. The hypoxia inducible factors 1...... including erythropoietin (EPO), transferrin and its receptor, as well as vascular endothelial growth factor (VEGF) and its receptor. Considering that several of these genes are also induced with exercise, we tested the hypothesis that the mRNA level of HIF-1alpha and HIF-2alpha subunits increases...

CBFA1 and topoisomerase I mRNA levels decline during cellular aging of human trabecular osteoblasts

DEFF Research Database (Denmark)

Christiansen, Mette; Kveiborg, M.; Kassem, M.

2000-01-01

In order to understand the reasons for age-related impairment of the function of bone forming osteoblasts, we have examined the steady-state mRNA levels of the transcription factor CBFA1 and topoisomerase I during cellular aging of normal human trabecular osteoblasts, by the use of semiquantitati...

Epstein-Barr virus nuclear antigen 3C stabilizes Gemin3 to block p53-mediated apoptosis.

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Qiliang Cai

2011-12-01

Full Text Available The Epstein-Barr nuclear antigen 3C (EBNA3C, one of the essential latent antigens for Epstein-Barr virus (EBV-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103 is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs. Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities.

The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli.

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Esquerré, Thomas; Bouvier, Marie; Turlan, Catherine; Carpousis, Agamemnon J; Girbal, Laurence; Cocaign-Bousquet, Muriel

2016-04-26

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

mRNA transfection of mouse and human neural stem cell cultures.

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Samuel McLenachan

Full Text Available The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

mRNA Transfection of Mouse and Human Neural Stem Cell Cultures

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J.; Chen, Fred K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages. PMID:24386231

mRNA transfection of mouse and human neural stem cell cultures.

Science.gov (United States)

McLenachan, Samuel; Zhang, Dan; Palomo, Ana Belén Alvarez; Edel, Michael J; Chen, Fred K

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has not been previously reported. Here we sought to establish an efficient method for delivering mRNA to primary neurosphere cultures. Neurospheres derived from the subventricular zone of adult mice or from human embryonic stem cells were transfected with EGFP mRNA by lipofection and electroporation. Transfection efficiency and expression levels were monitored by flow cytometry. Cell survival following transfection was examined using live cell counting and the MTT assay. Both lipofection and electroporation provided high efficiency transfection of neurospheres. In comparison with lipofection, electroporation resulted in increased transfection efficiencies, but lower expression per cell and shorter durations of expression. Additional rounds of lipofection renewed EGFP expression in neurospheres, suggesting this method may be suitable for reprogramming applications. In summary, we have developed a protocol for achieving high efficiency transfection rates in mouse and human neurosphere cell culture that can be applied for future studies of gene function studies in neural stem cells, such as defining efficient differentiation protocols for glial and neuronal linages.

Generation of human induced pluripotent stem cells using non-synthetic mRNA.

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Rohani, L; Fabian, C; Holland, H; Naaldijk, Y; Dressel, R; Löffler-Wirth, H; Binder, H; Arnold, A; Stolzing, A

2016-05-01

Here we describe some of the crucial steps to generate induced pluripotent stem cells (iPSCs) using mRNA transfection. Our approach uses a V. virus-derived capping enzyme instead of a cap-analog, ensuring 100% proper cap orientation for in vitro transcribed mRNA. V. virus' 2'-O-Methyltransferase enzyme creates a cap1 structure found in higher eukaryotes and has higher translation efficiency compared to other methods. Use of the polymeric transfection reagent polyethylenimine proved superior to other transfection methods. The mRNA created via this method did not trigger an intracellular immune response via human IFN-gamma (hIFN-γ) or alpha (hIFN-α) release, thus circumventing the use of suppressors. Resulting mRNA and protein were expressed at high levels for over 48h, thus obviating daily transfections. Using this method, we demonstrated swift activation of pluripotency associated genes in human fibroblasts. Low oxygen conditions further facilitated colony formation. Differentiation into different germ layers was confirmed via teratoma assay. Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Evaluation of ascitic soluble human leukocyte antigen-G for distinguishing malignant ascites from benign ascites.

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Sun, Juan; Chang, Yan-Xiang; Niu, Chun-Yan

2017-11-01

The overexpression of soluble human leukocyte antigen-G is associated with malignant tumours. The purpose of our study was to detect soluble human leukocyte antigen-G concentrations in ascites and to evaluate the value of ascitic soluble human leukocyte antigen-G for the diagnosis of malignant ascites. Enzyme-linked immunosorbent assay was used to detect soluble human leukocyte antigen-G levels in 64 patients with malignant ascites and 30 patients with benign ascites. Receiver operating characteristic curves were used to evaluate the diagnostic efficacy of ascitic soluble human leukocyte antigen-G for the detection of malignant ascites. Ascitic soluble human leukocyte antigen-G levels were significantly higher in the malignant ascites group than in the benign ascites group (20.718 ± 3.215 versus 12.467 ± 3.678 µg/L, t = 7.425, p human leukocyte antigen-G was 0.957 (95% confidence interval, 0.872-0.992). At a cut-off value of 19.60 µg/L, the sensitivity and specificity of ascitic soluble human leukocyte antigen-G were 87.5% (95% confidence interval, 71.0%-96.5%) and 100% (95% confidence interval, 88.4%-100%), respectively. With respect to area under the receiver operating characteristic curve, sensitivity and specificity, ascitic carcinoembryonic antigen (0.810, 68.75% and 83.33%, respectively) and carbohydrate antigen 19-9 (0.710, 65.63% and 70%, respectively) significantly differed (all p human leukocyte antigen-G was 75%, which was higher than the corresponding rates for ascitic carcinoembryonic antigen (31.25%) and carbohydrate antigen 19-9 (6.25%; both p human leukocyte antigen-G exhibited good performance for diagnosing malignant ascites, and particularly those that were cytology-negative and biopsy-positive.

A leukocyte antigen, Leu-13, is involved in induction of resistance of human cells to x-ray cell killing by interferon-α

International Nuclear Information System (INIS)

Kita, Kazuko; Zhai, Ling; Sugaya, Shigeru; Suzuki, Nobuo

2003-01-01

We previously reported on human interferon (HuIFN)-induced resistance of human cells to X-ray and UV cell killing. In this study, we searched for the genes whose expression is responsible for the resistance, using a PCR-based mRNA differential display method and Northern blotting analysis. RSa cells were used for this analysis, because they show increased resistance to X-ray- and UV-caused cell killing by HuIFN-α treatment prior to irradiation. Messenger RNA expression levels for Leu-13, a leukocyte antigen, were markedly up-regulated in RSa cells after HuIFN-α treatment. Furthermore, pretreatment of RSa cells with antisense oligonucleotides for Leu-13 mRNA resulted in the suppression of the HuIFN-α-induced resistance of the cells to X-ray cell killing, but did not modulate HuIFN-α-induced resistance to UV cell killing. These results suggest that Leu-13 is involved in HuIFN-α-induced resistance of human cells to X-ray cell killing, but not to UV cell killing. (author)

Efficient mRNA delivery with graphene oxide-polyethylenimine for generation of footprint-free human induced pluripotent stem cells.

Science.gov (United States)

Choi, Hye Yeon; Lee, Tae-Jin; Yang, Gwang-Mo; Oh, Jaesur; Won, Jihye; Han, Jihae; Jeong, Gun-Jae; Kim, Jongpil; Kim, Jin-Hoi; Kim, Byung-Soo; Cho, Ssang-Goo

2016-08-10

Clinical applications of induced pluripotent stem cells (iPSCs) require development of technologies for the production of "footprint-free" (gene integration-free) iPSCs, which avoid the potential risk of insertional mutagenesis in humans. Previously, several studies have shown that mRNA transfer can generate "footprint-free" iPSCs, but these studies did not use a delivery vehicle and thus repetitive daily transfection was required because of mRNA degradation. Here, we report an mRNA delivery system employing graphene oxide (GO)-polyethylenimine (PEI) complexes for the efficient generation of "footprint-free" iPSCs. GO-PEI complexes were found to be very effective for loading mRNA of reprogramming transcription factors and protection from mRNA degradation by RNase. Dynamic suspension cultures of GO-PEI/RNA complexes-treated cells dramatically increased the reprogramming efficiency and successfully generated rat and human iPSCs from adult adipose tissue-derived fibroblasts without repetitive daily transfection. The iPSCs showed all the hallmarks of pluripotent stem cells including expression of pluripotency genes, epigenetic reprogramming, and differentiation into the three germ layers. These results demonstrate that mRNA delivery using GO-PEI-RNA complexes can efficiently generate "footprint-free" iPSCs, which may advance the translation of iPSC technology into the clinical settings. Copyright © 2016. Published by Elsevier B.V.

Women's attitude towards routine human platelet antigen-screening in pregnancy.

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Winkelhorst, Dian; Loeff, Rosanne M; van den Akker-Van Marle, M Elske; de Haas, Masja; Oepkes, Dick

2017-08-01

Fetal and neonatal alloimmune thrombocytopenia is a potentially life-threatening disease with excellent preventative treatment available for subsequent pregnancies. To prevent index cases, the effectiveness of a population-based screening program has been suggested repeatedly. Therefore, we aimed to evaluate women's attitude towards possible future human platelet antigen-screening in pregnancy. We performed a cross-sectional questionnaire study among healthy pregnant women receiving prenatal care in one of seven participating midwifery practices. Attitude was assessed using a questionnaire based on the validated Multidimensional Measurement of Informed Choice model, containing questions assessing knowledge, attitude and intention to participate. A total of 143 of the 220 women (65%) completed and returned the questionnaire. A positive attitude towards human platelet antigen-screening was expressed by 91% of participants, of which 94% was based on sufficient knowledge. Attitude was more likely to be negatively influenced by the opinion that screening can be frightening. Informed choices were made in 87% and occurred significantly less in women from non-European origin, 89% in European women vs. 60% in non-European women (p = 0.03). Pregnant women in the Netherlands expressed a positive attitude towards human platelet antigen-screening in pregnancy. We therefore expect a high rate of informed uptake when human platelet antigen-screening is implemented. In future counseling on human platelet antigen-screening, ethnicity and possible anxiety associated with a screening test need to be specifically addressed. © 2017 Nordic Federation of Societies of Obstetrics and Gynecology.

Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue.

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Borges, Marcus Nascimento; Facina, Gil; Silva, Ismael Dale Cotrin Guerreiro; Waitzberg, Angela Flávia Logullo; Nazario, Afonso Celso Pinto

2011-12-01

Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13) in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75) was statistically larger (P fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

Alternative splicing of cyclooxygenase-1 mRNA in the human iris

NARCIS (Netherlands)

Dröge, M.J; van Sorge, A.A; van Haeringen, N.J; Quax, Wim; Zaagsma, Hans; Droge, MJ

2003-01-01

dIn homogenates of the human iris, the nonsteroidal antiinflammatory drug (NSAID) S(+)flurbiprofen has been reported to inhibit cyclooxygenase-1 (COX-1) 70-fold more potently than in human whole blood. We hypothesized that this difference may be due to alternative splicing of COX-1 mRNA in the human

Activation of pluripotency genes in human fibroblast cells by a novel mRNA based approach.

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Jordan R Plews

2010-12-01

Full Text Available Several methods have been used to induce somatic cells to re-enter the pluripotent state. Viral transduction of reprogramming genes yields higher efficiency but involves random insertions of viral sequences into the human genome. Although induced pluripotent stem (iPS cells can be obtained with the removable PiggyBac transposon system or an episomal system, both approaches still use DNA constructs so that resulting cell lines need to be thoroughly analyzed to confirm they are free of harmful genetic modification. Thus a method to change cell fate without using DNA will be very useful in regenerative medicine.In this study, we synthesized mRNAs encoding OCT4, SOX2, cMYC, KLF4 and SV40 large T (LT and electroporated them into human fibroblast cells. Upon transfection, fibroblasts expressed these factors at levels comparable to, or higher than those in human embryonic stem (ES cells. Ectopically expressed OCT4 localized to the cell nucleus within 4 hours after mRNA introduction. Transfecting fibroblasts with a mixture of mRNAs encoding all five factors significantly increased the expression of endogenous OCT4, NANOG, DNMT3β, REX1 and SALL4. When such transfected fibroblasts were also exposed to several small molecules (valproic acid, BIX01294 and 5'-aza-2'-deoxycytidine and cultured in human embryonic stem cell (ES medium they formed small aggregates positive for alkaline phosphatase activity and OCT4 protein within 30 days.Our results demonstrate that mRNA transfection can be a useful approach to precisely control the protein expression level and short-term expression of reprogramming factors is sufficient to activate pluripotency genes in differentiated cells.

BAG3 directly stabilizes Hexokinase 2 mRNA and promotes aerobic glycolysis in pancreatic cancer cells.

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An, Ming-Xin; Li, Si; Yao, Han-Bing; Li, Chao; Wang, Jia-Mei; Sun, Jia; Li, Xin-Yu; Meng, Xiao-Na; Wang, Hua-Qin

2017-12-04

Aerobic glycolysis, a phenomenon known historically as the Warburg effect, is one of the hallmarks of cancer cells. In this study, we characterized the role of BAG3 in aerobic glycolysis of pancreatic ductal adenocarcinoma (PDAC) and its molecular mechanisms. Our data show that aberrant expression of BAG3 significantly contributes to the reprogramming of glucose metabolism in PDAC cells. Mechanistically, BAG3 increased Hexokinase 2 (HK2) expression, the first key enzyme involved in glycolysis, at the posttranscriptional level. BAG3 interacted with HK2 mRNA, and the degree of BAG3 expression altered recruitment of the RNA-binding proteins Roquin and IMP3 to the HK2 mRNA. BAG3 knockdown destabilized HK2 mRNA via promotion of Roquin recruitment, whereas BAG3 overexpression stabilized HK2 mRNA via promotion of IMP3 recruitment. Collectively, our results show that BAG3 promotes reprogramming of glucose metabolism via interaction with HK2 mRNA in PDAC cells, suggesting that BAG3 may be a potential target in the aerobic glycolysis pathway for developing novel anticancer agents. © 2017 An et al.

In human granulosa cells from small antral follicles, androgen receptor mRNA and androgen levels in follicular fluid correlate with FSH receptor mRNA

DEFF Research Database (Denmark)

Nielsen, M. E.; Rasmussen, I. A.; Kristensen, S. G.

2011-01-01

significantly with the expression of AMHRII, but did not correlate with any of the hormones in the follicular fluid. These data demonstrate an intimate association between AR expression in immature granulosa cells, and the expression of FSHR in normal small human antral follicles and between the follicular......Human small antral follicles (diameter 3-9 mm) were obtained from ovaries surgically removed for fertility preservation. From the individual aspirated follicles, granulosa cells and the corresponding follicular fluid were isolated in 64 follicles, of which 55 were available for mRNA analysis (24...... and to the follicular fluid concentrations of AMH, inhibin-B, progesterone and estradiol. AR mRNA expression in granulosa cells and the follicular fluid content of androgens both showed a highly significant positive association with the expression of FSHR mRNA in granulosa cells. AR mRNA expression also correlated...

Conserved CPEs in the p53 3' untranslated region influence mRNA stability and protein synthesis

DEFF Research Database (Denmark)

Rosenstierne, Maiken W; Vinther, Jeppe; Mittler, Gerhard

2008-01-01

CaT skin and MCF-7 breast cancer cell lines were established. Quantitative PCR and an enzymatic assay were used to quantify the reporter mRNA and protein levels, respectively. Proteins binding to the CPEs were identified by RNA-immunoprecipitation (IP) and quantitative mass spectroscopy. RESULTS: The wild...... irradiation. Several proteins (including GAPDH, heterogeneous nuclear ribonucleoprotein (hnRNP) D and A/B) were identified from the MCF-7 cytoplasmic extracts that bound specifically to the CPEs. CONCLUSION: Two conserved CPEs in the p53 3'UTR regulate stability and translation of a reporter mRNA in non...

Effects of radiation on the expression of antigens on the membranes of human adenocarcinoma cells

International Nuclear Information System (INIS)

Hareyama, Masato; Imai, Kozo; Oouchi, Atsushi; Shidou, Mitsuo; Takahashi, Hiroki; Koshiba, Hirohumi; Yachi, Akira; Morita, Kazuo

1992-01-01

We have investigated the effects of irradiation on the membranes of tumor cells. X-ray irradiation caused remarkable increases in the expression of tumor-associated antigens (YH206, CEA) and c-erbB-2 protein by flow cytometry, whereas IFN had no obvious effect on the expression of tumor-associated antigens and c-erbB-2 protein. On the other hand, the expression of MHC Class I and ICAM-1 antigen on the membrane by flow cytometry was enhanced by both irradiation and IFN. In addition, the irradiated cells, when analyzed using a CEA specific probe, showed remarkable increases in the CEA mRNA compared to IFN-treated cells. It is possible that enhancement of the expression of tumor-associated antigens and c-erbB-2 protein, together with the enhancement of that of MHC-Class I and ICAM-1, would help cytotoxic killer cells recognize the tumor cells. (author)

Shark Variable New Antigen Receptor (VNAR Single Domain Antibody Fragments: Stability and Diagnostic Applications

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Stewart Nuttall

2013-01-01

Full Text Available The single variable new antigen receptor domain antibody fragments (VNARs derived from shark immunoglobulin new antigen receptor antibodies (IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds. As single domains, they demonstrate favorable size and cryptic epitope recognition properties, making them attractive in diagnosis and therapy of numerous disease states. Here, we examine the stability of VNAR domains with a focus on a family of VNARs specific for apical membrane antigen 1 (AMA-1 from Plasmodium falciparum. The VNARs are compared to traditional monoclonal antibodies (mAbs in liquid, lyophilized and immobilized nitrocellulose formats. When maintained in various formats at 45 °C, VNARs have improved stability compared to mAbs for periods of up to four weeks. Using circular dichroism spectroscopy we demonstrate that VNAR domains are able to refold following heating to 80 °C. We also demonstrate that VNAR domains are stable during incubation under potential in vivo conditions such as stomach acid, but not to the protease rich environment of murine stomach scrapings. Taken together, our results demonstrate the suitability of shark VNAR domains for various diagnostic platforms and related applications.

Analysis of CD83 antigen expression in human breast fibroadenoma and adjacent tissue

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Marcus Nascimento Borges

Full Text Available CONTEXT AND OBJECTIVE: Dendritic cell maturation is considered essential for starting an immune response. The CD83 antigen is an important marker of dendritic cell maturation. The objectives here were to analyze CD83 antigen expression in human breast fibroadenoma and breast tissue adjacent to the lesion and to identify clinical factors that might influence this expression. DESIGN AND SETTING: This was a retrospective study at a public university hospital, in which 29 histopathological samples of breast fibroadenoma and adjacent breast tissue, from 28 women of reproductive age, were analyzed. METHODS: The immunohistochemistry method was used to analyze the cell expression of the antigen. The antigen expression in the cells was evaluated by means of random manual counting using an optical microscope. RESULTS: Positive expression of the CD83 antigen in the epithelial cells of the fibroadenoma (365.52; standard deviation ± 133.13 in relation to the adjacent breast tissue cells (189.59; standard deviation ± 140.75 was statistically larger (P < 0.001. Several clinical features were analyzed, but only parity was shown to influence CD83 antigen expression in the adjacent breast tissue, such that positive expression was more evident in nulliparous women (P = 0.042. CONCLUSIONS: The expression of the CD83 antigen in the fibroadenoma was positive and greater than in the adjacent breast tissue. Positive expression of the antigen in the adjacent breast tissue was influenced by parity, and was significantly more evident in nulliparous women.

Human Tumor Antigens Yesterday, Today, and Tomorrow.

Science.gov (United States)

Finn, Olivera J

2017-05-01

The question of whether human tumors express antigens that can be recognized by the immune system has been answered with a resounding YES. Most were identified through spontaneous antitumor humoral and cellular immune responses found in cancer patients and include peptides, glycopeptides, phosphopeptides, viral peptides, and peptides resulting from common mutations in oncogenes and tumor-suppressor genes, or common gene fusion events. Many have been extensively tested as candidates for anticancer vaccines. More recently, attention has been focused on the potentially large number of unique tumor antigens, mutated neoantigens, that are the predicted products of the numerous mutations revealed by exome sequencing of primary tumors. Only a few have been confirmed as targets of spontaneous immunity and immunosurveillance, and even fewer have been tested in preclinical and clinical settings. The field has been divided for a long time on the relative importance of shared versus mutated antigens in tumor surveillance and as candidates for vaccines. This question will eventually need to be answered in a head to head comparison in well-designed clinical trials. One advantage that shared antigens have over mutated antigens is their potential to be used in vaccines for primary cancer prevention. Cancer Immunol Res; 5(5); 347-54. ©2017 AACR . ©2017 American Association for Cancer Research.

Transcription factor fos-related antigen-2 induces progressive peripheral vasculopathy in mice closely resembling human systemic sclerosis.

Science.gov (United States)

Maurer, Britta; Busch, Nicole; Jüngel, Astrid; Pileckyte, Margarita; Gay, Renate E; Michel, Beat A; Schett, Georg; Gay, Steffen; Distler, Jörg; Distler, Oliver

2009-12-08

Microvascular damage is one of the first pathological changes in systemic sclerosis. In this study, we investigated the role of Fos-related antigen-2 (Fra-2), a transcription factor of the activator protein-1 family, in the peripheral vasculopathy of systemic sclerosis and examined the underlying mechanisms. Expression of Fra-2 protein was significantly increased in skin biopsies of systemic sclerosis patients compared with healthy controls, especially in endothelial and vascular smooth muscle cells. Fra-2 transgenic mice developed a severe loss of small blood vessels in the skin that was paralleled by progressive skin fibrosis at 12 weeks of age. The reduction in capillary density was preceded by a significant increase in apoptosis in endothelial cells at week 9 as detected by immunohistochemistry. Similarly, suppression of Fra-2 by small interfering RNA prevented human microvascular endothelial cells from staurosporine-induced apoptosis and improved both the number of tubes and the cumulative tube lengths in the tube formation assay. In addition, cell migration in the scratch assay and vascular endothelial growth factor-dependent chemotaxis in a modified Boyden chamber assay were increased after transfection of human microvascular endothelial cells with Fra-2 small interfering RNA, whereas proliferation was not affected. Fra-2 is present in human systemic sclerosis and may contribute to the development of microvasculopathy by inducing endothelial cell apoptosis and by reducing endothelial cell migration and chemotaxis. Fra-2 transgenic mice are a promising preclinical model to study the mechanisms and therapeutic approaches of the peripheral vasculopathy in systemic sclerosis.

Ochratoxin A inhibits the production of tissue factor and plasminogen activator inhibitor-2 by human blood mononuclear cells: Another potential mechanism of immune-suppression

International Nuclear Information System (INIS)

Rossiello, Maria R.; Rotunno, Crescenzia; Coluccia, Addolorata; Carratu, Maria R.; Di Santo, Angelomaria; Evangelista, Virgilio; Semeraro, Nicola; Colucci, Mario

2008-01-01

The mycotoxin ochratoxin A (OTA), an ubiquitous contaminant of food products endowed with a wide spectrum of toxicity, affects several functions of mononuclear leukocytes. Monocytes/macrophages play a major role in fibrin accumulation associated with immune-inflammatory processes through the production of tissue factor (TF) and plasminogen activator inhibitor 2 (PAI-2). We studied the effect of OTA on TF and PAI-2 production by human blood mononuclear cells (MNC). The cells were incubated for 3 or 18 h at 37 deg. C with non toxic OTA concentrations in the absence and in the presence of lipopolysaccharide (LPS) or other inflammatory agents. TF activity was measured by a one-stage clotting test. Antigen assays were performed by specific ELISAs in cell extracts or conditioned media and specific mRNAs were assessed by RT-PCR. OTA had no direct effect on TF and PAI-2 production by MNC. However, OTA caused a dose-dependent reduction in LPS-induced TF (activity, antigen and mRNA) and PAI-2 (antigen and mRNA) production with > 85% inhibition at 1 μg/ml. Similar results were obtained when monocyte-enriched preparations were used instead of MNC. TF production was also impaired by OTA (1 μg/ml) when MNC were stimulated with phorbol myristate acetate (98% inhibition), IL-1β (83%) or TNF-α (62%). The inhibition of TF and PAI-2 induction might represent a hitherto unrecognized mechanism whereby OTA exerts immunosuppressant activity

New insights into the interplay between the translation machinery and nonsense-mediated mRNA decay factors.

Science.gov (United States)

Raimondeau, Etienne; Bufton, Joshua C; Schaffitzel, Christiane

2018-06-19

Faulty mRNAs with a premature stop codon (PTC) are recognized and degraded by nonsense-mediated mRNA decay (NMD). Recognition of a nonsense mRNA depends on translation and on the presence of NMD-enhancing or the absence of NMD-inhibiting factors in the 3'-untranslated region. Our review summarizes our current understanding of the molecular function of the conserved NMD factors UPF3B and UPF1, and of the anti-NMD factor Poly(A)-binding protein, and their interactions with ribosomes translating PTC-containing mRNAs. Our recent discovery that UPF3B interferes with human translation termination and enhances ribosome dissociation in vitro , whereas UPF1 is inactive in these assays, suggests a re-interpretation of previous experiments and modification of prevalent NMD models. Moreover, we discuss recent work suggesting new functions of the key NMD factor UPF1 in ribosome recycling, inhibition of translation re-initiation and nascent chain ubiquitylation. These new findings suggest that the interplay of UPF proteins with the translation machinery is more intricate than previously appreciated, and that this interplay quality-controls the efficiency of termination, ribosome recycling and translation re-initiation. © 2018 The Author(s).

Human Tregs Made Antigen Specific by Gene Modification: The Power to Treat Autoimmunity and Antidrug Antibodies with Precision

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Patrick R. Adair

2017-09-01

Full Text Available Human regulatory CD4+ T cells (Tregs are potent immunosuppressive lymphocytes responsible for immune tolerance and homeostasis. Since the seminal reports identifying Tregs, vast research has been channeled into understanding their genesis, signature molecular markers, mechanisms of suppression, and role in disease. This research has opened the doors for Tregs as a potential therapeutic for diseases and disorders such as multiple sclerosis, type I diabetes, transplantation, and immune responses to protein therapeutics, like factor VIII. Seminal clinical trials have used polyclonal Tregs, but the frequency of antigen-specific Tregs among polyclonal populations is low, and polyclonal Tregs may risk non-specific immunosuppression. Antigen-specific Treg therapy, which uses genetically modified Tregs expressing receptors specific for target antigens, greatly mitigates this risk. Building on the principles of T-cell receptor cloning, chimeric antigen receptors (CARs, and a novel CAR derivative, called B-cell antibody receptors, our lab has developed different types of antigen-specific Tregs. This review discusses the current research and optimization of gene-modified antigen-specific human Tregs in our lab in several disease models. The preparations and considerations for clinical use of such Tregs also are discussed.

mRNA related to insulin family in human placenta

International Nuclear Information System (INIS)

Younes, M.A.; D'Agostino, J.B.; Frazier, M.L.; Besch, P.K.

1986-01-01

The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A + ) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A + ) RNA templates. Five hundred transformants were initially screened by colony hybridization using a 32 P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II

mRNA related to insulin family in human placenta

Energy Technology Data Exchange (ETDEWEB)

Younes, M.A.; D' Agostino, J.B.; Frazier, M.L.; Besch, P.K.

1986-03-01

The authors have previously reported that human term placenta contains mRNA displaying sequence homology to a rat preproinsulin I cDNA clone (p119). When placental poly(A/sup +/) RNA was analyzed for homology to p119 by RNA/DNA blot hybridization, prominent hybridization was observed which was found by densitometric analysis to be three-fold higher than control. To further characterize this insulin-like message, a cDNA library was generated (approx.7000 transformants) using normal term cesarean-sectioned tissue to prepare placental poly(A/sup +/) RNA templates. Five hundred transformants were initially screened by colony hybridization using a /sup 32/P-labeled rat preproinsulin I cDNA as probe. Of the ten initial positives obtained, three were found to be true positives based on Southern hybridization analyses of the recombinant plasmids. Using Taq I digested pBr322 as a size marker, the cDNAs were found to be approximately 300 bp in length. Preliminary DNA sequencing using the Sanger dideoxy chain termination method has revealed that one of these clones displays significant homology to the 5' region of human insulin-like growth factors I and II.

Antigenic and genetic variability of human metapneumoviruses

NARCIS (Netherlands)

S. Herfst (Sander); L. Sprong; P.A. Cane; E. Forleo-Neto; A.D.M.E. Osterhaus (Albert); R.A.M. Fouchier (Ron); R.L. de Swart (Rik); B.G. van den Hoogen (Bernadette)

2004-01-01

textabstractHuman metapneumovirus (HMPV) is a member of the subfamily Pneumovirinae within the family Paramyxo- viridae. Other members of this subfamily, respiratory syncytial virus and avian pneumovirus, can be divided into subgroups on the basis of genetic or antigenic differences or both. For

Healthy human T-Cell Responses to Aspergillus fumigatus antigens.

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Neelkamal Chaudhary

2010-02-01

Full Text Available Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H1-T(H17 and destructive allergic (T(H2 immunity. How Aspergillus allergens (Asp f proteins participate in the development of allergic sensitization is unknown.To determine whether Asp f proteins are strictly associated with T(H2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17 to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H1-responses to Asp f3 (a putative peroxismal membrane protein, Asp f9/16 (cell wall glucanase, Asp f11 (cyclophilin type peptidyl-prolyl isomerase and Asp f22 (enolase. Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals.Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

Human antibody and antigen response to IncA antibody of Chlamydia trachomatis.

Science.gov (United States)

Tsai, P Y; Hsu, M C; Huang, C T; Li, S Y

2007-01-01

The high prevalence of C. trachomatis worldwide has underscored the importance of identifying specific immunogenic antigens in facilitating diagnosis as well as vaccine development. The aim of this study is to evaluate IncA antibody and antigen production in natural human infections. Our temporal expression study showed that IncA transcription and protein expression could be detected as early as 4 hours after the start of infection. Antibody responses could be detected in urine and genital swab samples from C. trachomatis-positive patients. It is especially interesting to note that the IncA antigen could be detected in urine. In conclusion, we have identified IncA as an important antigen in human. The potential applicability of the IncA antibody or antigen in the diagnosis as well as to vaccine development for C. trachomatis is also discussed.

Variable Domain N-Linked Glycans Acquired During Antigen-Specific Immune Responses Can Contribute to Immunoglobulin G Antibody Stability

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Fleur S. van de Bovenkamp

2018-04-01

Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

Science.gov (United States)

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Human antibody recognition of antigenic site IV on Pneumovirus fusion proteins.

Science.gov (United States)

Mousa, Jarrod J; Binshtein, Elad; Human, Stacey; Fong, Rachel H; Alvarado, Gabriela; Doranz, Benjamin J; Moore, Martin L; Ohi, Melanie D; Crowe, James E

2018-02-01

Respiratory syncytial virus (RSV) is a major human pathogen that infects the majority of children by two years of age. The RSV fusion (F) protein is a primary target of human antibodies, and it has several antigenic regions capable of inducing neutralizing antibodies. Antigenic site IV is preserved in both the pre-fusion and post-fusion conformations of RSV F. Antibodies to antigenic site IV have been described that bind and neutralize both RSV and human metapneumovirus (hMPV). To explore the diversity of binding modes at antigenic site IV, we generated a panel of four new human monoclonal antibodies (mAbs) and competition-binding suggested the mAbs bind at antigenic site IV. Mutagenesis experiments revealed that binding and neutralization of two mAbs (3M3 and 6F18) depended on arginine (R) residue R429. We discovered two R429-independent mAbs (17E10 and 2N6) at this site that neutralized an RSV R429A mutant strain, and one of these mAbs (17E10) neutralized both RSV and hMPV. To determine the mechanism of cross-reactivity, we performed competition-binding, recombinant protein mutagenesis, peptide binding, and electron microscopy experiments. It was determined that the human cross-reactive mAb 17E10 binds to RSV F with a binding pose similar to 101F, which may be indicative of cross-reactivity with hMPV F. The data presented provide new concepts in RSV immune recognition and vaccine design, as we describe the novel idea that binding pose may influence mAb cross-reactivity between RSV and hMPV. Characterization of the site IV epitope bound by human antibodies may inform the design of a pan-Pneumovirus vaccine.

Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

1985-12-01

Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae.

Mycobacterium leprae antigens involved in human immune responses. I. Identification of four antigens by monoclonal antibodies

International Nuclear Information System (INIS)

Britton, W.J.; Hellqvist, L.; Basten, A.; Raison, R.L.

1985-01-01

Four distinct antigens were identified in soluble sonicates of Mycobacterium leprae by using a panel of 11 monoclonal antibodies. Cross-reactivity studies with other mycobacterial species were conducted by using ELISA and immunoblot assays, and demonstrated that determinants on two of the antigens were present in many mycobacteria, whereas the other two were limited in distribution. Competitive inhibition experiments with radiolabeled monoclonal antibodies showed cross-inhibition between antibodies identifying two of the four antigenicbands. These two bands, of M/sub tau/ 4.5 to 6 KD and 30 to 40 KD, were resistant to protease treatment after immunoblotting. In contrast the two other bands of 16 and 70 KD were protease-sensitive. Although all four bands reacted with some human lepromatous leprosy sera in immunoblots, the 4.5 to 6 KD and 30 to 40 KD bands were most prominent. Lepromatous leprosy sera also inhibited the binding of radiolabeled monoclonal antibodies to each of the four antigens, with the mean titer causing 50% inhibition being higher for antibodies reacting with the 4.5 to 6 KD and 30 to 40 KD bands. These findings indicated that all four antigens were involved in the human B cell response to M. leprae

Stability-Diversity Tradeoffs Impose Fundamental Constraints on Selection of Synthetic Human VH/VL Single-Domain Antibodies from In Vitro Display Libraries.

Science.gov (United States)

Henry, Kevin A; Kim, Dae Young; Kandalaft, Hiba; Lowden, Michael J; Yang, Qingling; Schrag, Joseph D; Hussack, Greg; MacKenzie, C Roger; Tanha, Jamshid

2017-01-01

Human autonomous V H /V L single-domain antibodies (sdAbs) are attractive therapeutic molecules, but often suffer from suboptimal stability, solubility and affinity for cognate antigens. Most commonly, human sdAbs have been isolated from in vitro display libraries constructed via synthetic randomization of rearranged V H /V L domains. Here, we describe the design and characterization of three novel human V H /V L sdAb libraries through a process of: (i) exhaustive biophysical characterization of 20 potential V H /V L sdAb library scaffolds, including assessment of expression yield, aggregation resistance, thermostability and tolerance to complementarity-determining region (CDR) substitutions; (ii) in vitro randomization of the CDRs of three V H /V L sdAb scaffolds, with tailored amino acid representation designed to promote solubility and expressibility; and (iii) systematic benchmarking of the three V H /V L libraries by panning against five model antigens. We isolated ≥1 antigen-specific human sdAb against four of five targets (13 V H s and 7 V L s in total); these were predominantly monomeric, had antigen-binding affinities ranging from 5 nM to 12 µM (average: 2-3 µM), but had highly variable expression yields (range: 0.1-19 mg/L). Despite our efforts to identify the most stable V H /V L scaffolds, selection of antigen-specific binders from these libraries was unpredictable (overall success rate for all library-target screens: ~53%) with a high attrition rate of sdAbs exhibiting false positive binding by ELISA. By analyzing V H /V L sdAb library sequence composition following selection for monomeric antibody expression (binding to protein A/L followed by amplification in bacterial cells), we found that some V H /V L sdAbs had marked growth advantages over others, and that the amino acid composition of the CDRs of this set of sdAbs was dramatically restricted (bias toward Asp and His and away from aromatic and hydrophobic residues). Thus, CDR sequence

An MHC-restricted antibody-based chimeric antigen receptor requires TCR-like affinity to maintain antigen specificity

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Marcela V Maus

2016-01-01

Full Text Available Chimeric antigen receptors (CARs are synthetic receptors that usually redirect T cells to surface antigens independent of human leukocyte antigen (HLA. Here, we investigated a T cell receptor-like CAR based on an antibody that recognizes HLA-A*0201 presenting a peptide epitope derived from the cancer-testis antigen NY-ESO-1. We hypothesized that this CAR would efficiently redirect transduced T cells in an HLA-restricted, antigen-specific manner. However, we found that despite the specificity of the soluble Fab, the same antibody in the form of a CAR caused moderate lysis of HLA-A2 expressing targets independent of antigen owing to T cell avidity. We hypothesized that lowering the affinity of the CAR for HLA-A2 would improve its specificity. We undertook a rational approach of mutating residues that, in the crystal structure, were predicted to stabilize binding to HLA-A2. We found that one mutation (DN lowered the affinity of the Fab to T cell receptor-range and restored the epitope specificity of the CAR. DN CAR T cells lysed native tumor targets in vitro, and, in a xenogeneic mouse model implanted with two human melanoma lines (A2+/NYESO+ and A2+/NYESO−, DN CAR T cells specifically migrated to, and delayed progression of, only the HLA-A2+/NY-ESO-1+ melanoma. Thus, although maintaining MHC-restricted antigen specificity required T cell receptor-like affinity that decreased potency, there is exciting potential for CARs to expand their repertoire to include a broad range of intracellular antigens.

Large Scale Immune Profiling of Infected Humans and Goats Reveals Differential Recognition of Brucella melitensis Antigens

Science.gov (United States)

Liang, Li; Leng, Diana; Burk, Chad; Nakajima-Sasaki, Rie; Kayala, Matthew A.; Atluri, Vidya L.; Pablo, Jozelyn; Unal, Berkay; Ficht, Thomas A.; Gotuzzo, Eduardo; Saito, Mayuko; Morrow, W. John W.; Liang, Xiaowu; Baldi, Pierre; Gilman, Robert H.; Vinetz, Joseph M.; Tsolis, Renée M.; Felgner, Philip L.

2010-01-01

Brucellosis is a widespread zoonotic disease that is also a potential agent of bioterrorism. Current serological assays to diagnose human brucellosis in clinical settings are based on detection of agglutinating anti-LPS antibodies. To better understand the universe of antibody responses that develop after B. melitensis infection, a protein microarray was fabricated containing 1,406 predicted B. melitensis proteins. The array was probed with sera from experimentally infected goats and naturally infected humans from an endemic region in Peru. The assay identified 18 antigens differentially recognized by infected and non-infected goats, and 13 serodiagnostic antigens that differentiate human patients proven to have acute brucellosis from syndromically similar patients. There were 31 cross-reactive antigens in healthy goats and 20 cross-reactive antigens in healthy humans. Only two of the serodiagnostic antigens and eight of the cross-reactive antigens overlap between humans and goats. Based on these results, a nitrocellulose line blot containing the human serodiagnostic antigens was fabricated and applied in a simple assay that validated the accuracy of the protein microarray results in the diagnosis of humans. These data demonstrate that an experimentally infected natural reservoir host produces a fundamentally different immune response than a naturally infected accidental human host. PMID:20454614

Pattern of distribution of blood group antigens on human epidermal cells during maturation

DEFF Research Database (Denmark)

Dabelsteen, Erik; Buschard, Karsten; Hakomori, Sen-Itiroh

1984-01-01

The distribution in human epidermis of A, B, and H blood group antigens and of a precursor carbohydrate chain, N-acetyl-lactosamine, was examined using immunofluorescence staining techniques. The material included tissue from 10 blood group A, 4 blood group B, and 9 blood group O persons. Murine...... on the lower spinous cells whereas H antigen was seen predominantly on upper spinous cells or on the granular cells. Epithelia from blood group A or B persons demonstrated A or B antigens, respectively, but only if the tissue sections were trypsinized before staining. In such cases A or B antigens were found...... monoclonal antibodies were used to identify H antigen (type 2 chain) and N-acetyl-lactosamine. Human antisera were used to identify A and B antigens. In all groups N-acetyl-lactosamine and H antigen were found on the cell membranes of the spinous cell layer. N-acetyl-lactosamine was present mainly...

Human seminal proteinase and prostate-specific antigen are the ...

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/033/02/0195-0207. Keywords. Kallikrein; prostate cancer biomarker; proteinase activity; seminal plasma; tumour proliferation and metastasis; therapeutic target. Abstract. Human seminal proteinase and prostate-specific antigen (PSA) were each isolated from human seminal fluid and ...

Bat Caliciviruses and Human Noroviruses Are Antigenically Similar and Have Overlapping Histo-Blood Group Antigen Binding Profiles.

Science.gov (United States)

Kocher, Jacob F; Lindesmith, Lisa C; Debbink, Kari; Beall, Anne; Mallory, Michael L; Yount, Boyd L; Graham, Rachel L; Huynh, Jeremy; Gates, J Edward; Donaldson, Eric F; Baric, Ralph S

2018-05-22

Emerging zoonotic viral diseases remain a challenge to global public health. Recent surveillance studies have implicated bats as potential reservoirs for a number of viral pathogens, including coronaviruses and Ebola viruses. Caliciviridae represent a major viral family contributing to emerging diseases in both human and animal populations and have been recently identified in bats. In this study, we blended metagenomics, phylogenetics, homology modeling, and in vitro assays to characterize two novel bat calicivirus (BtCalV) capsid sequences, corresponding to strain BtCalV/A10/USA/2009, identified in Perimyotis subflavus near Little Orleans, MD, and bat norovirus. We observed that bat norovirus formed virus-like particles and had epitopes and receptor-binding patterns similar to those of human noroviruses. To determine whether these observations stretch across multiple bat caliciviruses, we characterized a novel bat calicivirus, BtCalV/A10/USA/2009. Phylogenetic analysis revealed that BtCalV/A10/USA/2009 likely represents a novel Caliciviridae genus and is most closely related to "recoviruses." Homology modeling revealed that the capsid sequences of BtCalV/A10/USA/2009 and bat norovirus resembled human norovirus capsid sequences and retained host ligand binding within the receptor-binding domains similar to that seen with human noroviruses. Both caliciviruses bound histo-blood group antigens in patterns that overlapped those seen with human and animal noroviruses. Taken together, our results indicate the potential for bat caliciviruses to bind histo-blood group antigens and overcome a significant barrier to cross-species transmission. Additionally, we have shown that bat norovirus maintains antigenic epitopes similar to those seen with human noroviruses, providing further evidence of evolutionary descent. Our results reiterate the importance of surveillance of wild-animal populations, especially of bats, for novel viral pathogens. IMPORTANCE Caliciviruses are

Protein structure of fetal antigen 1 (FA1). A novel circulating human epidermal-growth-factor-like protein expressed in neuroendocrine tumors and its relation to the gene products of dlk and pG2

DEFF Research Database (Denmark)

Jensen, Charlotte Harken; Krogh, Thomas N; Højrup, Peter

1994-01-01

The present paper describes the primary structure, glycosylation and tissue localization of fetal antigen 1 (FA1) isolated from second-trimester human amniotic fluid. FA1 is a single-chained, heterogeneous glycoprotein of 225-262 amino acid residues. FA1 has six well conserved epidermal...... extends with minor corrections to the human adrenal-specific mRNA, pG2 as well. Immunohistochemical analysis demonstrated the presence of FA1 in 10 out of 14 lung tumors containing neuroendocrine elements, and in the placental villi where FA1 was exclusively seen in stromal cells in close contact...... to the vascular structure. In the pancreas, FA1 co-localized with insulin in the insulin secretory granules of the beta cells within the islets of Langerhans. Our findings suggest that FA1 is synthesized as a membrane anchored protein and released into the circulation after enzymic cleavage, and that circulating...

Antigenic stability of pecan [Carya illinoinensis (Wangenh.) K. Koch] proteins: effects of thermal treatments and in vitro digestion.

Science.gov (United States)

Venkatachalam, Mahesh; Teuber, Suzanne S; Peterson, W Rich; Roux, Kenneth H; Sathe, Shridhar K

2006-02-22

Rabbit polyclonal antibody-based inhibition ELISA as well as immunoblotting analyses of proteins extracted from variously processed pecans (cv. Desirable) indicate that pecan proteins are antigenically stable. Pecan antigens were more sensitive to moist heat than dry heat processing treatments. SDS-PAGE and immunoblotting analysis of the native and heat-denatured proteins that were previously subjected to in vitro simulated gastric fluid digestions indicate that stable antigenic peptides were produced. Both enzyme-to-substrate ratio and digestion time were influential in determining the stability of pecan polypeptides. The stable antigenic polypeptides may serve as useful markers in developing assays suitable for the detection of trace amounts of pecans in foods.

Quantitative PCR--new diagnostic tool for quantifying specific mRNA and DNA molecules

DEFF Research Database (Denmark)

Schlemmer, B O; Sorensen, B S; Overgaard, J

2004-01-01

of a subset of ligands from the EGF system is increased in bladder cancer. Furthermore, measurement of the mRNA concentration gives important information such as the expression of these ligands correlated to the survival of the patients. In addition to the alterations at the mRNA level, changes also can occur...... at the DNA level in the EGF system. Thus, it has been demonstrated that the number of genes coding for the human epidermal growth factor receptor 2 (HER2) is increased in a number of breast tumors. It is now possible to treat breast cancer patients with a humanized antibody reacting with HER2...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression...

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination.

Science.gov (United States)

Revaud, Julien; Unterfinger, Yves; Rol, Nicolas; Suleman, Muhammad; Shaw, Julia; Galea, Sandra; Gavard, Françoise; Lacour, Sandrine A; Coulpier, Muriel; Versillé, Nicolas; Havenga, Menzo; Klonjkowski, Bernard; Zanella, Gina; Biacchesi, Stéphane; Cordonnier, Nathalie; Corthésy, Blaise; Ben Arous, Juliette; Richardson, Jennifer P

2018-01-01

To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.

Firewalls Prevent Systemic Dissemination of Vectors Derived from Human Adenovirus Type 5 and Suppress Production of Transgene-Encoded Antigen in a Murine Model of Oral Vaccination

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Julien Revaud

2018-01-01

Full Text Available To define the bottlenecks that restrict antigen expression after oral administration of viral-vectored vaccines, we tracked vectors derived from the human adenovirus type 5 at whole body, tissue, and cellular scales throughout the digestive tract in a murine model of oral delivery. After intragastric administration of vectors encoding firefly luciferase or a model antigen, detectable levels of transgene-encoded protein or mRNA were confined to the intestine, and restricted to delimited anatomical zones. Expression of luciferase in the form of multiple small bioluminescent foci in the distal ileum, cecum, and proximal colon suggested multiple crossing points. Many foci were unassociated with visible Peyer's patches, implying that transduced cells lay in proximity to villous rather than follicle-associated epithelium, as supported by detection of transgene-encoded antigen in villous epithelial cells. Transgene-encoded mRNA but not protein was readily detected in Peyer's patches, suggesting that post-transcriptional regulation of viral gene expression might limit expression of transgene-encoded antigen in this tissue. To characterize the pathways by which the vector crossed the intestinal epithelium and encountered sentinel cells, a fluorescent-labeled vector was administered to mice by the intragastric route or inoculated into ligated intestinal loops comprising a Peyer's patch. The vector adhered selectively to microfold cells in the follicle-associated epithelium, and, after translocation to the subepithelial dome region, was captured by phagocytes that expressed CD11c and lysozyme. In conclusion, although a large number of crossing events took place throughout the intestine within and without Peyer's patches, multiple firewalls prevented systemic dissemination of vector and suppressed production of transgene-encoded protein in Peyer's patches.

Typing of human fetal organs for the histocompatibility antigens A, B and DR.

Science.gov (United States)

Tuch, B E; Doran, T J; Messel, N; Turtle, J R

1985-01-01

In the transplantation of human fetal pancreatic explants into diabetic man, the importance of matching the histocompatibility antigens of donor and recipient to decrease the chances of rejection is unknown. Before this question can be answered human fetuses must be tissue typed. We have shown that lymphocytes harvested from fetal liver, thymus, bone marrow and spleen can be successfully HLA DR typed in 64% and A and B typed in 57% of 58 fetuses aged 15 wk or more. Typing should ideally be carried out on unseparated T and B cells. Best results were achieved if all four of the above organs were available and more than one million viable cells were able to be harvested for typing. Whilst the DR antigens could be typed from all tissues, the A and B antigens could be typed, with few exceptions only from thymus, spleen and bone marrow. The efficacy of matching the histocompatibility antigens of recipient and donor fetuses, especially the DR antigens can now be tested in the human diabetic being transplanted with pancreatic explants.

Analysis of a cDNA clone expressing a human autoimmune antigen: full-length sequence of the U2 small nuclear RNA-associated B antigen

International Nuclear Information System (INIS)

Habets, W.J.; Sillekens, P.T.G.; Hoet, M.H.; Schalken, J.A.; Roebroek, A.J.M.; Leunissen, J.A.M.; Van de Ven, W.J.M.; Van Venrooij, W.J.

1987-01-01

A U2 small nuclear RNA-associated protein, designated B'', was recently identified as the target antigen for autoimmune sera from certain patients with systemic lupus erythematosus and other rheumatic diseases. Such antibodies enabled them to isolate cDNA clone λHB''-1 from a phage λgt11 expression library. This clone appeared to code for the B'' protein as established by in vitro translation of hybrid-selected mRNA. The identity of clone λHB''-1 was further confirmed by partial peptide mapping and analysis of the reactivity of the recombinant antigen with monospecific and monoclonal antibodies. Analysis of the nucleotide sequence of the 1015-base-pair cDNA insert of clone λHB''-1 revealed a large open reading frame of 800 nucleotides containing the coding sequence for a polypeptide of 25,457 daltons. In vitro transcription of the λHB''-1 cDNA insert and subsequent translation resulted in a protein product with the molecular size of the B'' protein. These data demonstrate that clone λHB''-1 contains the complete coding sequence of this antigen. The deduced polypeptide sequence contains three very hydrophilic regions that might constitute RNA binding sites and/or antigenic determinants. These findings might have implications both for the understanding of the pathogenesis of rheumatic diseases as well as for the elucidation of the biological function of autoimmune antigens

Estrogens and growth factors induce the mRNA of the 52K-pro-cathepsin-D secreted by breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Cavailles, V; Augereau, P; Garcia, M; Rochefort, H

1988-03-25

The estrogen-induced 52K protein secreted by human breast cancer cells is a lysosomal protease recently identified as a pro-cathepsin D by sequencing several cDNA clones isolated from MCF/sub 7/ cells. Using one of these clones, the authors detected, in MCF/sub 7/ cells a 2.2 kb mRNA whose level was rapidly increased 4- to 10-fold by estradiol, but not by other classes of steroids. Other mitogens, such as epidermal growth factor and insulin, also induced the 2.2 kb mRNA in a dose-dependent manner. Induction with epidermal growth factor was as rapid but was 2- to 3-fold lower than with estradiol. Antiestrogens had no effect on the 52K-cathepsin-D mRNA in MCF/sub 7/ cells, but became estrogen agonists in two antiestrogen-resistant sublines R/sub 27/ and LY2. The use of transcription and translation inhibitors and nuclear run-on experiments indicate that estradiol enhances transcription of the 52K-cathepsin-D gene in MCF/sub 7/ cells.

FINANCIAL STABILITY AS A FACTOR ECONOMIC SECURITY

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A. V. Endovitskaya

2015-01-01

Full Text Available Summary. The article examines the linkages between financial stability and the level of its economic security. Considered the content of financial stability, represented by its own definition, we studied the basic conditions to achieve it. The logic diagram showing the location of financial stability and financial security to ensure the economic security of the business entity. A system of internal and external factors affecting the financial stability and endanger financial stability and financial security company. It has been established that it is the internal factors such as the availability of financial resources and financial position, capital structure, the company's ability to generate profits determine the level of economic security and its ability to withstand the negative impact of external and internal threats. The necessity of improving the financial sustainability in order to improve the economic security of the enterprise. On the basis of the research proposed matrix of risks affecting the financial stability and economic security, which allows to determine the probability of their occurrence and impact. It presents the economic, social, human, financial, organizational, economic, innovative and productive tools to increase the stability and financial security of an economic entity. List considered standard measures will make a plan of action to minimize the adverse impacts and enhance financial stability and security. Therefore, a prerequisite for the economic security of the enterprise is the attainment of financial stability.

Meningococcal factor H-binding protein vaccines with decreased binding to human complement factor H have enhanced immunogenicity in human factor H transgenic mice.

Science.gov (United States)

Rossi, Raffaella; Granoff, Dan M; Beernink, Peter T

2013-11-04

Factor H-binding protein (fHbp) is a component of a meningococcal vaccine recently licensed in Europe for prevention of serogroup B disease, and a second vaccine in clinical development. The protein specifically binds human factor H (fH), which down-regulates complement activation and enhances resistance to bactericidal activity. There are conflicting data from studies in human fH transgenic mice on whether binding of human fH to fHbp vaccines decreases immunogenicity, and whether mutant fHbp vaccines with decreased fH binding have enhanced immunogenicity. fHbp can be classified into two sub-families based on sequence divergence and immunologic cross-reactivity. Previous studies of mutant fHbp vaccines with low fH binding were from sub-family B, which account for approximately 60% of serogroup B case isolates. In the present study, we evaluated the immunogenicity of two mutant sub-family A fHbp vaccines containing single substitutions, T221A or D211A, which resulted in 15- or 30-fold lower affinity for human fH, respectively, than the corresponding control wild-type fHbp vaccine. In transgenic mice with high serum concentrations of human fH, both mutant vaccines elicited significantly higher IgG titers and higher serum bactericidal antibody responses than the control fHbp vaccine that bound human fH. Thus, mutations introduced into a sub-family A fHbp antigen to decrease fH binding can increase protective antibody responses in human fH transgenic mice. Collectively the data suggest that mutant fHbp antigens with decreased fH binding will result in superior vaccines in humans. Copyright © 2013 Elsevier Ltd. All rights reserved.

PKCα expression regulated by Elk-1 and MZF-1 in human HCC cells

International Nuclear Information System (INIS)

Hsieh, Y.-H.; Wu, T.-T.; Tsai, J.-H.; Huang, C.-Y.; Hsieh, Y.-S.; Liu, J.-Y.

2006-01-01

Our previous study found that PKCα was highly expressed in the poor-differentiated human HCC cells and associated with cell migration and invasion. In this study, we further investigated the gene regulation of this enzyme. We showed that PKCα expression enhancement in the poor-differentiated human HCC cells was found neither by DNA amplification nor by increasing mRNA stability using differential PCR and mRNA decay assays. After screening seven transcription factors in the putative cis-acting regulatory elements of human PKCα promoters, only Elk-1 and MZF-1 antisense oligonucleotide showed a significant reduction in the PKCα mRNA level. They also reduced cell proliferation, cell migratory and invasive capabilities, and DNA binding activities in the PKCα promoter region. Over-expression assay confirmed that the PKCα expression may be modulated by these two factors at the transcriptional level. Therefore, these results may provide a novel mechanism for PKCα expression regulation in human HCC cells

Kinetics of anti-carcinoembryonic antigen antibody internalization: effects of affinity, bivalency, and stability

Science.gov (United States)

Schmidt, Michael M.; Thurber, Greg M.

2010-01-01

Theoretical analyses suggest that the cellular internalization and catabolism of bound antibodies contribute significantly to poor penetration into tumors. Here we quantitatively assess the internalization of antibodies and antibody fragments against the commonly targeted antigen carcinoembryonic antigen (CEA). Although CEA is often referred to as a non-internalizing or shed antigen, anti-CEA antibodies and antibody fragments are shown to be slowly endocytosed by LS174T cells with a half-time of 10–16 h, a time scale consistent with the metabolic turnover rate of CEA in the absence of antibody. Anti-CEA single chain variable fragments (scFvs) with significant differences in affinity, stability against protease digestion, and valency exhibit similar uptake rates of bound antibody. In contrast, one anti-CEA IgG exhibits unique binding and trafficking properties with twice as many molecules bound per cell at saturation and significantly faster cellular internalization after binding. The internalization rates measured herein can be used in simple computational models to predict the microdistribution of these antibodies in tumor spheroids. PMID:18408925

Codon optimization of the HIV-1 vpu and vif genes stabilizes their mRNA and allows for highly efficient Rev-independent expression

International Nuclear Information System (INIS)

Nguyen, Kim-Lien; Llano, Manuel; Akari, Hirofumi; Miyagi, Eri; Poeschla, Eric M.; Strebel, Klaus; Bour, Stephan

2004-01-01

Two HIV-1 accessory proteins, Vpu and Vif, are notoriously difficult to express autonomously in the absence of the viral Tat and Rev proteins. We examined whether the codon bias observed in the vpu and vif genes relative to highly expressed human genes contributes to the Rev dependence and low expression level outside the context of the viral genome. The entire vpu gene as well as the 5' half of the vif gene were codon optimized and the resulting open reading frames (ORFs) (vphu and hvif, respectively) were cloned in autonomous expression vectors under the transcriptional control of the CMV promoter. Codon optimization efficiently removed the expression block observed in the native genes and allowed high levels of Rev- and Tat-independent expression of Vpu and Vif. Most of the higher protein levels detected are accounted for by enhanced steady-state levels of the mRNA encoding the optimized species. Nuclear run-on experiments show for the first time that codon optimization has no effect on the rate of transcriptional initiation or elongation of the vphu mRNA. Instead, optimization of the vpu gene was found to stabilize the vphu mRNA in the nucleus and enhance its export to the cytoplasm. This was achieved by allowing the optimized mRNA to use a new CRM1-independent nuclear export pathway. This work provides a better understanding of the molecular mechanisms underlying the process of codon optimization and introduces novel tools to study the biological functions of the Vpu and Vif proteins independently of other viral proteins

Tumor-associated antigens identified by mRNA expression profiling induce protective anti-tumor immunity

DEFF Research Database (Denmark)

Mathiassen, S; Lauemøller, S L; Ruhwald, M

2001-01-01

Defined tumor-associated antigens (TAA) are attractive targets for anti-tumor immunotherapy. Here, we describe a novel genome-wide approach to identify multiple TAA from any given tumor. A panel of transplantable thymomas was established from an inbred p53-/- mouse strain. The resulting tumors were...... of autoimmune reactions were observed. Thus, it appears possible to evaluate the entire metabolism of any given tumor and use this information rationally to identify multiple epitopes of value in the generation of tumor-specific immunotherapy. We expect that human tumors express similar tumor-specific metabolic...

Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

Directory of Open Access Journals (Sweden)

Minji Sim

Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

The natural antibody repertoire of sharks and humans recognizes the potential universe of antigens.

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Adelman, Miranda K; Schluter, Samuel F; Marchalonis, John J

2004-02-01

In ancestral sharks, a rapid emergence in the evolution of the immune system occurred, giving jawed-vertebrates the necessary components for the combinatorial immune response (CIR). To compare the natural antibody (NAb) repertoires of the most divergent vertebrates with the capacity to produce antibodies, we isolated NAbs to the same set of antigens by affinity chromatography from two species of Carcharhine sharks and from human polyclonal IgG and IgM antibody preparations. The activities of the affinity-purified anti-T-cell receptor (anti-TCR) NAbs were compared with those of monoclonal anti-TCR NAbs that were generated from a systemic lupus erythematosus patient. We report that sharks and humans, representing the evolutionary extremes of vertebrate species sharing the CIR, have NAbs to human TCRs, Igs, the human senescent cell antigen, and to numerous retroviral antigens, indicating that essential features of the combinatorial repertoire and the capacity to recognize the potential universe of antigens is shared among all jawed-vertebrates.

The expression of apoB mRNA editing factors is not the sole determinant for the induction of editing in differentiating Caco-2 cells

International Nuclear Information System (INIS)

Galloway, Chad A.; Smith, Harold C.

2010-01-01

Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is ∼80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.

Cellular localization of transforming growth factor-alpha mRNA in rat forebrain.

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Seroogy, K B; Lundgren, K H; Lee, D C; Guthrie, K M; Gall, C M

1993-05-01

The cellular localization of transforming growth factor-alpha (TGF alpha) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a 35S-labeled cRNA probe. TGF alpha cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocampal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGF alpha cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGF alpha in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGF alpha in the development of several forebrain systems. Our results demonstrating the prominent and wide-spread expression of TGF alpha mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGF alpha is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.

MRN1 implicates chromatin remodeling complexes and architectural factors in mRNA maturation

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Düring, Louis; Thorsen, Michael; Petersen, Darima

2012-01-01

A functional relationship between chromatin structure and mRNA processing events has been suggested, however, so far only a few involved factors have been characterized. Here we show that rsc nhp6¿¿ mutants, deficient for the function of the chromatin remodeling factor RSC and the chromatin....... Genetic interactions are observed between 2 µm-MRN1 and the splicing deficient mutants snt309¿, prp3, prp4, and prp22, and additional genetic analyses link MRN1, SNT309, NHP6A/B, SWI/SNF, and RSC supporting the notion of a role of chromatin structure in mRNA processing....

Application of Pharmacokinetics Modelling to Predict Human Exposure of a Cationic Liposomal Subunit Antigen Vaccine System

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Raj K. S. Badhan

2017-12-01

Full Text Available The pharmacokinetics of a liposomal subunit antigen vaccine system composed of the cationic lipid dimethyldioctadecylammonium bromide (DDA and the immunostimulatory agent trehalose 6,6-dibehenate (TDB (8:1 molar ratio combined with the Ag85B-ESAT-6 (H1 antigen were modelled using mouse in-vivo data. Compartment modelling and physiologically based pharmacokinetics (PBPK were used to predict the administration site (muscle and target site (lymph temporal concentration profiles and factors governing these. Initial estimates using compartmental modelling established that quadriceps pharmacokinetics for the liposome demonstrated a long half-life (22.6 days compared to the associated antigen (2.62 days. A mouse minimal-PBPK model was developed and successfully predicted quadriceps liposome and antigen pharmacokinetics. Predictions for the popliteal lymph node (PLN aligned well at earlier time-points. A local sensitivity analysis highlighted that the predicted AUCmuscle was sensitive to the antigen degradation constant kdeg (resulting in a 3-log change more so than the fraction escaping the quadriceps (fe (resulting in a 10-fold change, and the predicted AUCPLN was highly sensitive to fe. A global sensitivity analysis of the antigen in the muscle demonstrated that model predictions were within the 50th percentile for predictions and showed acceptable fits. To further translate in-vitro data previously generated by our group, the mouse minimal-PBPK model was extrapolated to humans and predictions made for antigen pharmacokinetics in muscle and PLN. Global analysis demonstrated that both kdeg and fe had a minimal impact on the resulting simulations in the muscle but a greater impact in the PLN. In summary, this study has predicted the in-vivo fate of DDA:TDB:H1 in humans and demonstrated the roles that formulation degradation and fraction escaping the depot site can play upon the overall depot effect within the site of administration.

Post-transcriptional regulation of vascular endothelial growth factor: Implications for tumor angiogenesis

Institute of Scientific and Technical Information of China (English)

Peter S Yoo; Abby L Mulkeen; Charles H Cha

2006-01-01

Vascular endothelial growth factor (VEGF) is a potent secreted mitogen critical for physiologic and tumor angiogenesis. Regulation of VEGF occurs at several levels, including transcription, mRNA stabilization,translation, and differential cellular localization of various isoforms. Recent advances in our understanding of posttranscriptional regulation of VEGF include identification of the stabilizing mRNA binding protein, HuR, and the discovery of internal ribosomal entry sites in the 5'UTR of the VEGF mRNA. Monoclonal anti-VEGF antibody was recently approved for use in humans, but suffers from the need for high systemic doses. RNA interference (RNAi)technology is being used in vitro and in animal models with promising results. Here, we review the literature on post-transcriptional regulation of VEGF and describe recent progress in targeting these mechanisms for therapeutic benefit.

Characterization of DNA polymerase. beta. mRNA: cell-cycle growth response in cultured human cells

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Zmudzka, B Z; Fornace, A; Collins, J; Wilson, S H

1988-10-25

DNA polymerase ..beta.. (..beta..-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. The authors used a cDNA probe to study abundance of ..beta..-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the ..beta..-polymerase transcript is low abundance and is neither cell-cycles nor growth phase responsive.

TCA cycle rewiring fosters metabolic adaptation to oxygen restriction in skeletal muscle from rodents and humans.

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Capitanio, Daniele; Fania, Chiara; Torretta, Enrica; Viganò, Agnese; Moriggi, Manuela; Bravatà, Valentina; Caretti, Anna; Levett, Denny Z H; Grocott, Michael P W; Samaja, Michele; Cerretelli, Paolo; Gelfi, Cecilia

2017-08-29

In mammals, hypoxic stress management is under the control of the Hypoxia Inducible Factors, whose activity depends on the stabilization of their labile α subunit. In particular, the skeletal muscle appears to be able to react to changes in substrates and O 2 delivery by tuning its metabolism. The present study provides a comprehensive overview of skeletal muscle metabolic adaptation to hypoxia in mice and in human subjects exposed for 7/9 and 19 days to high altitude levels. The investigation was carried out combining proteomics, qRT-PCR mRNA transcripts analysis, and enzyme activities assessment in rodents, and protein detection by antigen antibody reactions in humans and rodents. Results indicate that the skeletal muscle react to a decreased O 2 delivery by rewiring the TCA cycle. The first TCA rewiring occurs in mice in 2-day hypoxia and is mediated by cytosolic malate whereas in 10-day hypoxia the rewiring is mediated by Idh1 and Fasn, supported by glutamine and HIF-2α increments. The combination of these specific anaplerotic steps can support energy demand despite HIFs degradation. These results were confirmed in human subjects, demonstrating that the TCA double rewiring represents an essential factor for the maintenance of muscle homeostasis during adaptation to hypoxia.

Assessment of cancer and virus antigens for cross-reactivity in human tissues.

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Jaravine, Victor; Raffegerst, Silke; Schendel, Dolores J; Frishman, Dmitrij

2017-01-01

Cross-reactivity (CR) or invocation of autoimmune side effects in various tissues has important safety implications in adoptive immunotherapy directed against selected antigens. The ability to predict CR (on-target and off-target toxicities) may help in the early selection of safer therapeutically relevant target antigens. We developed a methodology for the calculation of quantitative CR for any defined peptide epitope. Using this approach, we performed assessment of 4 groups of 283 currently known human MHC-class-I epitopes including differentiation antigens, overexpressed proteins, cancer-testis antigens and mutations displayed by tumor cells. In addition, 89 epitopes originating from viral sources were investigated. The natural occurrence of these epitopes in human tissues was assessed based on proteomics abundance data, while the probability of their presentation by MHC-class-I molecules was modelled by the method of Keşmir et al. which combines proteasomal cleavage, TAP affinity and MHC-binding predictions. The results of these analyses for many previously defined peptides are presented as CR indices and tissue profiles. The methodology thus allows for quantitative comparisons of epitopes and is suggested to be suited for the assessment of epitopes of candidate antigens in an early stage of development of adoptive immunotherapy. Our method is implemented as a Java program, with curated datasets stored in a MySQL database. It predicts all naturally possible self-antigens for a given sequence of a therapeutic antigen (or epitope) and after filtering for predicted immunogenicity outputs results as an index and profile of CR to the self-antigens in 22 human tissues. The program is implemented as part of the iCrossR webserver, which is publicly available at http://webclu.bio.wzw.tum.de/icrossr/ CONTACT: d.frishman@wzw.tum.deSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press

LMKB/MARF1 localizes to mRNA processing bodies, interacts with Ge-1, and regulates IFI44L gene expression.

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Donald B Bloch

Full Text Available The mRNA processing body (P-body is a cellular structure that regulates the stability of cytoplasmic mRNA. MARF1 is a murine oocyte RNA-binding protein that is associated with maintenance of mRNA homeostasis and genomic stability. In this study, autoantibodies were used to identify Limkain B (LMKB, the human orthologue of MARF1, as a P-body component. Indirect immunofluorescence demonstrated that Ge-1 (a central component of the mammalian core-decapping complex co-localized with LMKB in P-bodies. Two-hybrid and co-immunoprecipitation assays were used to demonstrate interaction between Ge-1 and LMKB. The C-terminal 120 amino acids of LMKB mediated interaction with Ge-1 and the N-terminal 1094 amino acids of Ge-1 were required for interaction with LMKB. LMKB is the first protein identified to date that interacts with this portion of Ge-1. LMKB was expressed in human B and T lymphocyte cell lines; depletion of LMKB increased expression of IFI44L, a gene that has been implicated in the cellular response to Type I interferons. The interaction between LMKB/MARF1, a protein that contains RNA-binding domains, and Ge-1, which interacts with core-decapping proteins, suggests that LMKB has a role in the regulation of mRNA stability. LMKB appears to have different functions in different cell types: maintenance of genomic stability in developing oocytes and possible dampening of the inflammatory response in B and T cells.

Human intronless genes: Functional groups, associated diseases, evolution, and mRNA processing in absence of splicing

International Nuclear Information System (INIS)

Grzybowska, Ewa A.

2012-01-01

Highlights: ► Functional characteristics of intronless genes (IGs). ► Diseases associated with IGs. ► Origin and evolution of IGs. ► mRNA processing without splicing. -- Abstract: Intronless genes (IGs) constitute approximately 3% of the human genome. Human IGs are essentially different in evolution and functionality from the IGs of unicellular eukaryotes, which represent the majority in their genomes. Functional analysis of IGs has revealed a massive over-representation of signal transduction genes and genes encoding regulatory proteins important for growth, proliferation, and development. IGs also often display tissue-specific expression, usually in the nervous system and testis. These characteristics translate into IG-associated diseases, mainly neuropathies, developmental disorders, and cancer. IGs represent recent additions to the genome, created mostly by retroposition of processed mRNAs with retained functionality. Processing, nuclear export, and translation of these mRNAs should be hampered dramatically by the lack of splice factors, which normally tightly cover mature transcripts and govern their fate. However, natural IGs manage to maintain satisfactory expression levels. Different mechanisms by which IGs solve the problem of mRNA processing and nuclear export are discussed here, along with their possible impact on reporter studies.

Acclimatization to 4100 m does not change capillary density or mRNA expression of potential angiogenesis regulatory factors in human skeletal muscle

DEFF Research Database (Denmark)

Lundby, Carsten; Pilegaard, Henriette; Andersen, Jesper L.

2004-01-01

growth factor (VEGF), a known target gene for hypoxia inducible factor 1 (HIF-1). We hypothesised that prolonged exposure to high altitude increases muscle capillary density and that this can be explained by an enhanced HIF-1alpha expression inducing an increase in VEGF expression. We measured mRNA...... or VEGF mRNA was not changed with prolonged hypoxic exposure in SLR, and both genes were similarly expressed in SLR and HAN. In SLR, whole body mass, mean muscle fibre area and capillary to muscle fibre ratio remained unchanged during acclimatization. The capillary to fibre ratio was lower in HAN than...... in SLR (2.4+/-0.1 vs 3.6+/-0.2; PRNA expression and capillary density are not significantly increased by 8 weeks of exposure to high altitude and are not increased in Aymara high-altitude natives compared with sea level residents....

Identification of "tumor-associated" nucleolar antigens in human urothelial cancer.

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Yu, D; Pietro, T; Jurco, S; Scardino, P T

1987-09-01

Nucleoli isolated from HeLa S3 cells were used to produce rabbit antisera capable of binding nucleoli of transitional cell carcinomas (TCCa) of the bladder. Cross-reactivity of the rabbit antiserum with normal nucleoli was reduced by absorption with fetal calf serum, normal human serum, and human placental nucleoli. This antinucleolar antiserum exhibited strong reactivity in immunoperoxidase assays performed on specimens of human bladder cancer. In frozen tissue sections of 24 patients with TCCa and eight individuals without tumor, nucleolar staining was observed in all malignant specimens, but was not observed in seven of the normal specimens. Cytologic examination of bladder washing specimens from 47 normal individuals showed absence of nucleolar staining in 43 (91%) of 47 normal specimens while 12 (86%) of 14 specimens from patients with TCCa were positive. These results suggest that there are antigens associated with the nucleoli of HeLa cells and transitional cell carcinomas which are generally absent (or in low concentration) in normal human urothelial cells, and that antisera to these antigens may be useful in the cytologic diagnosis of human transitional cell carcinoma.

Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand.

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Sarah L James

2016-06-01

Full Text Available Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development.This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation, in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera.Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.

Cloning of the DNA-binding subunit of human nuclear factor κB: The level of its mRNA is strongly regulated by phorbol ester or tumor necrosis factor α

International Nuclear Information System (INIS)

Meyer, R.; Hatada, E.N.; Bartsch, C.; Scheidereit, C.; Hohmann, H.P.; Haiker, M.; Roethlisberger, U.; Lahm, H.W.; Schlaeger, E.J.; van Loon, A.P.G.M.

1991-01-01

The DNA binding subunit of nuclear factor κB (NF-κB), a B-cell protein that interacts with the immunoglobulin κ light-chain gene enhancer, has been purified from nuclei of human HL-60 cells stimulated with tumor necrosis factor α (TNFα), and internal peptide sequences were obtained. Overlapping cDNA clones were isolated and sequenced. The encoded open reading frame of about 105 kDa contained at its N-terminal half all six tryptic peptide sequences, suggesting that the 51-kDa NF-κB protein is processed from a 105-kDa precursor. An in vitro synthesized protein containing most of the N-terminal half of the open reading frame bound specifically to an NF-κB binding site. This region also showed high homology to a domain shared by the Drosophila dorsal gene and the avian and mammalian rel (proto)oncogene products. The level of the 3.8-kilobase mRNA was strongly increased after stimulation with TNFα or phorbol ester. Thus, both factors not only activate NF-κB protein, as described previously, but also induce expression of the gene encoding the DNA-binding subunit of NF-κB

Regulation of human lung fibroblast C1q-receptors by transforming growth factor-beta and tumor necrosis factor-alpha.

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Lurton, J; Soto, H; Narayanan, A S; Raghu, G

1999-03-01

Transforming growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) are two polypeptide mediators which are believed to play a role in the evolution of idiopathic pulmonary fibrosis (IPF). We have evaluated the effect of these two substances on the expression of receptors for collagen (cC1q-R) and globular (gC1q-R) domains of C1q and on type I collagen in human lung fibroblasts. Two fibroblast subpopulations differing in C1q receptor expression were obtained by culturing human lung explants in medium containing fresh human serum and heated plasma-derived serum and separating them based on C1q binding [Narayanan, Lurton and Raghu: Am J Resp Cell Mol Biol. 1998; 17:84]. The cells, referred to as HH and NL cells, respectively, were exposed to TGF-beta and TNF-alpha in serum-free conditions. The levels of mRNA were assessed by in situ hybridization and Northern analysis, and protein levels compared after SDS-polyacrylamide gel electrophoresis and Western blotting. NL cells exposed to TGF-beta and TNF-alpha contained 1.4 and 1.6 times as much cC1q-R mRNA, respectively, whereas in HH cells cC1q-R mRNA increased 2.0- and 2.4-fold. The gC1q-R mRNA levels increased to a lesser extent in both cells. These increases were not reflected in protein levels of CC1q-R and gC1q-R, which were similar to or less than controls. Both TGF-beta and TNF-alpha also increased procollagen [I] mRNA levels in both cells. Overall, TNF-alpha caused a greater increase and the degree of response by HH fibroblasts to both TGF-beta and TNF-alpha was higher than NL cells. These results indicated that TGF-beta and TNF-alpha upregulate the mRNA levels for cC1q-R and collagen and that they do not affect gC1q-R mRNA levels significantly. They also indicated different subsets of human lung fibroblasts respond differently to inflammatory mediators.

Identification of derlin-1 as a novel growth factor-responsive endothelial antigen by suppression subtractive hybridization

International Nuclear Information System (INIS)

Ran Yuliang; Jiang Yangfu; Zhong Xing; Zhou Zhuan; Liu Haiyan; Hu Hai; Lou Jinning; Yang Zhihua

2006-01-01

Endothelial cells play an important regulatory role in embryonic development, reproductive functions, tumor growth and progression. In the present study, the suppression subtractive hybridization (SSH) method was employed to identify differentially expressed genes between non-stimulated endothelial cells and activated endothelial cells. Following mRNA isolation of non-stimulated and hepatocellular carcinoma homogenate-stimulated cells, cDNAs of both populations were prepared and subtracted by suppressive PCR. Sequencing of the enriched cDNAs identified a couple of genes differentially expressed, including derlin-1. Derlin-1 was significantly up-regulated by tumor homogenates, VEGF, and endothelial growth supplements in a dose-dependent manner. Knock-down of derlin-1 triggered endothelial cell apoptosis, inhibited endothelial cell proliferation, and blocked the formation of a network of tubular-like structures. Our data reveal that derlin-1 is a novel growth factor-responsive endothelial antigen that promotes endothelial cell survival and growth

NCYM, a Cis-antisense gene of MYCN, encodes a de novo evolved protein that inhibits GSK3β resulting in the stabilization of MYCN in human neuroblastomas.

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Yusuke Suenaga

2014-01-01

Full Text Available The rearrangement of pre-existing genes has long been thought of as the major mode of new gene generation. Recently, de novo gene birth from non-genic DNA was found to be an alternative mechanism to generate novel protein-coding genes. However, its functional role in human disease remains largely unknown. Here we show that NCYM, a cis-antisense gene of the MYCN oncogene, initially thought to be a large non-coding RNA, encodes a de novo evolved protein regulating the pathogenesis of human cancers, particularly neuroblastoma. The NCYM gene is evolutionally conserved only in the taxonomic group containing humans and chimpanzees. In primary human neuroblastomas, NCYM is 100% co-amplified and co-expressed with MYCN, and NCYM mRNA expression is associated with poor clinical outcome. MYCN directly transactivates both NCYM and MYCN mRNA, whereas NCYM stabilizes MYCN protein by inhibiting the activity of GSK3β, a kinase that promotes MYCN degradation. In contrast to MYCN transgenic mice, neuroblastomas in MYCN/NCYM double transgenic mice were frequently accompanied by distant metastases, behavior reminiscent of human neuroblastomas with MYCN amplification. The NCYM protein also interacts with GSK3β, thereby stabilizing the MYCN protein in the tumors of the MYCN/NCYM double transgenic mice. Thus, these results suggest that GSK3β inhibition by NCYM stabilizes the MYCN protein both in vitro and in vivo. Furthermore, the survival of MYCN transgenic mice bearing neuroblastoma was improved by treatment with NVP-BEZ235, a dual PI3K/mTOR inhibitor shown to destabilize MYCN via GSK3β activation. In contrast, tumors caused in MYCN/NCYM double transgenic mice showed chemo-resistance to the drug. Collectively, our results show that NCYM is the first de novo evolved protein known to act as an oncopromoting factor in human cancer, and suggest that de novo evolved proteins may functionally characterize human disease.

Human Langerhans cells use an IL-15R-α/IL-15/pSTAT5-dependent mechanism to break T-cell tolerance against the self-differentiation tumor antigen WT1.

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Romano, Emanuela; Cotari, Jesse W; Barreira da Silva, Rosa; Betts, Brian C; Chung, David J; Avogadri, Francesca; Fink, Mitsu J; St Angelo, Erin T; Mehrara, Babak; Heller, Glenn; Münz, Christian; Altan-Bonnet, Gregoire; Young, James W

2012-05-31

Human CD34(+) progenitor-derived Langerhans-type dendritic cells (LCs) are more potent stimulators of T-cell immunity against tumor and viral antigens in vitro than are monocyte-derived DCs (moDCs). The exact mechanisms have remained elusive until now, however. LCs synthesize the highest amounts of IL-15R-α mRNA and protein, which binds IL-15 for presentation to responder lymphocytes, thereby signaling the phosphorylation of signal transducer and activator of transcription 5 (pSTAT5). LCs electroporated with Wilms tumor 1 (WT1) mRNA achieve sufficiently sustained presentation of antigenic peptides, which together with IL-15R-α/IL-15, break tolerance against WT1 by stimulating robust autologous, WT1-specific cytolytic T-lymphocytes (CTLs). These CTLs develop from healthy persons after only 7 days' stimulation without exogenous cytokines and lyse MHC-restricted tumor targets, which include primary WT1(+) leukemic blasts. In contrast, moDCs require exogenous rhuIL-15 to phosphorylate STAT5 and attain stimulatory capacity comparable to LCs. LCs therefore provide a more potent costimulatory cytokine milieu for T-cell activation than do moDCs, thus accounting for their superior stimulation of MHC-restricted Ag-specific CTLs without need for exogenous cytokines. These data support the use of mRNA-electroporated LCs, or moDCs supplemented with exogenous rhuIL-15, as vaccines for cancer immunotherapy to break tolerance against self-differentiation antigens shared by tumors.

Generation of human induced pluripotent stem cells using non-synthetic mRNA

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L. Rohani

2016-05-01

Reprogramming with non-synthetic mRNA holds great promise for safe generation of iPSCs of human origin. Using the protocols described herein we hope to make this method more accessible to other groups as a fast, inexpensive, and non-viral reprogramming approach.

Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

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Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

1992-09-01

The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

The Many Faces of Human Leukocyte Antigen-G

DEFF Research Database (Denmark)

Dahl, Mette; Djurisic, Snezana; Hviid, Thomas Vauvert F

2014-01-01

Pregnancy is an immunological paradox, where fetal antigens encoded by polymorphic genes inherited from the father do not provoke a maternal immune response. The fetus is not rejected as it would be theorized according to principles of tissue transplantation. A major contribution to fetal tolerance...... is the human leukocyte antigen (HLA)-G, a nonclassical HLA protein displaying limited polymorphism, restricted tissue distribution, and a unique alternative splice pattern. HLA-G is primarily expressed in placenta and plays multifaceted roles during pregnancy, both as a soluble and a membrane-bound molecule......, differences in HLA-G isoform expression, and possible differences in functional activity. Furthermore, we highlight important observations regarding HLA-G genetics and expression in preeclampsia that future research should address....

Monoclonal antibodies to human factor VII: a one step immunoradiometric assay for VII:Ag.

OpenAIRE

Takase, T; Tuddenham, E G; Chand, S; Goodall, A H

1988-01-01

Three mouse monoclonal antibodies (RFF-VII/1, RFF-VII/2, and RFF-VII/3) which bind specifically to different epitopes on human factor VII antigen were raised. Two of the antibodies, RFF-VII/1 and RFF-VII/2, bound strongly to factor VII antigen (VII:Ag), but only RFF-VII/1 and RFF-VII/3 were potent inhibitors of factor VII coagulation activity (VII:C). RFF-VII/1 and RFF-VII/2 were used in a one step, double monoclonal immunoradiometric assay for VII:Ag. This was highly reproducible and detecte...

Determination of an unrelated donor pool size for human leukocyte antigen-matched platelets in Brazil

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Carolina Bonet Bub

2016-02-01

Full Text Available ABSTRACT Background: Successful transfusion of platelet refractory patients is a challenge. Many potential donors are needed to sustain human leukocyte antigen matched-platelet transfusion programs because of the different types of antigens and the constant needs of these patients. For a highly mixed population such as the Brazilian population, the pool size required to provide adequate platelet support is unknown. Methods: A mathematical model was created to estimate the appropriate size of an unrelated donor pool to provide human leukocyte antigen-compatible platelet support for a Brazilian population. A group of 154 hematologic human leukocyte antigen-typed patients was used as the potential patient population and a database of 65,500 human leukocyte antigen-typed bone marrow registered donors was used as the donor population. Platelet compatibility was based on the grading system of Duquesnoy. Results: Using the mathematical model, a pool containing 31,940, 1710 and 321 donors would be necessary to match more than 80% of the patients with at least five completely compatible (no cross-reactive group, partial compatible (one cross-reactive group or less compatible (two cross-reactive group donors, respectively. Conclusion: The phenotypic diversity of the Brazilian population has probably made it more difficulty to find completely compatible donors. However, this heterogeneity seems to have facilitated finding donors when cross-reactive groups are accepted as proposed by the grading system of Duquesnoy. The results of this study may help to establish unrelated human leukocyte antigen-compatible platelet transfusions, a procedure not routinely performed in most Brazilian transfusion services.

Expression of Leukemia/Lymphoma-Related Factor (LRF/POKEMON) in Human Breast Carcinoma and Other Cancers

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Aggarwal, Anshu; Hunter, William J.; Aggarwal, Himanshu; Silva, Edibaldo D.; Davey, Mary S.; Murphy, Richard F.; Agrawal, Devendra K.

2010-01-01

The POK family of proteins plays an important role in not only embryonic development and cell differentiation, but also in oncogenesis. Leukemia/lymphoma-related factor (LRF) belongs to the POK family of transcriptional repressors and is also known as POK erythroid myeloid ontogenic factor (POKEMON), which binds to short transcripts of HIV-1 (FBI-1) and TTF-1 interacting peptide (TIP21). Its oncogenic role is known only in lymphoma, non-small cell lung carcinoma, and malignant gliomas. The functional expression of LRF in human breast carcinoma has not yet been confirmed. The aim of this study was to investigate and compare the expression of LRF in human breast cancer tissues and other human tumors. The expression of LRF mRNA transcripts and protein was observed in twenty human benign and malignant breast biopsy tissues. Expression of LRF was observed in several formalin-fixed tissues by immunohistochemistry and immunofluorescence. All malignant breast tissues expressed mRNA transcripts and protein for LRF. However, 40% and 15% benign breast biopsy tissues expressed LRF mRNA transcripts and protein, respectively. The overall expression of LRF mRNA transcripts and total protein was significantly more in malignant breast tissues than the benign breast tissues. LRF expression was also observed in the nuclei of human colon, renal, lung, hepatocellular carcinomas and thymoma tumor cells. In general, a significantly higher expression of LRF was seen in malignant tissues than in the corresponding benign or normal tissue. Further studies are warranted to determine the malignant role of LRF in human breast carcinoma. PMID:20471975

Detrimental ELAVL-1/HuR-dependent GSK3β mRNA stabilization impairs resolution in acute respiratory distress syndrome.

Directory of Open Access Journals (Sweden)

Olivia Hoffman

Full Text Available A hallmark of acute respiratory distress syndrome (ARDS is accumulation of protein-rich edema in the distal airspaces and its removal is critical for patient survival. Previous studies have shown a detrimental role of Glycogen Synthase Kinase (GSK 3β during ARDS via inhibition of alveolar epithelial protein transport. We hypothesized that post-transcriptional regulation of GSK3β could play a functional role in ARDS resolution. To address this hypothesis, we performed an in silico analysis to identify regulatory genes whose expression correlation to GSK3β messenger RNA utilizing two lung cancer cell line array datasets. Among potential regulatory partners of GSK3β, these studies identified the RNA-binding protein ELAVL-1/HuR (Embryonic Lethal, Abnormal Vision, Drosophila-Like as a central component in a likely GSK3β signaling network. ELAVL-1/HuR is a RNA-binding protein that selectively binds to AU-rich elements of mRNA and enhances its stability thereby increasing target gene expression. Subsequent studies with siRNA suppression of ELAVL-1/HuR demonstrated deceased GSK3β mRNA and protein expression and improved clearance of FITC-albumin in A549 cells. Conversely, stabilization of ELAVL-1/HuR with the proteasome inhibitor MG-132 resulted in induction of GSK3β at mRNA and protein level and attenuated FITC-albumin clearance. Utilizing ventilator-induced lung injury or intra-tracheal installation of hydrochloric acid to induce ARDS in mice, we observed increased mRNA and protein expression of ELAVL-1/HuR and GSK3β. Together, our findings indicate a previously unknown interaction between GSK3β and ELAV-1 during ARDS, and suggest the inhibition of the ELAV-1- GSK3β pathways as a novel ARDS treatment approach.

Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.

Science.gov (United States)

Zhao, Yangbing; Moon, Edmund; Carpenito, Carmine; Paulos, Chrystal M; Liu, Xiaojun; Brennan, Andrea L; Chew, Anne; Carroll, Richard G; Scholler, John; Levine, Bruce L; Albelda, Steven M; June, Carl H

2010-11-15

Redirecting T lymphocyte antigen specificity by gene transfer can provide large numbers of tumor-reactive T lymphocytes for adoptive immunotherapy. However, safety concerns associated with viral vector production have limited clinical application of T cells expressing chimeric antigen receptors (CAR). T lymphocytes can be gene modified by RNA electroporation without integration-associated safety concerns. To establish a safe platform for adoptive immunotherapy, we first optimized the vector backbone for RNA in vitro transcription to achieve high-level transgene expression. CAR expression and function of RNA-electroporated T cells could be detected up to a week after electroporation. Multiple injections of RNA CAR-electroporated T cells mediated regression of large vascularized flank mesothelioma tumors in NOD/scid/γc(-/-) mice. Dramatic tumor reduction also occurred when the preexisting intraperitoneal human-derived tumors, which had been growing in vivo for >50 days, were treated by multiple injections of autologous human T cells electroporated with anti-mesothelin CAR mRNA. This is the first report using matched patient tumor and lymphocytes showing that autologous T cells from cancer patients can be engineered to provide an effective therapy for a disseminated tumor in a robust preclinical model. Multiple injections of RNA-engineered T cells are a novel approach for adoptive cell transfer, providing flexible platform for the treatment of cancer that may complement the use of retroviral and lentiviral engineered T cells. This approach may increase the therapeutic index of T cells engineered to express powerful activation domains without the associated safety concerns of integrating viral vectors. Copyright © 2010 AACR.

Pattern-Recognition Receptor Signaling Regulator mRNA Expression in Humans and Mice, and in Transient Inflammation or Progressive Fibrosis

Science.gov (United States)

Günthner, Roman; Kumar, Vankayala Ramaiah Santhosh; Lorenz, Georg; Anders, Hans-Joachim; Lech, Maciej

2013-01-01

The cell type-, organ-, and species-specific expression of the pattern-recognition receptors (PRRs) are well described but little is known about the respective expression profiles of their negative regulators. We therefore determined the mRNA expression levels of A20, CYLD, DUBA, ST2, CD180, SIGIRR, TANK, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, SHP1, SHP2, TOLLIP, IRF4, SIKE, NLRX1, ERBIN, CENTB1, and Clec4a2 in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. Additionally, we characterized their expression profiles in mononuclear blood cells upon bacterial endotoxin, which showed a consistent induction of A20, SOCS3, IRAK-M, and Clec4a2 in human and murine cells. Furthermore, we studied the expression pattern in transient kidney ischemia-reperfusion injury versus post-ischemic atrophy and fibrosis in mice. A20, CD180, ST2, SOCS1, SOCS3, SHIP, IRAK-M, DOK1, DOK2, IRF4, CENTB1, and Clec4a2 were all induced, albeit at different times of injury and repair. Progressive fibrosis was associated with a persistent induction of these factors. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to PRR-mediated innate immunity, which seems to be involved in tissue injury, tissue regeneration and in progressive tissue scarring. PMID:24009023

Alternative Polyadenylation and Nonsense-Mediated Decay Coordinately Regulate the Human HFE mRNA Levels

Science.gov (United States)

Martins, Rute; Proença, Daniela; Silva, Bruno; Barbosa, Cristina; Silva, Ana Luísa; Faustino, Paula; Romão, Luísa

2012-01-01

Nonsense-mediated decay (NMD) is an mRNA surveillance pathway that selectively recognizes and degrades defective mRNAs carrying premature translation-termination codons. However, several studies have shown that NMD also targets physiological transcripts that encode full-length proteins, modulating their expression. Indeed, some features of physiological mRNAs can render them NMD-sensitive. Human HFE is a MHC class I protein mainly expressed in the liver that, when mutated, can cause hereditary hemochromatosis, a common genetic disorder of iron metabolism. The HFE gene structure comprises seven exons; although the sixth exon is 1056 base pairs (bp) long, only the first 41 bp encode for amino acids. Thus, the remaining downstream 1015 bp sequence corresponds to the HFE 3′ untranslated region (UTR), along with exon seven. Therefore, this 3′ UTR encompasses an exon/exon junction, a feature that can make the corresponding physiological transcript NMD-sensitive. Here, we demonstrate that in UPF1-depleted or in cycloheximide-treated HeLa and HepG2 cells the HFE transcripts are clearly upregulated, meaning that the physiological HFE mRNA is in fact an NMD-target. This role of NMD in controlling the HFE expression levels was further confirmed in HeLa cells transiently expressing the HFE human gene. Besides, we show, by 3′-RACE analysis in several human tissues that HFE mRNA expression results from alternative cleavage and polyadenylation at four different sites – two were previously described and two are novel polyadenylation sites: one located at exon six, which confers NMD-resistance to the corresponding transcripts, and another located at exon seven. In addition, we show that the amount of HFE mRNA isoforms resulting from cleavage and polyadenylation at exon seven, although present in both cell lines, is higher in HepG2 cells. These results reveal that NMD and alternative polyadenylation may act coordinately to control HFE mRNA levels, possibly varying its

Viperin mRNA is a novel target for the human RNase MRP/RNase P endoribonuclease.

Science.gov (United States)

Mattijssen, Sandy; Hinson, Ella R; Onnekink, Carla; Hermanns, Pia; Zabel, Bernhard; Cresswell, Peter; Pruijn, Ger J M

2011-07-01

RNase MRP is a conserved endoribonuclease, in humans consisting of a 267-nucleotide RNA associated with 7-10 proteins. Mutations in its RNA component lead to several autosomal recessive skeletal dysplasias, including cartilage-hair hypoplasia (CHH). Because the known substrates of mammalian RNase MRP, pre-ribosomal RNA, and RNA involved in mitochondrial DNA replication are not likely involved in CHH, we analyzed the effects of RNase MRP (and the structurally related RNase P) depletion on mRNAs using DNA microarrays. We confirmed the upregulation of the interferon-inducible viperin mRNA by RNAi experiments and this appeared to be independent of the interferon response. We detected two cleavage sites for RNase MRP/RNase P in the coding sequence of viperin mRNA. This is the first study providing direct evidence for the cleavage of a mRNA by RNase MRP/RNase P in human cells. Implications for the involvement in the pathophysiology of CHH are discussed.

Tissue distribution of histo-blood group antigens

DEFF Research Database (Denmark)

Ravn, V; Dabelsteen, Erik

2000-01-01

carrier carbohydrate chains. Histo-blood group antigens are found in most epithelial tissues. Meanwhile, several factors influence the type, the amount, and the histological distribution of histoblood group antigens, i.e. the ABO, Lewis, and saliva-secretor type of the individual, and the cell- and tissue......The introduction of immunohistochemical techniques and monoclonal antibodies to specific carbohydrate epitopes has made it possible to study in detail the tissue distribution of histo-blood group antigens and related carbohydrate structures. The present paper summarizes the available data...... concerning the histological distribution of histo-blood group antigens and their precursor structures in normal human tissues. Studies performed have concentrated on carbohydrate antigens related to the ABO, Lewis, and TTn blood group systems, i.e. histo-blood group antigens carried by type 1, 2, and 3 chain...

Human apolipoprotein B (apoB) mRNA: Identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine

International Nuclear Information System (INIS)

Higuchi, K.; Hospattankar, A.V.; Law, S.W.; Meglin, N.; Cortright, J.; Brewer, H.B. Jr.

1988-01-01

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6,538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2,153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides, designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine

Carcinoma-associated antigens

International Nuclear Information System (INIS)

Bartorelli, A.; Accinni, R.

1981-01-01

This invention relates to novel antigens associated with breast carcinoma, anti-sera specific to said antigens, 125 I-labeled forms of said antigens and methods of detecting said antigens in serum or plasma. The invention also relates to a diagnostic kit containing standardised antigens or antisera or marked forms thereof for the detection of said antigens in human blood, serum or plasma. (author)

Human leukocyte antigen (HLA) polymorphism and type 1 diabetes ...

African Journals Online (AJOL)

Insulin-dependent diabetes mellitus or type 1 diabetes is an autoimmune multifactorial disease which has a great socio-economic impact. In Morocco, less is known about the contribution of Human leukocyte antigen (HLA) alleles to type 1 diabetes susceptibility. Our study focused on evaluating the distribution of class II ...

VDR mRNA overexpression is associated with worse prognostic factors in papillary thyroid carcinoma

Directory of Open Access Journals (Sweden)

June Young Choi

2017-03-01

Full Text Available The purpose of this study was to assess the relationship between vitamin D receptor gene (VDR expression and prognostic factors in papillary thyroid cancer (PTC. mRNA sequencing and somatic mutation data from The Cancer Genome Atlas (TCGA were analyzed. VDR mRNA expression was compared to clinicopathologic variables by linear regression. Tree-based classification was applied to find cutoff and patients were split into low and high VDR group. Logistic regression, Kaplan–Meier analysis, differentially expressed gene (DEG test and pathway analysis were performed to assess the differences between two VDR groups. VDR mRNA expression was elevated in PTC than that in normal thyroid tissue. VDR expressions were high in classic and tall-cell variant PTC and lateral neck node metastasis was present. High VDR group was also associated with classic and tall cell subtype, AJCC stage IV and lower recurrence-free survival. DEG test reveals that 545 genes were upregulated in high VDR group. Thyroid cancer-related pathways were enriched in high VDR group in pathway analyses. VDR mRNA overexpression was correlated with worse prognostic factors such as subtypes of papillary thyroid carcinoma that are known to be worse prognosis, lateral neck node metastasis, advanced stage and recurrence-free survival.

Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray.

Directory of Open Access Journals (Sweden)

Daniel S Chen

2005-10-01

Full Text Available In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect.In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-gamma (IFNgamma and tumor necrosis factor-alpha (TNFalpha in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNgamma and TNFalpha did so.Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome.

Antigen-presenting cells transfected with Hsp65 messenger RNA fail to treat experimental tuberculosis

Energy Technology Data Exchange (ETDEWEB)

Rocha, C.D.; Trombone, A.P.F.; Lorenzi, J.C.C.; Almeida, L.P.; Gembre, A.F.; Padilha, E. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Ramos, S.G. [Departamento de Patologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil); Silva, C.L.; Coelho-Castelo, A.A.M. [Departamento de Bioquímica e Imunologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP (Brazil)

2012-09-21

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Prevalence of human immunodeficiency virus type 1 p24 antigen in U.S. blood donors--an assessment of the efficacy of testing in donor screening. The HIV-Antigen Study Group.

Science.gov (United States)

Alter, H J; Epstein, J S; Swenson, S G; VanRaden, M J; Ward, J W; Kaslow, R A; Menitove, J E; Klein, H G; Sandler, S G; Sayers, M H

1990-11-08

We performed a multicenter study in 1989 to determine whether screening whole-blood donors for human immunodeficiency virus type 1 (HIV-1) p24 antigen would improve transfusion safety by identifying carriers of the virus who are seronegative for HIV-1 antibody. More than 500,000 donations were tested at 13 U.S. blood centers with test kits from two manufacturers. Units found repeatedly reactive were retested in a central laboratory; if the results were positive, they were confirmed by a neutralization assay. A subgroup of units was also tested for HIV-1 by the polymerase chain reaction. Selected donors confirmed or not confirmed as having p24 antigen were contacted for follow-up interviews to identify risk factors and undergo retesting for HIV-1 markers. Positive tests for p24 antigen were confirmed by neutralization in five donors (0.001 percent of all donations tested), all of whom were also positive for HIV-1 antibody and HIV-1 by polymerase chain reaction. Three of the antigen-positive donors had other markers of infectious disease that would have resulted in the exclusion of their blood; two had risk factors for HIV-1 that should have led to self-exclusion. Of 220 blood units with repeatedly reactive p24 antigen whose presence could not be confirmed by neutralization (0.04 percent of the donations studied), none were positive for HIV-1 antibody, HIV-1 by polymerase chain reaction (120 units tested), or virus culture (76 units tested)--attesting to the specificity of confirmatory neutralization. The finding that no donation studied was positive for p24 antigen and negative for HIV-1 antibody suggests that screening donors for p24 antigen with tests of the current level of sensitivity would not add substantially to the safety of the U.S. blood supply.

Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII.

OpenAIRE

Broze, G J; Hickman, S; Miletich, J P

1985-01-01

Several murine monoclonal anti-human Factor VII antibodies were produced using hybridoma technology. Two noncompetitive monoclonal antibodies were used to examine by Western blotting the Factor VII cross-reactive material (CRM) in normal human plasma and three commercially available congenitally Factor VII-deficient plasmas, and to construct a facile "sandwich" immunoassay for plasma Factor VII. A second, previously undescribed, form of Factor VII CRM was detected in human plasma, which on We...

Immunochemical identification of human trophoblast membrane antigens using monoclonal antibodies

Energy Technology Data Exchange (ETDEWEB)

Brown, P J; Molloy, C M; Johnson, P M [Liverpool Univ. (UK). Dept. of Immunology

1983-11-01

Human trophoblast membrane antigens recognised by monoclonal antibodies (H310, H315, H316 and H317) have been identified using combinations of radioimmunoprecipitation, SDS-PAGE, electroblotting, chromatographic and ELISA-type techniques. H317 is known to identify heat-stable placental-type alkaline phosphatase and accordingly was shown to react with a protein of subunit Msub(r) of 68000. H310 and H316 both recognise an antigen with a subunit Msub(r) of 34000 under reducing conditions. In non-reducing conditions, the H310/316 antigen gave oligomers of a component of Msub(r) 62000. It is unknown whether this 62000 dalton component is a dimer of the 34000 dalton protein with either itself or a second protein chain of presumed Msub(r) around 28000. H315 recognises an antigen with subunit Msub(r) of 36000; in non-reducing conditions this component readily associates to oligomeric structures. The epitope recognised by H315 may be sensitive to SDS. The two proteins recognised by H310/316 and H315 have been termed the p34 and p36 trophoblast membrane proteins, respectively.

Transcription factor Fos-Related Antigen-2 induces progressive peripheral vasculopathy in mice closely resembling human systemic sclerosis

OpenAIRE

Maurer, B; Busch, N; Jüngel, A; Pileckyte, M; Gay, R E; Michel, B A; Schett, G; Gay, S; Distler, J; Distler, O

2009-01-01

BACKGROUND: -Microvascular damage is one of the first pathological changes in systemic sclerosis. In this study, we investigated the role of Fos-related antigen-2 (Fra-2), a transcription factor of the activator protein-1 family, in the peripheral vasculopathy of systemic sclerosis and examined the underlying mechanisms. Methods and Results-Expression of Fra-2 protein was significantly increased in skin biopsies of systemic sclerosis patients compared with healthy controls, especially in endo...

mRNA: From a chemical blueprint for protein production to an off-the-shelf therapeutic.

Science.gov (United States)

Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

2013-02-01

Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and "naked mRNA" for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters.

Allopurinol reduces antigen-specific and polyclonal activation of human T cells

Directory of Open Access Journals (Sweden)

Damián ePérez-Mazliah

2012-09-01

Full Text Available Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

Radioimmunoassay and some properties of human antibodies to hepatitis B core antigen

Energy Technology Data Exchange (ETDEWEB)

Neurath, A R; Szmuness, W; Stevens, C E; Strick, N; Harley, E J [New York Blood Center, N.Y. (USA)

1978-03-01

A solid-phase radioimmunoassay for antibodies to hepatitis B core antigen (anti-HBsub(c)) is described. Polystyrene beads coated with anti-HBsub(c), hepatitis B core antigen prepared from pooled sera of humans infected with hepatitis B virus (HBV) and /sup 125/I-labelled anti-HBsub(c) were used for the test. Distinct patterns of development and changes of anti-HBsub(c) and their immunological properties are all related to variations of other markers specific for HBV infections. Knowledge concerning the detailed features of the immune response to hepatitis B core antigen may provide deeper insight into the pathogenesis of HBV infections.

[Search for a new antigen associated with oncornavirus D in human breast cancer tumors].

Science.gov (United States)

Kosiakov, P N; Korosteleva, V S; Pavliuchenkova, R P; Kosiakova, N; Nabokov, Iu S

1979-01-01

An antigen similar in its specificity to a nonvirion antigen emerging in stationary tissue culture cells spontaneously or experimentally infected with oncornavirus D was found in mammary gland cancer tumour tissues of 9 out of 54 examined patients. This virus-associated antigen was absent in 17 examined specimens of benign tumours of the same localization (fibroadenomas, mastopathies) or in the organs of a normal adult man or human embryo.

Mini-review: Can non-human leucocyte antigen genes determine susceptibility to severe dengue syndromes?

Science.gov (United States)

Ng, Dorothy; Ghosh, Aparna; Jit, Mark; Seneviratne, Suranjith L

2017-09-01

Dengue viral infections are endemic or epidemic in virtually all tropical countries. Among individuals infected with the dengue virus, severe dengue syndromes (i.e., dengue haemorrhagic fever and dengue shock syndromes) tend to affect only some and this may be due to a combination of host genetic susceptibility and viral factors. In this review article we analyse and discuss the present knowledge of non-human leucocyte antigen host genetic susceptibility to severe dengue syndromes. The relevance of genetic polymorphisms in the pathways of antigen recognition, uptake, processing and presentation, activation of interferon α responses, mast cell and complement activation and T cell activation and dengue disease severity has been reviewed and analysed. © The Author(s) 2018. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

Science.gov (United States)

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

Nogo-receptor gene activity: cellular localization and developmental regulation of mRNA in mice and humans.

Science.gov (United States)

Josephson, Anna; Trifunovski, Alexandra; Widmer, Hans Ruedi; Widenfalk, Johan; Olson, Lars; Spenger, Christian

2002-11-18

Nogo (reticulon-4) is a myelin-associated protein that is expressed in three different splice variants, Nogo-A, Nogo-B, and Nogo-C. Nogo-A inhibits neurite regeneration in the central nervous system. Messenger RNA encoding Nogo is expressed in oligodendrocytes and central and peripheral neurons, but not in astrocytes or Schwann cells. Nogo is a transmembraneous protein; the extracellular domain is termed Nogo-66, and a Nogo-66-receptor (Nogo-R) has been identified. We performed in situ hybridization in human and mouse nervous tissues to map the cellular distribution of Nogo-R gene activity patterns in fetal and adult human spinal cord and sensory ganglia, adult human brain, and the nervous systems of developing and adult mice. In the human fetus Nogo-R was transcribed in the ventral horn of the spinal cord and in dorsal root ganglia. In adult human tissues Nogo-R gene activity was found in neocortex, hippocampus, amygdala, and a subset of large and medium-sized neurons of the dorsal root ganglia. Nogo-R mRNA was not expressed in the adult human spinal cord at detectable levels. In the fetal mouse, Nogo-R was diffusely expressed in brain, brainstem, trigeminal ganglion, spinal cord, and dorsal root ganglia at all stages. In the adult mouse strong Nogo-R mRNA expression was found in neurons in neocortex, hippocampus, amygdala, habenula, thalamic nuclei, brainstem, the granular cell layer of cerebellum, and the mitral cell layer of the olfactory bulb. Neurons in the adult mouse striatum, the medial septal nucleus, and spinal cord did not express Nogo-R mRNA at detectable levels. In summary, Nogo-66-R mRNA expression in humans and mice was observed in neurons of the developing nervous system Expression was downregulated in the adult spinal cord of both species, and specific expression patterns were seen in the adult brain. Copyright 2002 Wiley-Liss, Inc.

Coordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

KAUST Repository

Arae, Toshihiro

2017-04-18

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

Structural analysis of inter-genus complexes of V-antigen and its regulator and their stabilization by divalent metal ions.

Science.gov (United States)

Basu, Abhishek; Das, Atanu; Mondal, Abhisek; Datta, Saumen

2016-03-01

Gram-negative bacteria like Yersinia, Pseudomonas, and Aeromonas need type III secretion system (T3SS) for their pathogenicity. V-antigen and its regulator are essential for functioning of T3SS. There is significant functional conservation amongst V-antigen and its regulator belonging to the Ysc family. In this study, we have structurally characterized the inter-genus complexes of V-antigen and its regulator. ConSurf analysis demonstrates that V-antigens belonging to the Ysc family show high structural identity predominantly confined to the two long helical regions. The regulator of V-antigen shows high conservation in its first intramolecular coiled-coil domain, responsible for interaction with V-antigen. ∆LcrG(1-70) localizes within the groove formed by long helices of LcrV, as observed in PcrV-∆PcrG(13-72) interaction. Inter-genus complexes of LcrV-PcrG and PcrV-LcrG exhibited elongated conformation and 1:1 heterodimeric state like the native complex of PcrV-PcrG and LcrV-LcrG. Both native and inter-genus complexes showed rigid tertiary structure, solvent-exposed hydrophobic patches, and cooperative melting behavior with high melting temperature. LcrV-PcrG and PcrV-LcrG showed nanomolar affinity of interaction, identical to PcrV-PcrG interaction, but stronger than LcrV-LcrG interaction. Calcium (a secretion blocker of T3SS) propels all the complexes towards a highly monodisperse form. Calcium and magnesium increase the helicity of the native and inter-genus complexes, and causes helix-helix stabilization. Stabilization of helices leads to a slight increase in the melting temperature by 1.5-2.0 °C. However, calcium does not alter the affinity of interaction of V-antigen and its regulator, emphasizing the effect of divalent of cations at the structural level without any regulatory implications. Therefore, the structural conservation of these inter-genus complexes could be the basis for their functional complementation.

Identification of antigen-specific human monoclonal antibodies using high-throughput sequencing of the antibody repertoire.

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Liu, Ju; Li, Ruihua; Liu, Kun; Li, Liangliang; Zai, Xiaodong; Chi, Xiangyang; Fu, Ling; Xu, Junjie; Chen, Wei

2016-04-22

High-throughput sequencing of the antibody repertoire provides a large number of antibody variable region sequences that can be used to generate human monoclonal antibodies. However, current screening methods for identifying antigen-specific antibodies are inefficient. In the present study, we developed an antibody clone screening strategy based on clone dynamics and relative frequency, and used it to identify antigen-specific human monoclonal antibodies. Enzyme-linked immunosorbent assay showed that at least 52% of putative positive immunoglobulin heavy chains composed antigen-specific antibodies. Combining information on dynamics and relative frequency improved identification of positive clones and elimination of negative clones. and increase the credibility of putative positive clones. Therefore the screening strategy could simplify the subsequent experimental screening and may facilitate the generation of antigen-specific antibodies. Copyright © 2016 Elsevier Inc. All rights reserved.

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

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Hasanzadeh, Leila; Ghaznavi-Rad, Ehsanollah; Soufian, Safieh; Farjadi, Vahideh; Abtahi, Hamid

2013-01-01

Objective(s) : Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity. Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis . PMID:23997913

Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori

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Leila Hasanzadeh

2013-07-01

Full Text Available Objective(s: Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.   Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3 pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification. Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera. Conclusion : Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis .

Antigen-binding radioimmunoassays for human IgG antibodies to bovine ν-lactoglobulin

International Nuclear Information System (INIS)

Turner, M.W.; Paganelli, R.; Levinsky, R.J.; Williams, A.

1983-01-01

A double antibody antigen-binding assay for the detection of human IgG antibodies to the bovine milk allergen ν-lactoglobulin is described. The levels of such antibodies in patients with established cows' milk protein intolerance were significantly higher than the levels observed in a healthy control group (P<0.01). The assay showed excellent correlation with a solid phase antigen binding assay (rsub(s) = 0.8, P<0.001). (Auth.)

The involvement of mRNA processing factors TIA-1, TIAR, and PABP-1 during mammalian hibernation.

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Tessier, Shannon N; Audas, Timothy E; Wu, Cheng-Wei; Lee, Stephen; Storey, Kenneth B

2014-11-01

Mammalian hibernators survive low body temperatures, ischemia-reperfusion, and restricted nutritional resources via global reductions in energy-expensive cellular processes and selective increases in stress pathways. Consequently, studies that analyze hibernation uncover mechanisms which balance metabolism and support survival by enhancing stress tolerance. We hypothesized processing factors that influence messenger ribonucleic acid (mRNA) maturation and translation may play significant roles in hibernation. We characterized the amino acid sequences of three RNA processing proteins (T cell intracellular antigen 1 (TIA-1), TIA1-related (TIAR), and poly(A)-binding proteins (PABP-1)) from thirteen-lined ground squirrels (Ictidomys tridecemlineatus), which all displayed a high degree of sequence identity with other mammals. Alternate Tia-1 and TiaR gene variants were found in the liver with higher expression of isoform b versus a in both cases. The localization of RNA-binding proteins to subnuclear structures was assessed by immunohistochemistry and confirmed by subcellular fractionation; TIA-1 was identified as a major component of subnuclear structures with up to a sevenfold increase in relative protein levels in the nucleus during hibernation. By contrast, there was no significant difference in the relative protein levels of TIARa/TIARb in the nucleus, and a decrease was observed for TIAR isoforms in cytoplasmic fractions of torpid animals. Finally, we used solubility tests to analyze the formation of reversible aggregates that are associated with TIA-1/R function during stress; a shift towards the soluble fraction (TIA-1a, TIA-1b) was observed during hibernation suggesting enhanced protein aggregation was not present during torpor. The present study identifies novel posttranscriptional regulatory mechanisms that may play a role in reducing translational rates and/or mRNA processing under unfavorable environmental conditions.

Staphylococcus aureus RNAIII binds to two distant regions of coa mRNA to arrest translation and promote mRNA degradation.

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Clément Chevalier

2010-03-01

Full Text Available Staphylococcus aureus RNAIII is the intracellular effector of the quorum sensing system that temporally controls a large number of virulence factors including exoproteins and cell-wall-associated proteins. Staphylocoagulase is one major virulence factor, which promotes clotting of human plasma. Like the major cell surface protein A, the expression of staphylocoagulase is strongly repressed by the quorum sensing system at the post-exponential growth phase. Here we used a combination of approaches in vivo and in vitro to analyze the mechanism used by RNAIII to regulate the expression of staphylocoagulase. Our data show that RNAIII represses the synthesis of the protein through a direct binding with the mRNA. Structure mapping shows that two distant regions of RNAIII interact with coa mRNA and that the mRNA harbors a conserved signature as found in other RNAIII-target mRNAs. The resulting complex is composed of an imperfect duplex masking the Shine-Dalgarno sequence of coa mRNA and of a loop-loop interaction occurring downstream in the coding region. The imperfect duplex is sufficient to prevent the formation of the ribosomal initiation complex and to repress the expression of a reporter gene in vivo. In addition, the double-strand-specific endoribonuclease III cleaves the two regions of the mRNA bound to RNAIII that may contribute to the degradation of the repressed mRNA. This study validates another direct target of RNAIII that plays a role in virulence. It also illustrates the diversity of RNAIII-mRNA topologies and how these multiple RNAIII-mRNA interactions would mediate virulence regulation.

Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

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Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A.

1990-01-01

Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC

Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function.

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Kazemi-Lomedasht, Fatemeh; Muyldermans, Serge; Habibi-Anbouhi, Mahdi; Behdani, Mahdi

2018-03-01

Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem. Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody), evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity. The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis.

Lack of ADAM2, CALR3 and SAGE1 Cancer/Testis Antigen Expression in Lung and Breast Cancer

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Maheswaran, Emeaga; Pedersen, Christina B; Ditzel, Henrik J

2015-01-01

and antigenic properties, but the expression patterns of most of the more than 200 identified cancer/testis antigens in various cancers remain largely uncharacterized. In this study, we investigated the expression of the cancer/testis antigens ADAM2, CALR3 and SAGE1 in lung and breast cancer, the two most...... frequent human cancers, with the purpose of providing novel therapeutic targets for these diseases. We used a set of previously uncharacterized antibodies against the cancer/testis antigens ADAM2, CALR3 and SAGE1 to investigate their expression in a large panel of normal tissues as well as breast and lung...... cancers. Staining for the well-characterized MAGE-A proteins was included for comparison. Immunohistochemical staining confirmed previous mRNA analysis demonstrating that ADAM2, CALR3 and SAGE1 proteins are confined to testis in normal individuals. Negative tissues included plancenta, which express many...

Emotional stability components of human performance problems

International Nuclear Information System (INIS)

Wexler, R.H.

1987-01-01

Over half of all significant events that occur in nuclear plants involve human performance problems. There is increasing worldwide recognition that human performance problems have a significant impact on the safety, cost, and efficiency of nuclear plant operations. Emotional stability components have an important direct and indirect impact on human performance problems. This paper examines emotional stability components that are currently incorporated into human performance evaluation systems (HPES) in nuclear plants. It describes HPES programs being developed around the world, the emotional stability components that are currently referred to in these programs, and suggestions for improving HPES programs through a greater understanding of emotion stability components. A review of emotional stability components that may hinder or promote a plant environment that encourages the voluntary reporting and correction of human error is also presented

Antigenicity and diagnostic potential of vaccine candidates in human Chagas disease.

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Shivali Gupta

Full Text Available Chagas disease, caused by Trypanosoma cruzi, is endemic in Latin America and an emerging infectious disease in the US and Europe. We have shown TcG1, TcG2, and TcG4 antigens elicit protective immunity to T. cruzi in mice and dogs. Herein, we investigated antigenicity of the recombinant proteins in humans to determine their potential utility for the development of next generation diagnostics for screening of T. cruzi infection and Chagas disease.Sera samples from inhabitants of the endemic areas of Argentina-Bolivia and Mexico-Guatemala were analyzed in 1(st-phase for anti-T. cruzi antibody response by traditional serology tests; and in 2(nd-phase for antibody response to the recombinant antigens (individually or mixed by an ELISA. We noted similar antibody response to candidate antigens in sera samples from inhabitants of Argentina and Mexico (n=175. The IgG antibodies to TcG1, TcG2, and TcG4 (individually and TcG(mix were present in 62-71%, 65-78% and 72-82%, and 89-93% of the subjects, respectively, identified to be seropositive by traditional serology. Recombinant TcG1- (93.6%, TcG2- (96%, TcG4- (94.6% and TcG(mix- (98% based ELISA exhibited significantly higher specificity compared to that noted for T. cruzi trypomastigote-based ELISA (77.8% in diagnosing T. cruzi-infection and avoiding cross-reactivity to Leishmania spp. No significant correlation was noted in the sera levels of antibody response and clinical severity of Chagas disease in seropositive subjects.Three candidate antigens were recognized by antibody response in chagasic patients from two distinct study sites and expressed in diverse strains of the circulating parasites. A multiplex ELISA detecting antibody response to three antigens was highly sensitive and specific in diagnosing T. cruzi infection in humans, suggesting that a diagnostic kit based on TcG1, TcG2 and TcG4 recombinant proteins will be useful in diverse situations.

A novel strategy to improve antigen presentation for active immunotherapy in cancer. Fusion of the human papillomavirus type 16 E7 antigen to a cell penetrating peptide

International Nuclear Information System (INIS)

Granadillo, Milaid; Torrens, Isis; Guerra, Maribel

2012-01-01

Facilitating the delivery of exogenous antigens to antigen-presenting cells, ensuing processing and presentation via the major histocompatibility complex class I and induction of an effective immune response are fundamental for an effective therapeutic cancer vaccine. In this regard, we propose the use of cell-penetrating peptides fused to a tumor antigen. To demonstrate this concept we designed a fusion protein comprising a novel cell-penetrating and immunostimulatory peptide corresponding to residues 32 to 51 of the Limulus anti-lipopolysaccharide factor protein (LALF 32-51 ) linked to human papillomavirus 16 E7 antigen (LALF 32-51 -E7). In this work, we demonstrated that the immunization with LALF 32-51 -E7 using the TC-1 mouse model induces a potent and long-lasting anti-tumor response supported on an effective E7-specific CD8 +T -cell response. The finding that therapeutic immunization with LALF 32-51 or E7 alone, or an admixture of LALF32-51 and E7, does not induce significant tumor reduction indicates that covalent linkage between LALF 32-51 and E7 is required for the anti-tumor effect. These results support the use of this novel cell-penetrating peptide as an efficient means for delivering therapeutic targets into cellular compartments with the induction of a cytotoxic CD8 +T lymphocyte immune response. This approach is promissory for the treatment of tumors associated with the human papillomavirus 16, which is responsible for the 50% of cervical cancer cases worldwide and other malignancies. Furthermore, protein-based vaccines can circumvent the major histocompatibility complex specificity limitation associated with peptide vaccines providing a greater extent in their application

CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

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Vanesa Alonso-Camino

2013-01-01

Full Text Available A human single-chain variable fragment (scFv antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs. The repertoire was fused to a first-generation T cell receptor ζ (TCRζ-based chimeric antigen receptor (CAR. We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2 bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR and the selection context (cell synapse, which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells.

Human Parvovirus B19 Induced Apoptotic Bodies Contain Altered Self-Antigens that are Phagocytosed by Antigen Presenting Cells

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Thammasri, Kanoktip; Rauhamäki, Sanna; Wang, Liping; Filippou, Artemis; Kivovich, Violetta; Marjomäki, Varpu; Naides, Stanley J.; Gilbert, Leona

2013-01-01

Human parvovirus B19 (B19V) from the erythrovirus genus is known to be a pathogenic virus in humans. Prevalence of B19V infection has been reported worldwide in all seasons, with a high incidence in the spring. B19V is responsible for erythema infectiosum (fifth disease) commonly seen in children. Its other clinical presentations include arthralgia, arthritis, transient aplastic crisis, chronic anemia, congenital anemia, and hydrops fetalis. In addition, B19V infection has been reported to trigger autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. However, the mechanisms of B19V participation in autoimmunity are not fully understood. B19V induced chronic disease and persistent infection suggests B19V can serve as a model for viral host interactions and the role of viruses in the pathogenesis of autoimmune diseases. Here we investigate the involvement of B19V in the breakdown of immune tolerance. Previously, we demonstrated that the non-structural protein 1 (NS 1) of B19V induces apoptosis in non-permissive cells lines and that this protein can cleave host DNA as well as form NS1-DNA adducts. Here we provide evidence that through programmed cell death, apoptotic bodies (ApoBods) are generated by B19V NS1 expression in a non-permissive cell line. Characterization of purified ApoBods identified potential self-antigens within them. In particular, signature self-antigens such as Smith, ApoH, DNA, histone H4 and phosphatidylserine associated with autoimmunity were present in these ApoBods. In addition, when purified ApoBods were introduced to differentiated macrophages, recognition, engulfment and uptake occurred. This suggests that B19V can produce a source of self-antigens for immune cell processing. The results support our hypothesis that B19V NS1-DNA adducts, and nucleosomal and lysosomal antigens present in ApoBods created in non-permissive cell lines, are a source of self-antigens. PMID:23776709

Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.

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Barrera, C; Ye, G; Espejo, R; Gunasena, S; Almanza, R; Leary, J; Crowe, S; Ernst, P; Reyes, V E

2001-10-01

Helicobacter pylori infection is linked to chronic gastritis, peptic ulcer and gastric carcinoma. During H. pylori infection, class II MHC expression by the gastric epithelium increases, as does the number of local CD4(+) T cells, which appear to be important in the associated pathogenesis. These observations suggested that the epithelium might present antigens to T cells. Thus, we sought to determine whether gastric epithelial cells process antigens to establish their function as local antigen presenting cells (APC). We examined a panel of gastric epithelial cell lines for expression of the antigen processing cathepsins B (CB), L (CL), S (CS), and D (CD). The mRNA for these enzymes were detected by RT-PCR and the enzymes in the gastric epithelial cells were identified by various independent methods. We corroborated the expression of CB and CD on gastric epithelial cells from human biopsy samples. The functions of these proteases were confirmed by assessing their ability to digest ovalbumin, a conventional dietary antigen, and proteins from H. pylori. In summary, multiple lines of evidence suggest gastric epithelial cells process antigens for presentation to CD4(+) T cells. To our knowledge, these are the first studies to document the antigen processing capacity of human gastric epithelial cells.

Low levels of the AhR in chronic obstructive pulmonary disease (COPD)-derived lung cells increases COX-2 protein by altering mRNA stability.

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Zago, Michela; Sheridan, Jared A; Traboulsi, Hussein; Hecht, Emelia; Zhang, Yelu; Guerrina, Necola; Matthews, Jason; Nair, Parameswaran; Eidelman, David H; Hamid, Qutayba; Baglole, Carolyn J

2017-01-01

Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

Human Leukocyte Antigen-G Is Frequently Expressed in a Multicentric Study on Glioblastoma and May Be Induced in Vitro by Combined 5-aza-2'-deoxycytidine and Interferon-γ Treatments

DEFF Research Database (Denmark)

Wastowski, Isabela J; Simões, Renata T; Yaghi, Layale

2012-01-01

-G protein expression was associated with a better long-term survival rate. The mechanisms underlying HLA-G gene expression were investigated in glioma cell lines U251MG, D247MG, and U138MG. Induction of HLA-G transcriptional activity was dependent of 5-aza-2'-deoxycytidine treatment and enhanced......Human leukocyte antigen-G (HLA-G) is a nonclassical major histocompatibility complex (MHC) class I molecule involved in immune tolerance processes, playing an important role in the maintenance of the semi-allogeneic fetus. Although HLA-G expression is restricted in normal tissues, it is broadly...... expressed in malignant tumors and may favor tumor immune escape. We analyzed HLA-G protein and mRNA expression in tumor samples from patients with glioblastoma collected in France, Denmark, and Brazil. We found HLA-G protein expression in 65 of 108 samples and mRNA in 20 of 21 samples. The absence of HLA...

Analysis of mechanism for human γδ T cell recognition of nonpeptide antigens

International Nuclear Information System (INIS)

Yamashita, Seiji; Tanaka, Yoshimasa; Tsutsumi, Shuichi; Aburatani, Hiroyuki; Minato, Nagahiro; Ihara, Sigeo

2005-01-01

Whereas human γδ T cells respond to nonpeptide antigens like pyrophosphomonoesters and alkyl amines in the primary reactions, only pyrophosphomonoesters provoke proliferative responses in the secondary responses. To elucidate the differences in stimulatory activity between the two groups of nonpeptide antigens, we systematically analyzed time courses of gene expressions by microarray analyses. While 253 genes were induced by stimulation with 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP), only 35 genes were detected after stimulation with isobutyl amine. Then, γδ T cells expressed various cytokines like XCL1-2, CCL3-4, TNF-α, and IFN-γ in response to 2M3B1PP in a time-dependent manner, while transient expressions were observed in IBA during the time period. The differences in such responsiveness are likely to originate from the activation state of NFAT, which is involved in the expression of transcription factors, EGR1-3 and NR4A1-2, and might play a crucial role in effector functions of γδ T cells

Design of a humanized anti vascular endothelial growth factor nanobody and evaluation of its in vitro function

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Fatemeh Kazemi-Lomedasht

2018-03-01

Full Text Available Objective(s: Nanobodies, the single domain antigen binding fragments of heavy chain-only antibodies occurring naturally in camelid sera, are the smallest intact antigen binding entities. Their minimal size assists in reaching otherwise largely inaccessible regions of antigens. However, their camelid origin raises a possible concern of immunogenicity when used for human therapy. Humanization is a promising approach to overcome the problem.   Materials and Methods: Here, we designed a humanized version of previously developed nanobody (anti vascular endothelial growth factor nanobody, evaluated and compared its predicted 3D structure, affinity and biological activity with its original wild type nanobody. Results: Our in silico results revealed an identical 3D structure of the humanized nanobody as compare to original nanobody. In vitro studies also demonstrated that the humanization had no significant visible effect on the nanobody affinity or on its biological activity.  Conclusion: The humanized nanobody could be developed and proposed as a promising lead to target pathologic angiogenesis.

Conserved epitope on several human vitamin K-dependent proteins: location of the antigenic site and influence of metal ions on antibody binding

International Nuclear Information System (INIS)

Church, W.R.; Messier, T.; Howard, P.R.; Amiral, J.; Meyer, D.; Mann, K.G.

1988-01-01

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125 I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 x 10 -8 to 1 x 10 -6 M. Chemical treatment of prothrombin with a variety of agents did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. Increasing concentrations of Ca 2+ , Mg 2+ , or Mn 2+ partially inhibited binding of 125 I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively. The antigenic site thus recognized by monoclonal antibody H-11 is located at the amino-terminal region in the highly conserved γ-carboxyglutamic acid-containing domains of several, but not all, vitamin K-dependent proteins

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

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Isabel Correa

2018-03-01

Full Text Available Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1 specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells.

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Correa, Isabel; Ilieva, Kristina M; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F; Tutt, Andrew N J; Nestle, Frank O; Karagiannis, Panagiotis; Lacy, Katie E; Karagiannis, Sophia N

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires.

Evaluation of Antigen-Conjugated Fluorescent Beads to Identify Antigen-Specific B Cells

Science.gov (United States)

Correa, Isabel; Ilieva, Kristina M.; Crescioli, Silvia; Lombardi, Sara; Figini, Mariangela; Cheung, Anthony; Spicer, James F.; Tutt, Andrew N. J.; Nestle, Frank O.; Karagiannis, Panagiotis; Lacy, Katie E.; Karagiannis, Sophia N.

2018-01-01

Selection of single antigen-specific B cells to identify their expressed antibodies is of considerable interest for evaluating human immune responses. Here, we present a method to identify single antibody-expressing cells using antigen-conjugated fluorescent beads. To establish this, we selected Folate Receptor alpha (FRα) as a model antigen and a mouse B cell line, expressing both the soluble and the membrane-bound forms of a human/mouse chimeric antibody (MOv18 IgG1) specific for FRα, as test antibody-expressing cells. Beads were conjugated to FRα using streptavidin/avidin-biotin bridges and used to select single cells expressing the membrane-bound form of anti-FRα. Bead-bound cells were single cell-sorted and processed for single cell RNA retrotranscription and PCR to isolate antibody heavy and light chain variable regions. Variable regions were then cloned and expressed as human IgG1/k antibodies. Like the original clone, engineered antibodies from single cells recognized native FRα. To evaluate whether antigen-coated beads could identify specific antibody-expressing cells in mixed immune cell populations, human peripheral blood mononuclear cells (PBMCs) were spiked with test antibody-expressing cells. Antigen-specific cells could comprise up to 75% of cells selected with antigen-conjugated beads when the frequency of the antigen-positive cells was 1:100 or higher. In PBMC pools, beads conjugated to recombinant antigens FRα and HER2 bound antigen-specific anti-FRα MOv18 and anti-HER2 Trastuzumab antibody-expressing cells, respectively. From melanoma patient-derived B cells selected with melanoma cell line-derived protein-coated fluorescent beads, we generated a monoclonal antibody that recognized melanoma antigen-coated beads. This approach may be further developed to facilitate analysis of B cells and their antibody profiles at the single cell level and to help unravel humoral immune repertoires. PMID:29628923

Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

OpenAIRE

Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

2011-01-01

An oligonucleotide representing a regulatory element of human thymidylate synthase mRNA has been crystallized as a dimer. The structure of the asymmetric dimer has been determined at 1.97 Å resolution.

Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

Science.gov (United States)

Canuto, K. S.; Teixeira, A. F.; Rodrigues, J. A.; Paoli, F.; Nogueira, E. M.; Mencalha, A. L.; Fonseca, A. S.

2017-06-01

MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

Effects of UVB irradiation on keratinocyte growth factor (KGF) and receptor (KGFR) expression in cultured human keratinocytes

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Zhou, Y.; Lee, H.S.T.; Kooshesh, F.; Fujisawa, H.; Sauder, D.N.; Kondo, S. [Univ. of Toronto, Sunnybrook Health Science Centre, Div. of Dermatology, Toronto (Canada)

1996-06-01

Keratinocyte growth factor (KGF) and its receptor (KGFR) are thought to play important roles in normal keratinocyte growth and differentiation. Since UVB radiation is known to influence keratinocyte growth, we sought to determine whether UVB would alter the expression of KGF and KGFR. Using a reverse-transcription coupled polymerase chain reaction (RT-PCR), the present study examined the expression of KGF and KGFR mRNA in cultured normal human keratinocytes exposed to UVB irradiation. Total cellular RNA was extracted from cultured keratinocytes at various time points after irradiation, reverse transcribed and used for PCR amplification using primers specific for KGF and KGFR. Constitutive expression of KGFR mRNA, but not KGF mRNA, was detected in normal cultured human keratinocytes. After UVB irradiation at 300 J/m{sup 2}, the KGF mRNA remained undetectable while the KGFR mRNA level was significantly decreased. The down-regulation of KGFR mRNA expression was also confirmed by Northern blot analysis. Immunohistochemical studies demonstrated a decreased positive signal of KGFR in human keratinocytes after UVB irradiation. Our results suggest a possible role for the KGF-KGFR signalling pathway in the skin after exposure to UVB, and that UVB-induced growth inhibition of keratinocytes in hyperproliferative skin disorders may be related to downregulation of KGFR. (au) 39 refs.

Peroxisome proliferator-activated receptor α (PPARα mRNA expression in human hepatocellular carcinoma tissue and non-cancerous liver tissue

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Kurokawa Tsuyoshi

2011-12-01

Full Text Available Abstract Background Peroxisome proliferator-activated receptor α (PPARα regulates lipid metabolism in the liver. It is unclear, however, how this receptor changes in liver cancer tissue. On the other hand, mouse carcinogenicity studies showed that PPARα is necessary for the development of liver cancer induced by peroxisome proliferators, and the relationship between PPARα and the development of liver cancer have been the focus of considerable attention. There have been no reports, however, demonstrating that PPARα is involved in the development of human liver cancer. Methods The subjects were 10 patients who underwent hepatectomy for hepatocellular carcinoma. We assessed the expression of PPARα mRNA in human hepatocellular carcinoma tissue and non-cancerous tissue, as well as the expression of target genes of PPARα, carnitine palmitoyltransferase 1A and cyclin D1 mRNAs. We also evaluated glyceraldehyde 3-phosphate dehydrogenase, a key enzyme in the glycolytic system. Results The amounts of PPARα, carnitine palmitoyltransferase 1A and glyceraldehyde 3-phosphate dehydrogenase mRNA in cancerous sections were significantly increased compared to those in non-cancerous sections. The level of cyclin D1 mRNA tends to be higher in cancerous than non-cancerous sections. Although there was a significant correlation between the levels of PPARα mRNA and cyclin D1 mRNA in both sections, however the correlation was higher in cancerous sections. Conclusion The present investigation indicated increased expression of PPARα mRNA and mRNAs for PPARα target genes in human hepatocellular carcinoma. These results might be associated with its carcinogenesis and characteristic features of energy production.

Enhancement of Human Antigen-Specific Memory T-Cell Responses by Interleukin-7 May Improve Accuracy in Diagnosing Tuberculosis▿ †

Science.gov (United States)

Feske, Marsha; Nudelman, Rodolfo J.; Medina, Miguel; Lew, Justin; Singh, Manisha; Couturier, Jacob; Graviss, Edward A.; Lewis, Dorothy E.

2008-01-01

Children and immunocompromised adults are at an increased risk of tuberculosis (TB), but diagnosis is more challenging. Recently developed gamma interferon (IFN-γ) release assays provide increased sensitivity and specificity for diagnosis of latent TB, but their use is not FDA approved in immunocompromised or pediatric populations. Both populations have reduced numbers of T cells, which are major producers of IFN-γ. Interleukin 7 (IL-7), a survival cytokine, stabilizes IFN-γ message and increases protein production. IL-7 was added to antigen-stimulated lymphocytes to improve IFN-γ responses as measured by enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunospot (ELISPOT) assay. Antigens used were tetanus toxoid (n = 10), p24 (from human immunodeficiency virus [HIV], n = 9), and TB peptides (n = 15). Keyhole limpet hemocyanin was used as a negative control, and phytohemagglutinin was the positive control. IL-7 improved antigen-specific responses to all antigens tested including tetanus toxoid, HIV type 1 p24, and TB peptides (ESAT-6 and CFP-10) with up to a 14-fold increase (mean = 3.8), as measured by ELISA. Increased IFN-γ responses from controls, HIV-positive patients, and TB patients were statistically significant, with P values of <0.05, 0.01, and 0.05, respectively. ELISPOT assay results confirmed ELISA findings (P values of <0.01, 0.02, and 0.03, respectively), with a strong correlation between the two tests (R2 = 0.82 to 0.99). Based on average background levels, IL-7 increased detection of IFN-γ by 39% compared to the level with antigen alone. Increased production of IFN-γ induced by IL-7 improves sensitivity of ELISA and ELISPOT assays for all antigens tested. Further enhancement of IFN-γ-based assays might improve TB diagnosis in those populations at highest risk for TB. PMID:18753334

Visfatin mRNA expression in human subcutaneous adipose tissue is regulated by exercise

DEFF Research Database (Denmark)

Frydelund-Larsen, Lone; Åkerström, Thorbjörn; Nielsen, Søren

2006-01-01

in abdominal subcutaneous adipose tissue and skeletal muscle biopsies obtained from healthy young men at time points 0, 3, 4.5, 6, 9, and 24 h in relation to either 3 h of ergometer cycle exercise at 60% of Vo(2 max) or rest. Adipose tissue visfatin mRNA expression increased threefold at the time points 3, 4......Visfatin [pre-beta-cell colony-enhancing factor (PBEF)] is a novel adipokine that is produced by adipose tissue, skeletal muscle, and liver and has insulin-mimetic actions. Regular exercise enhances insulin sensitivity. In the present study, we therefore examined visfatin mRNA expression.......5, and 6 h in response to exercise (n = 8) compared with preexercise samples and compared with the resting control group (n = 7, P = 0.001). Visfatin mRNA expression in skeletal muscle was not influenced by exercise. The exercise-induced increase in adipose tissue visfatin was, however, not accompanied...

Immunity to tumour antigens.

Science.gov (United States)

Li, Geng; Ali, Selman A; McArdle, Stephanie E B; Mian, Shahid; Ahmad, Murrium; Miles, Amanda; Rees, Robert C

2005-01-01

During the last decade, a large number of human tumour antigens have been identified. These antigens are classified as tumour-specific shared antigens, tissue-specific differentiation antigens, overexpressed antigens, tumour antigens resulting from mutations, viral antigens and fusion proteins. Antigens recognised by effectors of immune system are potential targets for antigen-specific cancer immunotherapy. However, most tumour antigens are self-proteins and are generally of low immunogenicity and the immune response elicited towards these tumour antigens is not always effective. Strategies to induce and enhance the tumour antigen-specific response are needed. This review will summarise the approaches to discovery of tumour antigens, the current status of tumour antigens, and their potential application to cancer treatment.

Metabolic activity and mRNA levels of human cardiac CYP450s involved in drug metabolism.

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Veronique Michaud

2010-12-01

Full Text Available Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68 of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7, samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

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Wouter Rozemeijer

Full Text Available Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag vaccine comprising capsular polysaccharide (types 5 and 8 conjugates, clumping factor A (ClfA and manganese transporter C (MntC is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27 or bloodstream (24 infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

Science.gov (United States)

Rozemeijer, Wouter; Fink, Pamela; Rojas, Eduardo; Jones, C Hal; Pavliakova, Danka; Giardina, Peter; Murphy, Ellen; Liberator, Paul; Jiang, Qin; Girgenti, Douglas; Peters, Remco P H; Savelkoul, Paul H M; Jansen, Kathrin U; Anderson, Annaliesa S; Kluytmans, Jan

2015-01-01

Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

Integrated Analysis of Dysregulated ncRNA and mRNA Expression Profiles in Humans Exposed to Carbon Nanotubes.

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Anna A Shvedova

Full Text Available As the application of carbon nanotubes (CNT in consumer products continues to rise, studies have expanded to determine the associated risks of exposure on human and environmental health. In particular, several lines of evidence indicate that exposure to multi-walled carbon nanotubes (MWCNT could pose a carcinogenic risk similar to asbestos fibers. However, to date the potential markers of MWCNT exposure are not yet explored in humans.In the present study, global mRNA and ncRNA expression profiles in the blood of exposed workers, having direct contact with MWCNT aerosol for at least 6 months (n = 8, were compared with expression profiles of non-exposed (n = 7 workers (e.g., professional and/or technical staff from the same manufacturing facility.Significant changes in the ncRNA and mRNA expression profiles were observed between exposed and non-exposed worker groups. An integrative analysis of ncRNA-mRNA correlations was performed to identify target genes, functional relationships, and regulatory networks in MWCNT-exposed workers. The coordinated changes in ncRNA and mRNA expression profiles revealed a set of miRNAs and their target genes with roles in cell cycle regulation/progression/control, apoptosis and proliferation. Further, the identified pathways and signaling networks also revealed MWCNT potential to trigger pulmonary and cardiovascular effects as well as carcinogenic outcomes in humans, similar to those previously described in rodents exposed to MWCNTs.This study is the first to investigate aberrant changes in mRNA and ncRNA expression profiles in the blood of humans exposed to MWCNT. The significant changes in several miRNAs and mRNAs expression as well as their regulatory networks are important for getting molecular insights into the MWCNT-induced toxicity and pathogenesis in humans. Further large-scale prospective studies are necessary to validate the potential applicability of such changes in mRNAs and miRNAs as prognostic markers

Transforming growth factor-beta1 stimulates the production of insulin-like growth factor-I and insulin-like growth factor-binding protein-3 in human bone marrow stromal osteoblast progenitors

DEFF Research Database (Denmark)

Kveiborg, Marie; Flyvbjerg, Allan; Eriksen, E F

2001-01-01

While transforming growth factor-beta1 (TGF-beta1) regulates proliferation and differentiation of human osteoblast precursor cells, the mechanisms underlying these effects are not known. Several hormones and locally acting growth factors regulate osteoblast functions through changes in the insulin......-like growth factors (IGFs) and IGF-binding proteins (IGFBPs). Thus, we studied the effects of TGF-beta1 on IGFs and IGFBPs in human marrow stromal (hMS) osteoblast precursor cells. TGF-beta1 increased the steady-state mRNA level of IGF-I up to 8.5+/-0.6-fold (P...

Muscle-specific expression of hypoxia-inducible factor in human skeletal muscle

DEFF Research Database (Denmark)

Mounier, Rémi; Pedersen, Bente Klarlund; Plomgaard, Peter

2010-01-01

fibres that possess unique patterns of protein and gene expression, producing different capillarization and energy metabolism systems. In this work, we analysed HIF-1alpha mRNA and protein expression related to the fibre-type composition in untrained human skeletal muscle by obtaining muscle biopsies...... from triceps brachii (characterized by a high proportion of type II fibres), from soleus (characterized by a high proportion of type I fibres) and from vastus lateralis (characterized by an equal proportion of type I and II fibres). The hypothesis was that type I muscle fibres would have lower HIF-1......alpha protein level. Interestingly, none of the HIF-1alpha target genes, like the most studied angiogenic factor involved in muscle angiogenesis, vascular endothelial growth factor (VEGF), exhibited a muscle fibre-specific-related mRNA expression at rest in normoxia. However, soleus presented...

Binding of NUFIP2 to Roquin promotes recognition and regulation of ICOS mRNA.

Science.gov (United States)

Rehage, Nina; Davydova, Elena; Conrad, Christine; Behrens, Gesine; Maiser, Andreas; Stehklein, Jenny E; Brenner, Sven; Klein, Juliane; Jeridi, Aicha; Hoffmann, Anne; Lee, Eunhae; Dianzani, Umberto; Willemsen, Rob; Feederle, Regina; Reiche, Kristin; Hackermüller, Jörg; Leonhardt, Heinrich; Sharma, Sonia; Niessing, Dierk; Heissmeyer, Vigo

2018-01-19

The ubiquitously expressed RNA-binding proteins Roquin-1 and Roquin-2 are essential for appropriate immune cell function and postnatal survival of mice. Roquin proteins repress target mRNAs by recognizing secondary structures in their 3'-UTRs and by inducing mRNA decay. However, it is unknown if other cellular proteins contribute to target control. To identify cofactors of Roquin, we used RNA interference to screen ~1500 genes involved in RNA-binding or mRNA degradation, and identified NUFIP2 as a cofactor of Roquin-induced mRNA decay. NUFIP2 binds directly and with high affinity to Roquin, which stabilizes NUFIP2 in cells. Post-transcriptional repression of human ICOS by endogenous Roquin proteins requires two neighboring non-canonical stem-loops in the ICOS 3'-UTR. This unconventional cis-element as well as another tandem loop known to confer Roquin-mediated regulation of the Ox40 3'-UTR, are bound cooperatively by Roquin and NUFIP2. NUFIP2 therefore emerges as a cofactor that contributes to mRNA target recognition by Roquin.

A novel dendritic cell-based direct ex vivo assay for detection and enumeration of circulating antigen-specific human T cells.

Science.gov (United States)

Carrio, Roberto; Zhang, Ge; Drake, Donald R; Schanen, Brian C

2018-05-07

Although a variety of assays have been used to examine T cell responses in vitro, standardized ex vivo detection of antigen-specific CD4 + T cells from human circulatory PBMCs remains constrained by low-dimensional characterization outputs and the need for polyclonal, mitogen-induced expansion methods to generate detectable response signals. To overcome these limitations, we developed a novel methodology utilizing antigen-pulsed autologous human dendritic target cells in a rapid and sensitive assay to accurately enumerate antigen-specific CD4 + T cell precursor frequency by multiparametric flow cytometry. With this approach, we demonstrate the ability to reproducibly quantitate poly-functional T cell responses following both primary and recall antigenic stimulation. Furthermore, this approach enables more comprehensive phenotypic profiling of circulating antigen-specific CD4 + T cells, providing valuable insights into the pre-existing polarization of antigen-specific T cells in humans. Combined, this approach permits sensitive and detailed ex vivo detection of antigen-specific CD4 + T cells delivering an important tool for advancing vaccine, immune-oncology and other therapeutic studies.

Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

International Nuclear Information System (INIS)

Zambetti, G.; Stein, J.; Stein, G.

1987-01-01

The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

Effect of radiation on the expression of tumor-associated antigens of human lung adenocarcinoma cells

International Nuclear Information System (INIS)

Hareyama, Masato

1988-01-01

We studied the effects of irradiation on the expression of a tumor-associated antigen (YH206 antigen) of cultured human lung adenocarcinoma A549 cells by using enzyme-linked immunosorbent assay (ELISA) and flow cytometry. YH206 antigen is preferentially expressed on adenocarcinoma cells. Irradiation of A549 cells remarkably increased the expression of YH206 antigen on the cell surface and the level of the antigen in the culture supernatant as well as in the cell lysate, whereas it significantly affected the expression of HLA (MHC-class I) antigen on the same cells. The expression of HLA antigen on the cell was also increased after treatment of the cells with interferon-γ. In an additional experiment, cells were stained simultaneously for surface antigens (fluorescein coupled antibodies) and for DNA content (propidium iodide), and then dual parameter measurements were performed by flow cytometry to analyse the relationship between antigen levels and the cell cycle. YH206 antigen and HLA antigen increased more in the S and G 2 /M phases of the cell cycle than in G 0 /G 1 . The expression of YH206 antigen was enhanced in the S and G 2 /M phases by irradiation, whereas the expression of HLA antigen was enhanced in each phase of the cell cycle with irradiation or IFN. These results suggest that irradiation plays a key role in the change of the expression of certain tumor-associated antigens. (author)

Connective tissue growth factor linked to the E7 tumor antigen generates potent antitumor immune responses mediated by an antiapoptotic mechanism.

Science.gov (United States)

Cheng, W-F; Chang, M-C; Sun, W-Z; Lee, C-N; Lin, H-W; Su, Y-N; Hsieh, C-Y; Chen, C-A

2008-07-01

A novel method for generating an antigen-specific cancer vaccine and immunotherapy has emerged using a DNA vaccine. However, antigen-presenting cells (APCs) have a limited life span, which hinders their long-term ability to prime antigen-specific T cells. Connective tissue growth factor (CTGF) has a role in cell survival. This study explored the intradermal administration of DNA encoding CTGF with a model tumor antigen, human papilloma virus type 16 E7. Mice vaccinated with CTGF/E7 DNA exhibited a dramatic increase in E7-specific CD4(+) and CD8(+) T-cell precursors. They also showed an impressive antitumor effect against E7-expressing tumors compared with mice vaccinated with the wild-type E7 DNA. The delivery of DNA encoding CTGF and E7 or CTGF alone could prolong the survival of transduced dendritic cells (DCs) in vivo. In addition, CTGF/E7-transduced DCs could enhance a higher number of E7-specific CD8(+) T cells than E7-transduced DCs. By prolonging the survival of APCs, DNA vaccine encoding CTGF linked to a tumor antigen represents an innovative approach to enhance DNA vaccine potency and holds promise for cancer prophylaxis and immunotherapy.

Host factor I, Hfq, binds to Escherichia coli ompA mRNA in a growth rate-dependent fashion and regulates its stability

DEFF Research Database (Denmark)

Vytvytska, O; Jakobsen, J S; Balcunaite, G

1998-01-01

RNA. In hfq mutant cells with a deficient Hfq gene product, the RNA-binding activity is missing, and analysis of the ompA mRNA showed that the growth-rate dependence of degradation is lost. Furthermore, the half-life of the ompA mRNA is prolonged in the mutant cells, irrespective of growth rate. Hfq has...

Toll-Like Receptor and Accessory Molecule mRNA Expression in Humans and Mice as Well as in Murine Autoimmunity, Transient Inflammation, and Progressive Fibrosis

Science.gov (United States)

Ramaiah, Santhosh Kumar Vankayala; Günthner, Roman; Lech, Maciej; Anders, Hans-Joachim

2013-01-01

The cell type-, organ-, and species-specific expression of the Toll-like receptors (TLRs) are well described, but little is known about the respective expression profiles of their accessory molecules. We therefore determined the mRNA expression levels of LBP, MD2, CD36, CD14, granulin, HMGB1, LL37, GRP94, UNC93b1, TRIL, PRAT4A, AP3B1, AEP and the respective TLRs in human and mouse solid organs. Humans and mice displayed significant differences between their respective mRNA expression patterns of these factors. In addition, the expression profiles in transient tissue inflammation upon renal ischemia-reperfusion injury, in spleens and kidneys from mice with lupus-like systemic autoimmunity, and in progressive tissue fibrosis upon unilateral ureteral obstruction were studied. Several TLR co-factors were specifically regulated during the different phases of these disease entities, suggesting a functional involvement in the disease process. Thus, the organ- and species-specific expression patterns need to be considered in the design and interpretation of studies related to TLR-mediated innate immunity, which seems to be involved in the tissue injury phase, in the phase of tissue regeneration, and in progressive tissue remodelling. PMID:23803655

PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2.

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Zhongshuai Xin

Full Text Available Interferon-α (IFN-α is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b. However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.

Ubiquitous hazardous metal lead induces TNF-{alpha} in human phagocytic THP-1 cells: Primary role of ERK 1/2

Energy Technology Data Exchange (ETDEWEB)

Khan, Mohd Imran [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Islam, Najmul [Department of Biochemistry, J.N Medical College, Aligarh Muslim University, Aligarh (India); Sahasrabuddhe, Amogh A. [Molecular and Structural Biology Division, Central Drug Research Institute, Lucknow (India); Mahdi, Abbas Ali [Department of Biochemistry, C.S.M. Medical University, Lucknow (India); Siddiqui, Huma; Ashquin, Mohd [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India); Ahmad, Iqbal, E-mail: ahmadi@sify.com [Fiber Toxicology Division, Indian Institute of Toxicology Research, Council of Scientific and Industrial Research (CSIR), Mahatma Gandhi Marg, P.O Box 80, Lucknow 226001, U.P. (India)

2011-05-15

Induction of tumor necrosis factor-{alpha} (TNF-{alpha}) in response to lead (Pb) exposure has been implicated in its immunotoxicity. However, the molecular mechanism by which Pb upregulates the level of TNF-{alpha} is wagely known. An attempt was therefore made to elucidate the mechanistic aspect of TNF-{alpha} induction, mainly focusing transcriptional and post transcriptional regulation via mitogen activated protein kinases (MAPKs) activation. We observed that exposure of Pb to human monocytic THP-1 cells resulted in significant enhanced production of TNF-{alpha} m-RNA and protein secretion. Moreover, the stability of TNF-{alpha} m-RNA was also increased as indicated by its half life. Notably, activation of ERK 1/2, p38 and JNK in Pb exposed THP-1 was also evident. Specific inhibitor of ERK1/2, PD 98059 caused significant inhibition in production and stability of TNF-{alpha} m-RNA. However, SB 203580 partially inhibited production and stability of TNF-{alpha} m-RNA. Interestingly, a combined exposure of these two inhibitors completely blocked modulation of TNF-{alpha} m-RNA. Data tends to suggest that expression and stability of TNF-{alpha} induction due to Pb exposure is mainly regulated through ERK. Briefly, these observations are useful in understanding some mechanistic aspects of proinflammatory and immunotoxicity of Pb, a globally acknowledged key environmental contaminant.

Ubiquitous hazardous metal lead induces TNF-α in human phagocytic THP-1 cells: Primary role of ERK 1/2

International Nuclear Information System (INIS)

Khan, Mohd Imran; Islam, Najmul; Sahasrabuddhe, Amogh A.; Mahdi, Abbas Ali; Siddiqui, Huma; Ashquin, Mohd; Ahmad, Iqbal

2011-01-01

Induction of tumor necrosis factor-α (TNF-α) in response to lead (Pb) exposure has been implicated in its immunotoxicity. However, the molecular mechanism by which Pb upregulates the level of TNF-α is wagely known. An attempt was therefore made to elucidate the mechanistic aspect of TNF-α induction, mainly focusing transcriptional and post transcriptional regulation via mitogen activated protein kinases (MAPKs) activation. We observed that exposure of Pb to human monocytic THP-1 cells resulted in significant enhanced production of TNF-α m-RNA and protein secretion. Moreover, the stability of TNF-α m-RNA was also increased as indicated by its half life. Notably, activation of ERK 1/2, p38 and JNK in Pb exposed THP-1 was also evident. Specific inhibitor of ERK1/2, PD 98059 caused significant inhibition in production and stability of TNF-α m-RNA. However, SB 203580 partially inhibited production and stability of TNF-α m-RNA. Interestingly, a combined exposure of these two inhibitors completely blocked modulation of TNF-α m-RNA. Data tends to suggest that expression and stability of TNF-α induction due to Pb exposure is mainly regulated through ERK. Briefly, these observations are useful in understanding some mechanistic aspects of proinflammatory and immunotoxicity of Pb, a globally acknowledged key environmental contaminant.

A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

DEFF Research Database (Denmark)

Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.

2015-01-01

Background: Computational methods for T cell-based vaccine target discovery focus on selection of highly conserved peptides identified across pathogen variants, followed by prediction of their binding of human leukocyte antigen molecules. However, experimental studies have shown that T cells ofte...... or proteome using human leukocyte antigen binding predictions and made a web-accessible software implementation freely available at http://met-hilab.cbs.dtu.dk/blockcons/....

Comparative efficacy of antigen and antibody detection tests for human trichinellosis

International Nuclear Information System (INIS)

Ivanoska, D.; Cuperlovic, K.; Gamble, H.R.; Murrell, K.D.

1989-01-01

Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory products of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

Science.gov (United States)

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

Antibody reaction of human anti-Toxoplasma gondii positive and negative sera with Neospora caninum antigens

OpenAIRE

Nam, Ho-Woo; Kang, Seung-Won; Choi, Won-Young

1998-01-01

Anti-Neospora caninum antibody was detected in anti-Toxoplasma gondii positive and negative human sera by ELISA, western blot and immunofluorescence assay (IFA). Twelve cases out of 172 (6.7%) Toxoplasma-positive sera cross-reacted with both T. gondii and N. caninum antigens, and one out of 110 Toxoplasma-negative sera reacted with N. caninum antigen by ELISA. By western blot, all 12 sera reacted with T. gondii antigens with various banding patterns but specifically at 30 kDa (SAG1) and 22 kD...

Complex mutual regulation of facilitates chromatin transcription (FACT) subunits on both mRNA and protein levels in human cells.

Science.gov (United States)

Safina, Alfiya; Garcia, Henry; Commane, Mairead; Guryanova, Olga; Degan, Seamus; Kolesnikova, Kateryna; Gurova, Katerina V

2013-08-01

Facilitates chromatin transcription (FACT) is a chromatin remodeling complex with two subunits: SSRP1 and SPT16. Mechanisms controlling FACT levels are of interest, since the complex is not expressed in most differentiated cells, but is frequently upregulated in cancer, particularly in poorly differentiated, aggressive tumors. Moreover, inhibition of FACT expression or function in tumor cells interferes with their survival. Here we demonstrate that SSRP1 and SPT16 protein levels decline upon induction of cellular differentiation or senescence in vitro and that similar declines in protein levels for both SSRP1 and SPT16 occur upon RNAi-mediated knockdown of either SSRP1 or SPT16. The interdependence of SSRP1 and SPT16 protein levels was found to be due to their association with SSRP1 and SPT16 mRNAs, which stabilizes the proteins. In particular, presence of SSRP1 mRNA is critical for SPT16 protein stability. In addition, binding of SSRP1 and SPT16 mRNAs to the FACT complex increases the stability and efficiency of translation of the mRNAs. These data support a model in which the FACT complex is stable when SSRP1 mRNA is present, but quickly degrades when SSRP1 mRNA levels drop. In the absence of FACT complex, SSRP1 and SPT16 mRNAs are unstable and inefficiently translated, making reactivation of FACT function unlikely in normal cells. Thus, we have described a complex and unusual mode of regulation controlling cellular FACT levels that results in amplified and stringent control of FACT activity. The FACT dependence of tumor cells suggests that mechanisms controlling FACT levels could be targeted for anticancer therapy.

Tissue localization of human trefoil factors 1, 2, and 3

DEFF Research Database (Denmark)

Madsen, Jens; Nielsen, Ole; Tornøe, Ida

2007-01-01

Trefoil factors (TTFs) are small, compact proteins coexpressed with mucins in the gastrointestinal tract. Three trefoil factors are known in mammals: TFF1, TFF2, and TFF3. They are implicated to play diverse roles in maintenance and repair of the gastrointestinal channel. We compared the expression...... pattern of the three trefoil factors analyzing mRNA from a panel of 20 human tissues by conventional reverse transcriptase (RT) PCR and, in addition, by real-time PCR. These findings were supported by immunohistochemical analysis of paraffin-embedded human tissues using rabbit polyclonal antibodies raised...... against these factors. TFF1 showed highest expression in the stomach and colon, whereas TFF2 and TFF3 showed highest expression in stomach and colon, respectively. All three TFFs were found in the ducts of pancreas. Whereas TFF2 was found to be restricted to these two tissues, the structurally more...

Human cytomegalovirus TRS1 protein associates with the 7-methylguanosine mRNA cap and facilitates translation.

Science.gov (United States)

Ziehr, Benjamin; Lenarcic, Erik; Vincent, Heather A; Cecil, Chad; Garcia, Benjamin; Shenk, Thomas; Moorman, Nathaniel J

2015-06-01

Viruses rely on the host translation machinery for the synthesis of viral proteins. Human cells have evolved sensors that recognize viral RNAs and inhibit mRNA translation in order to limit virus replication. Understanding how viruses manipulate the host translation machinery to gain access to ribosomes and disable the antiviral response is therefore a critical aspect of the host/pathogen interface. In this study, we used a proteomics approach to identify human cytomegalovirus (HCMV) proteins that might contribute to viral mRNA translation. The HCMV TRS1 protein (pTRS1) associated with the 7-methylguanosine mRNA cap, increased the total level of protein synthesis, and colocalized with mRNAs undergoing translation initiation during infection. pTRS1 stimulated translation of a nonviral reporter gene and increased the translation of a reporter containing an HCMV 5' untranslated region (5'UTR) to a greater extent. The preferential effect of pTRS1 on translation of an mRNA containing a viral 5'UTR required the pTRS1 RNA and double-stranded RNA-dependent protein kinase (PKR)-binding domains, and was likely the result of PKR inhibition. However, pTRS1 also stimulated the total level of protein synthesis and translation directed by an HCMV 5'UTR in cells lacking PKR. Thus our results demonstrate that pTRS1 stimulates translation through both PKR-dependent and PKR-independent mechanisms. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Protamine-based nanoparticles as new antigen delivery systems.

Science.gov (United States)

González-Aramundiz, José Vicente; Peleteiro Olmedo, Mercedes; González-Fernández, África; Alonso Fernández, María José; Csaba, Noemi Stefánia

2015-11-01

The use of biodegradable nanoparticles as antigen delivery vehicles is an attractive approach to overcome the problems associated with the use of Alum-based classical adjuvants. Herein we report, the design and development of protamine-based nanoparticles as novel antigen delivery systems, using recombinant hepatitis B surface antigen as a model viral antigen. The nanoparticles, composed of protamine and a polysaccharide (hyaluronic acid or alginate), were obtained using a mild ionic cross-linking technique. The size and surface charge of the nanoparticles could be modulated by adjusting the ratio of the components. Prototypes with optimal physicochemical characteristics and satisfactory colloidal stability were selected for the assessment of their antigen loading capacity, antigen stability during storage and in vitro and in vivo proof-of-concept studies. In vitro studies showed that antigen-loaded nanoparticles induced the secretion of cytokines by macrophages more efficiently than the antigen in solution, thus indicating a potential adjuvant effect of the nanoparticles. Finally, in vivo studies showed the capacity of these systems to trigger efficient immune responses against the hepatitis B antigen following intramuscular administration, suggesting the potential interest of protamine-polysaccharide nanoparticles as antigen delivery systems. Copyright © 2015 Elsevier B.V. All rights reserved.

"It is the antigen(s), stupid" and other lessons from over a decade of vaccitherapy of human cancer.

Science.gov (United States)

Buckwalter, Matthew R; Srivastava, Pramod K

2008-10-01

The lessons are: (a) human cancers certainly respond to immunological manipulations. Efforts at human cancer immunotherapy are therefore worthwhile. (b) Prophylaxis is very different from therapy of pre-existing disease, and hence much enthusiasm should not be derived from successful prophylaxis studies. Even in case of infectious agents against which robust prophylaxis is routinely achieved, therapy is nearly impossible once the disease has established. (c) Studies with appropriate cancer models of mice and rats are useful. The notion that it is easy to cure cancers in mice is generally advanced the most confidently by those who have never cured a mouse of cancer by immunotherapy. (d) With a nod to James Carville, it is the antigen(s), stupid! We still do not know the identity of protective tumor antigens. If any lesson can be drawn at all, it may well be that cancer immunotherapy must move away from the one-shoe-fits-all therapeutic models of chemotherapy and must move to individualized approaches. (e) All targets are equal, but some are more equal than others. The key is specificity for cancer. That does not necessarily mean specificity for cancer cells. (f) Vaccitherapy must be attempted preferably in the minimal residual disease setting, even though this is certain to be time-taking and expensive. In the setting of bulky disease, vaccitherapy must be combined with blockade of inhibitory signals, or depletion of down-regulatory T cells. Inhibition of effector level suppression of immune response is a key. Vaccitherapy alone or immuno-modulation alone is unlikely to succeed in therapy of bulky metastatic disease.

Radioimmunoassay procedure using a stabilized complex

International Nuclear Information System (INIS)

Sultanian, I.V.; Irani, J.H.

1978-01-01

An improved radioimmunoassay procedure involves the use of a stabilized complex of labelled antigen and antibody which has an extended shelf life as compared to the same complex absent the stabilizers. Since the time needed to incubate the mixture of labelled antigen and antibody is eliminated, the time for completing the assay is considerably shortened and simplified. The components for carrying out the procedure are packaged in a kit basically including standard antigen for generation of a standard curve, a stabilized labelled antigen-antibody complex and reference serum, if used. A plurality of stabilizers are used in the complex to provide a shelf life of six weeks or more. 10 claims

Partially purified fraction antigen from adult Fasciola Gigantic a for the serodiagnosis of human fascioliasis using Dot-ELISA technique

International Nuclear Information System (INIS)

Dalimi, Abdolhossein; Hadighi, Ramtin; Madani, Rasool

2004-01-01

Human fascioliasis has been reported in many countries including Iran. Various techniques have been evaluated for diagnosis of human fascioliasis using different antigens. We evaluated fasciola gigantica partially purified fraction antigen (PPF) isolated from sheep's liver fluke for the diagnosis of human fascioliasis. 261 sera was collected from 104 patients living in an area endemic for human fascioliasis from 89 non-fascioliasis patients living in a non-endemic area and from 68 healthy individuals. Micro-ELISA ws used in the evaluation of the sensitivity and the specificity of dot-ELISA. With a 1:800 sera dilution as the cut-off titer, the sensitivity of Dot-ELISA test in diagnosis of human fascioliasis was 94.23% and the specificity was 99.36%.Dot-ELISA using PPF antigen is sensitive and specific method for diagnosis of human fascioliasisthat is also rapid and inexpensive. (author)

The staphylococcal accessory regulator, SarA, is an RNA-binding protein that modulates the mRNA turnover properties of late-exponential and stationary phase Staphylococcus aureus cells

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John M Morrison

2012-03-01

Full Text Available The modulation of mRNA turnover is gaining recognition as a mechanism by which Staphylococcus aureus regulates gene expression, but the factors that orchestrate alterations in transcript degradation are poorly understood. In that regard, we previously found that 138 mRNA species, including the virulence factors protein A (spa and collagen binding protein (cna, are stabilized in a sarA-dependent manner during exponential phase growth, suggesting that SarA protein may directly or indirectly effect the RNA turnover properties of these transcripts. Herein, we expanded our characterization of the effects of sarA on mRNA turnover during late exponential and stationary phases of growth. Results revealed that the locus affects the RNA degradation properties of cells during both growth phases. Further, using gel mobility shift assays and RIP-ChIP, it was found that SarA protein is capable of binding mRNA species that it stabilizes both in vitro and within bacterial cells. Taken together, these results suggest that SarA post-transcriptionally regulates S. aureus gene expression in a manner that involves binding to and consequently altering the mRNA turnover properties of target transcripts.

A murine model of type 2 autoimmune hepatitis: Xenoimmunization with human antigens.

Science.gov (United States)

Lapierre, Pascal; Djilali-Saiah, Idriss; Vitozzi, Susana; Alvarez, Fernando

2004-04-01

Autoimmune hepatitis (AIH) is characterized by an immune-mediated injury of the hepatic parenchyma of unknown pathogenesis. Type 2 AIH is identified by the presence of anti-liver-kidney microsomes type 1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1) autoantibodies. The current study shows that a murine model of AIH can be generated by DNA immunization against type 2 AIH self-antigens (P450 2D6 and formiminotransferase-cyclodeaminase). A pCMV plasmid containing the N-terminal region of mouse CTLA-4 and the antigenic region of human CYP2D6 (672-1,377 bp) and human formiminotransferase cyclodeaminase (FTCD; 1,232-1,668 bp) was used for DNA immunization of C57BL/6 female mice. Immunized mice showed elevated levels of alanine aminotransferase (ALT), with peaks at 4 and 7 months postinjection. Periportal, portal, and intralobular liver inflammatory infiltrates were observed at histology. Mainly CD4+ lymphocytes, but also CD8+ and B lymphocytes, were found in the liver. Cytotoxic-specific T cells were found in both the liver and spleen of these animals. Mice developed anti-LKM1 and anti-LC1 antibodies of immunoglobulin G2 (IgG2) subclass, against specific mouse autoantigens. The ALT levels correlated with both the presence of anti-LKM1/anti-LC1 antibodies and the presence of liver necroinflammation. In conclusion, in mice, DNA immunization against human autoantigens breaks tolerance and induces an autoimmune liver disease. Molecular mimicry between foreign and self-antigens explains the liver injury. This model of AIH resembles human type 2 AIH and will be helpful for the study of its pathogenesis.

Natural micropolymorphism in human leukocyte antigens provides a basis for genetic control of antigen recognition

Energy Technology Data Exchange (ETDEWEB)

Archbold, Julia K.; Macdonald, Whitney A.; Gras, Stephanie; Ely, Lauren K.; Miles, John J.; Bell, Melissa J.; Brennan, Rebekah M.; Beddoe, Travis; Wilce, Matthew C.J.; Clements, Craig S.; Purcell, Anthony W.; McCluskey, James; Burrows, Scott R.; Rossjohn, Jamie; (Monash); (Queensland Inst. of Med. Rsrch.); (Melbourne)

2009-07-10

Human leukocyte antigen (HLA) gene polymorphism plays a critical role in protective immunity, disease susceptibility, autoimmunity, and drug hypersensitivity, yet the basis of how HLA polymorphism influences T cell receptor (TCR) recognition is unclear. We examined how a natural micropolymorphism in HLA-B44, an important and large HLA allelic family, affected antigen recognition. T cell-mediated immunity to an Epstein-Barr virus determinant (EENLLDFVRF) is enhanced when HLA-B*4405 was the presenting allotype compared with HLA-B*4402 or HLA-B*4403, each of which differ by just one amino acid. The micropolymorphism in these HLA-B44 allotypes altered the mode of binding and dynamics of the bound viral epitope. The structure of the TCR-HLA-B*4405EENLLDFVRF complex revealed that peptide flexibility was a critical parameter in enabling preferential engagement with HLA-B*4405 in comparison to HLA-B*4402/03. Accordingly, major histocompatibility complex (MHC) polymorphism can alter the dynamics of the peptide-MHC landscape, resulting in fine-tuning of T cell responses between closely related allotypes.

Evaluation of a new set of recombinant antigens for the serological diagnosis of human and canine visceral leishmaniasis.

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Franklin B Magalhães

Full Text Available Current strategies for the control of zoonotic visceral leishmaniasis (VL rely on its efficient diagnosis in both human and canine hosts. The most promising and cost effective approach is based on serologic assays with recombinant proteins. However, no single antigen has been found so far which can be effectively used to detect the disease in both dogs and humans. In previous works, we identified Leishmania infantum antigens with potential for the serodiagnosis of VL. Here, we aimed to expand the panel of the available antigens for VL diagnosis through another screening of a genomic expression library. Seven different protein-coding gene fragments were identified, five of which encoding proteins which have not been previously studied in Leishmania and rich in repetitive motifs. Poly-histidine tagged polypeptides were generated from six genes and evaluated for their potential for diagnosis of VL by ELISA (Enzyme Linked ImmunoSorbent Assay with sera from infected humans and dogs. None of those was valid for the detection of human VL (26-52% sensitivity although their performance was increased in the canine sera (48-91% sensitivity, with one polypeptide useful for the diagnosis of canine leishmaniasis. Next, we assayed a mixture of three antigens, found to be best for human or canine VL, among 13 identified through different screenings. This "Mix" resulted in similar levels of sensitivity for both human (84% and canine (88% sera. With improvements, this validates the use of multiple proteins, including antigens identified here, as components of a single system for the diagnosis of both forms of leishmaniasis.

cDNA cloning, mRNA distribution and heterogeneity, chromosomal location, and RFLP analysis of human osteopontin (OPN)

DEFF Research Database (Denmark)

Young, M F; Kerr, J M; Termine, J D

1990-01-01

A human osteopontin (OP) cDNA was isolated from a library made from primary cultures of human bone cells. The distribution of osteopontin mRNA in human tissues was investigated by Northern analysis and showed that the human message was predominant in cultures of bone cells and in decidua cells...... osteopontin cDNA indicated that the gene is a single copy with an approximate length of 5.4-8.2 kb....

The human E48 antigen, highly homologous to the murine Ly-6 antigen ThB, is a GPI-anchored molecule apparently involved in keratinocyte cell-cell adhesion

NARCIS (Netherlands)

Brakenhoff, R H; Gerretsen, M; Knippels, E M; van Dijk, M.; van Essen, H; Weghuis, D O; Sinke, R J; Snow, G B; van Dongen, G A

The E48 antigen, a putative human homologue of the 20-kD protein present in desmosomal preparations of bovine muzzle, and formerly called desmoglein III (dg4), is a promising target antigen for antibody-based therapy of squamous cell carcinoma in man. To anticipate the effect of high antibody dose

Human peripheral blood monocytes display surface antigens recognized by monoclonal antinuclear antibodies

International Nuclear Information System (INIS)

Holers, V.M.; Kotzin, B.L.

1985-01-01

The authors used monoclonal anti-nuclear autoantibodies and indirect immunofluorescence to examine normal human peripheral blood mononuclear leukocytes for the presence of cell surface nuclear antigens. Only one monoclonal anti-histone antibody (MH-2) was found to bind to freshly isolated PBL, staining approximately 10% of large cells. However, after cells were placed into culture for 16-24 h, a high percentage (up to 60%) of large-sized cells were recognized by an anti-DNA (BWD-1) and several different antihistone monoclonal antibodies (BWH-1, MH-1, and MH-2). These antibodies recognize separate antigenic determinants on chromatin and histones extracted from chromatin. The histone antigen-positive cells were viable, and the monoclonal antibodies could be shown to be binding to the cell surface and not to the nucleus. Using monoclonal antibodies specific for monocytes and T cells, and complement-mediated cytotoxicity, the cells bearing histone antigens were shown to be primarily monocytes. The appearance of histone and DNA antigen-positive cells was nearly completely inhibited by the addition of low concentrations of cycloheximide at initiation of the cultures. In contrast, little effect on the percentage of positive cells was detected if cells were exposed to high doses of gamma irradiation before culture. These data further support the existence of cell surface nuclear antigens on selected cell subsets, which may provide insight into the immunopathogenesis of systemic lupus erythematosus and related autoimmune diseases

Statins Activate Human PPAR Promoter and Increase PPAR mRNA Expression and Activation in HepG2 Cells

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Makoto Seo

2008-01-01

Full Text Available Statins increase peroxisome proliferator-activated receptor (PPAR mRNA expression, but the mechanism of this increased PPAR production remains elusive. To examine the regulation of PPAR production, we examined the effect of 7 statins (atorvastatin, cerivastatin, fluvastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin on human PPAR promoter activity, mRNA expression, nuclear protein levels, and transcriptional activity. The main results are as follows. (1 Majority of statins enhanced PPAR promoter activity in a dose-dependent manner in HepG2 cells transfected with the human PPAR promoter. This enhancement may be mediated by statin-induced HNF-4. (2 PPAR mRNA expression was increased by statin treatment. (3 The PPAR levels in nuclear fractions were increased by statin treatment. (4 Simvastatin, pravastatin, and cerivastatin markedly enhanced transcriptional activity in 293T cells cotransfected with acyl-coenzyme A oxidase promoter and PPAR/RXR expression vectors. In summary, these data demonstrate that PPAR production and activation are upregulated through the PPAR promoter activity by statin treatment.

Role of the mRNA export factor Sus1 in oxidative stress tolerance in Candida albicans.

Science.gov (United States)

Xiao, Chenpeng; Yu, Qilin; Zhang, Bing; Li, Jianrong; Zhang, Dan; Li, Mingchun

2018-02-05

In eukaryotes, the nuclear export of mRNAs is essential for gene expression. However, little is known about the role of mRNA nuclear export in the important fungal pathogen, Candida albicans. In this study, we identified C. albicans Sus1, a nucleus-localized protein that is required for mRNA export. Interestingly, the sus1Δ/Δ displayed hyper-sensitivity to extracellular oxidative stress, enhanced ROS accumulation and severe oxidative stress-related cell death. More strikingly, although the mutant exhibited normal activation of the expression of oxidative stress response (OSR) genes, it had attenuated activity of ROS scavenging system, which may be attributed to the defect in OSR mRNA export in this mutant. In addition, the virulence of the sus1Δ/Δ was seriously attenuated. Taken together, our findings provide evidence that the mRNA export factor Sus1 plays an important role in oxidative stress tolerance and pathogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Prevalence of Human Papillomavirus Infection in Unselected SurePath Samples Using the APTIMA HPV mRNA Assay

DEFF Research Database (Denmark)

Rebolj, Matejka; Preisler, Sarah; Ejegod, Ditte M

2013-01-01

The APTIMA Human Papillomavirus (HPV) Assay detects E6/E7 mRNA from 14 human papillomavirus genotypes. Horizon was a population-based split-sample study among well-screened women, with an aim to compare APTIMA, Hybrid Capture 2 (HC2), and liquid-based cytology (LBC) using SurePath samples. APTIMA...

Human IgG repertoire of malaria antigen-immunized human immune system (HIS) mice.

Science.gov (United States)

Nogueira, Raquel Tayar; Sahi, Vincent; Huang, Jing; Tsuji, Moriya

2017-08-01

Humanized mouse models present an important tool for preclinical evaluation of new vaccines and therapeutics. Here we show the human variable repertoire of antibody sequences cloned from a previously described human immune system (HIS) mouse model that possesses functional human CD4+ T cells and B cells, namely HIS-CD4/B mice. We sequenced variable IgG genes from single memory B-cell and plasma-cell sorted from splenocytes or whole blood lymphocytes of HIS-CD4/B mice that were vaccinated with a human plasmodial antigen, a recombinant Plasmodium falciparum circumsporozoite protein (rPfCSP). We demonstrate that rPfCSP immunization triggers a diverse B-cell IgG repertoire composed of various human VH family genes and distinct V(D)J recombinations that constitute diverse CDR3 sequences similar to humans, although low hypermutated sequences were generated. These results demonstrate the substantial genetic diversity of responding human B cells of HIS-CD4/B mice and their capacity to mount human IgG class-switched antibody response upon vaccination. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Human NTH1 physically interacts with p53 and proliferating cell nuclear antigen

International Nuclear Information System (INIS)

Oyama, Masaki; Wakasugi, Mitsuo; Hama, Takashi; Hashidume, Hatsuho; Iwakami, Yasutaka; Imai, Rika; Hoshino, Sanae; Morioka, Hiroshi; Ishigaki, Yasuhito; Nikaido, Osamu; Matsunaga, Tsukasa

2004-01-01

Thymine glycol (Tg) is one of predominant oxidative DNA lesions caused by ionizing radiation and other oxidative stresses. Human NTH1 is a bifunctional enzyme with DNA glycosylase and AP lyase activities and removes Tg as the first step of base excision repair (BER). We have searched for the factors interacting with NTH1 by using a pull-down assay and found that GST-NTH1 fusion protein precipitates proliferating cell nuclear antigen (PCNA) and p53 as well as XPG from human cell-free extracts. GST-NTH1 also bound to recombinant FLAG-tagged XPG, PCNA, and (His) 6 -tagged p53 proteins, indicating direct protein-protein interaction between those proteins. Furthermore, His-p53 and FLAG-XPG, but not PCNA, stimulated the Tg DNA glycosylase/AP lyase activity of GST-NTH1 or NTH1. These results provide an insight into the positive regulation of BER reaction and also suggest a possible linkage between BER of Tg and other cellular mechanisms

Regulation of coagulation factor XI expression by microRNAs in the human liver.

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Salam Salloum-Asfar

Full Text Available High levels of factor XI (FXI increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK demonstrated a direct interaction between miR-181a-5p and 3'untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.

Directory of Open Access Journals (Sweden)

Sean A Gray

Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.

NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination.

Directory of Open Access Journals (Sweden)

Yoshie Kametani

Full Text Available Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD, which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg. After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2 cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP or keyhole limpet hemocyanin (KLH successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

Plastic downregulation of the transcriptional repressor BCL6 during maturation of human dendritic cells

International Nuclear Information System (INIS)

Pantano, Serafino; Jarrossay, David; Saccani, Simona; Bosisio, Daniela; Natoli, Gioacchino

2006-01-01

Dendritic cell (DC) maturation links peripheral events initiated by the encounter with pathogens to the activation and expansion of antigen-specific T lymphocytes in secondary lymphoid organs. Here, we describe an as yet unrecognized modulator of human DC maturation, the transcriptional repressor BCL6. We found that both myeloid and plasmacytoid DCs constitutively express BCL6, which is rapidly downregulated following maturation triggered by selected stimuli. Both in unstimulated and maturing DCs, control of BCL6 protein levels reflects the convergence of several mechanisms regulating BCL6 stability, mRNA transcription and nuclear export. By regulating the induction of several genes implicated in the immune response, including inflammatory cytokines, chemokines and survival genes, BCL6 may represent a pivotal modulator of the afferent branch of the immune response

The use of a synthetic antigen for the serological diagnosis of human trichinellosis

Directory of Open Access Journals (Sweden)

Bruschi F.

2001-06-01

Full Text Available Hosts infected with Trichinella produce antibodies specific for an epitope common to the TSL-1 family antigens. This epitope contained uncommon terminal 3, 6-dideoxy-D-arabinohexose (so called tyvelose residues. The disaccharide moiety was synthesized and an immunodiagnostic assay was developed, which was specific and sensitive in swine trichinellosis. We aimed to verify the specificity and sensitivity of this immunodiagnostic test in human trichinellosis. 15 sera from normal subjects, 12 from patients with other parasitic diseases and 50 from trichinellosis patients were tested. Indirect enzyme linked immunosorbent assay (ELISA for specific IgG and an amplified ELISA for specific IgE were performed using β-tyvelose-GalNAc-bovine serum albumin (BSA disaccharide conjugate or T. spiralis muscle larvae excretory/secretory (E/S products, as antigens. Neither control sera nor other parasitic infection sera resulted positive both for IgG and IgE when synthetic or E/S antigens were used. In trichinellosis patient sera, specific IgG were present in 100 % of cases, irrespective of the antigen used, but whereas specific IgE were detected in 78 % using E/S antigens, a 100% positivity rate was obtained, using the β-tyvelose- BSA conjugate.

Familial occurrence of subacute thyroiditis associated with human leukocyte antigen-B35

NARCIS (Netherlands)

Kramer, AB; Roozendaal, C; Dullaart, RPF

Subacute thyroiditis (SAT) is a spontaneously remitting inflammatory disorder of the thyroid, associated with human leukocyte antigen (HLA)-B35, and may be virally induced in genetically predisposed individuals. A 57-year-old Caucasian man presented with symptoms of hyperthyroidism as well as

mRNA transfection of mouse and human neural stem cell cultures

OpenAIRE

McLenachan, Samuel; Zhang, D.; Palomo, A.B.; Edel, Michael John; Chen, F.K.

2013-01-01

The use of synthetic mRNA as an alternative gene delivery vector to traditional DNA-based constructs provides an effective method for inducing transient gene expression in cell cultures without genetic modification. Delivery of mRNA has been proposed as a safer alternative to viral vectors in the induction of pluripotent cells for regenerative therapies. Although mRNA transfection of fibroblasts, dendritic and embryonic stem cells has been described, mRNA delivery to neurosphere cultures has ...

Efficacy of Omega Fatty Acid Supplementation on mRNA Expression Level of Tumor Necrosis Factor Alpha in Patients with Gastric Adenocarcinoma.

Science.gov (United States)

Hosseinzadeh, Asghar; Ardebili, Seyed Mojtaba Mohaddes

2016-09-01

Tumor necrosis factor alpha (TNF-α), a multifunctional cytokine, is involved in apoptosis, cell proliferation, cell survival, and inflammation. It plays a dual role in cancer development and progression. It has been revealed that polyunsaturated fatty acids (PUFAs) modulate the production and activity of TNF family cytokines. The objective of the present study was to evaluate the effect of PUFAs on messenger RNA expression levels of TNF-α in patients with gastric adenocarcinoma. Thirty-four chemotherapy-naive patients diagnosed with gastric adenocarcinoma were randomly divided into two groups. The first group (17 individuals) received cisplatin without supplements and the second group (17 individuals) received cisplatin plus orally administered PUFA supplements for 3 weeks, based on treatment strategies. The gastric biopsy samples were obtained from all participants before and after treatment, and TNF-α mRNA expression levels were evaluated by quantitative real-time PCR procedure. Our findings revealed that TNF-α mRNA expression is downregulated in group II, after receiving cisplatin and omega fatty acid supplement for 3 weeks. However, this difference is not statistically significant (p > 0.05). TNF-α mRNA expression did not show significant alteration in group I, after receiving cisplatin alone. Taken together, we concluded that omega fatty acids reduce TNF-α expression at the mRNA level in patients with gastric adenocarcinoma. These data suggest that TNF-α may act as a potential target for the therapy of human gastric adenocarcinoma.

Identification and quantification of mRNA for nerve growth factor in histological preparations

Energy Technology Data Exchange (ETDEWEB)

Wilson, P A; Rush, R A; Scott, J; Penschow, J; Coghlan, J

1986-03-14

Hybridization histochemistry has been used to detect mRNA for nerve growth factor (NGF) in histological preparations of mouse salivary glands and rat iris using a /sup 32/P-labelled cDNA probe and autoradiography. Label was visible over the tubular cells of the male mouse submaxillary gland but not the sublingual gland. A much lower label density was found over the tubular cells of the female submaxillary gland, whereas sections of liver and pancreas were negative. Quantitative autoradiography allowed the detection of low levels of mRNA for NGF in the rat iris which was elevated by prior culture of the tissue. The results provide direct histological evidence for the presence of specific NGF-mRNA in the mouse submaxillary gland and rat iris, with increased levels following culture. 15 refs.

TP53 and ATM mRNA expression in skin and skeletal muscle after low-level laser exposure.

Science.gov (United States)

Guedes de Almeida, Luciana; Sergio, Luiz Philippe da Silva; de Paoli, Flavia; Mencalha, Andre Luiz; da Fonseca, Adenilson de Souza

2017-08-01

Low-level lasers are widespread in regenerative medicine, but the molecular mechanisms involved in their biological effects are not fully understood, particularly those on DNA stability. Therefore, this study aimed to investigate mRNA expression of genes related to DNA genomic stability in skin and skeletal muscle tissue from Wistar rats exposed to low-level red and infrared lasers. For this, TP53 (Tumor Protein 53) and ATM (Ataxia Telangiectasia Mutated gene) mRNA expressions were evaluated by real-time quantitative PCR (RT-qPCR) technique 24 hours after low-level red and infrared laser exposure. Our data showed that relative TP53 mRNA expression was not significantly altered in both tissues exposed to lasers. For ATM, relative mRNA expression in skin tissue was not significantly altered, but in muscle tissue, laser exposure increased relative ATM mRNA expression. Low-level red and infrared laser radiations alter ATM mRNA expression related to DNA stability in skeletal muscle tissue.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes.

Science.gov (United States)

Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-09-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1alpha (HNF1alpha) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes

International Nuclear Information System (INIS)

Richert, Lysiane; Lamboley, Christelle; Viollon-Abadie, Catherine; Grass, Peter; Hartmann, Nicole; Laurent, Stephane; Heyd, Bruno; Mantion, Georges; Chibout, Salah-Dine; Staedtler, Frank

2003-01-01

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of L-fatty acid binding protein (L-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1α (HNF1α) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis

In silico and cell-based analyses reveal strong divergence between prediction and observation of T-cell-recognized tumor antigen T-cell epitopes.

Science.gov (United States)

Schmidt, Julien; Guillaume, Philippe; Dojcinovic, Danijel; Karbach, Julia; Coukos, George; Luescher, Immanuel

2017-07-14

Tumor exomes provide comprehensive information on mutated, overexpressed genes and aberrant splicing, which can be exploited for personalized cancer immunotherapy. Of particular interest are mutated tumor antigen T-cell epitopes, because neoepitope-specific T cells often are tumoricidal. However, identifying tumor-specific T-cell epitopes is a major challenge. A widely used strategy relies on initial prediction of human leukocyte antigen-binding peptides by in silico algorithms, but the predictive power of this approach is unclear. Here, we used the human tumor antigen NY-ESO-1 (ESO) and the human leukocyte antigen variant HLA-A*0201 (A2) as a model and predicted in silico the 41 highest-affinity, A2-binding 8-11-mer peptides and assessed their binding, kinetic complex stability, and immunogenicity in A2-transgenic mice and on peripheral blood mononuclear cells from ESO-vaccinated melanoma patients. We found that 19 of the peptides strongly bound to A2, 10 of which formed stable A2-peptide complexes and induced CD8 + T cells in A2-transgenic mice. However, only 5 of the peptides induced cognate T cells in humans; these peptides exhibited strong binding and complex stability and contained multiple large hydrophobic and aromatic amino acids. These results were not predicted by in silico algorithms and provide new clues to improving T-cell epitope identification. In conclusion, our findings indicate that only a small fraction of in silico -predicted A2-binding ESO peptides are immunogenic in humans, namely those that have high peptide-binding strength and complex stability. This observation highlights the need for improving in silico predictions of peptide immunogenicity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Epigenetic modulation of cancer-germline antigen gene expression in tumorigenic human mesenchymal stem cells: implications for cancer therapy

DEFF Research Database (Denmark)

Gjerstorff, Morten; Burns, Jorge S; Nielsen, Ole

2009-01-01

Cancer-germline antigens are promising targets for cancer immunotherapy, but whether such therapies will also eliminate the primary tumor stem cell population remains undetermined. We previously showed that long-term cultures of telomerized adult human bone marrow mesenchymal stem cells can...... spontaneously evolve into tumor-initiating, mesenchymal stem cells (hMSC-TERT20), which have characteristics of clinical sarcoma cells. In this study, we used the hMSC-TERT20 tumor stem cell model to investigate the potential of cancer-germline antigens to serve as tumor stem cell targets. We found...... of cancer-germline antigens in hMSC-TERT20 cells, while their expression levels in primary human mesenchymal stem cells remained unaffected. The expression pattern of cancer-germline antigens in tumorigenic mesenchymal stem cells and sarcomas, plus their susceptibility to enhancement by epigenetic...

Removal of a C-terminal serine residue proximal to the inter-chain disulfide bond of a human IgG1 lambda light chain mediates enhanced antibody stability and antibody dependent cell-mediated cytotoxicity

Science.gov (United States)

Shen, Yang; Zeng, Lin; Zhu, Aiping; Blanc, Tim; Patel, Dipa; Pennello, Anthony; Bari, Amtul; Ng, Stanley; Persaud, Kris; Kang, Yun (Kenneth); Balderes, Paul; Surguladze, David; Hindi, Sagit; Zhou, Qinwei; Ludwig, Dale L.; Snavely, Marshall

2013-01-01

Optimization of biophysical properties is a critical success factor for the developability of monoclonal antibodies with potential therapeutic applications. The inter-domain disulfide bond between light chain (Lc) and heavy chain (Hc) in human IgG1 lends structural support for antibody scaffold stability, optimal antigen binding, and normal Fc function. Recently, human IgG1λ has been suggested to exhibit significantly greater susceptibility to reduction of the inter Lc-Hc disulfide bond relative to the same disulfide bond in human IgG1κ. To understand the molecular basis for this observed difference in stability, the sequence and structure of human IgG1λ and human IgG1κ were compared. Based on this Lc comparison, three single mutations were made in the λ Lc proximal to the cysteine residue, which forms a disulfide bond with the Hc. We determined that deletion of S214 (dS) improved resistance of the association between Lc and Hc to thermal stress. In addition, deletion of this terminal serine from the Lc of IgG1λ provided further benefit, including an increase in stability at elevated pH, increased yield from transient transfection, and improved in vitro antibody dependent cell-mediated cytotoxicity (ADCC). These observations support the conclusion that the presence of the terminal serine of the λ Lc creates a weaker inter-chain disulfide bond between the Lc and Hc, leading to slightly reduced stability and a potential compromise in IgG1λ function. Our data from a human IgG1λ provide a basis for further investigation of the effects of deleting terminal serine from λLc on the stability and function of other human IgG1λ antibodies. PMID:23567210

Dichotomy of the human T cell response to Leishmania antigens. I. Th1-like response to Leishmania major promastigote antigens in individuals recovered from cutaneous leishmaniasis

DEFF Research Database (Denmark)

Kemp, M; Hey, A S; Kurtzhals, J A

1994-01-01

of skin lesions, and in Danes without known exposure to Leishmania parasites. Proliferation and production of interferon-gamma (IFN-gamma) and IL-4 in antigen-stimulated cultures was measured. Lymphocytes from individuals with a history of CL proliferated vigorously and produced IFN-gamma after...... the unexposed Danes were not activated by gp63. The cells from Danish donors produced either IFN-gamma or IL-4, but not both cytokines after incubation with the crude preparation of L. major antigens. The data show that the T cell response to Leishmania antigens in humans who have had uncomplicated CL...... stimulation with either a crude preparation of L. major antigens or the major surface protease gp63. These cultures produced no or only little IL-4. Also cells from leishmanin skin test-positive donors with no history of CL produced IFN-gamma and no IL-4 in response to L. major antigens. Cells from...

2'-O-methylation in mRNA disrupts tRNA decoding during translation elongation.

Science.gov (United States)

Choi, Junhong; Indrisiunaite, Gabriele; DeMirci, Hasan; Ieong, Ka-Weng; Wang, Jinfan; Petrov, Alexey; Prabhakar, Arjun; Rechavi, Gideon; Dominissini, Dan; He, Chuan; Ehrenberg, Måns; Puglisi, Joseph D

2018-03-01

Chemical modifications of mRNA may regulate many aspects of mRNA processing and protein synthesis. Recently, 2'-O-methylation of nucleotides was identified as a frequent modification in translated regions of human mRNA, showing enrichment in codons for certain amino acids. Here, using single-molecule, bulk kinetics and structural methods, we show that 2'-O-methylation within coding regions of mRNA disrupts key steps in codon reading during cognate tRNA selection. Our results suggest that 2'-O-methylation sterically perturbs interactions of ribosomal-monitoring bases (G530, A1492 and A1493) with cognate codon-anticodon helices, thereby inhibiting downstream GTP hydrolysis by elongation factor Tu (EF-Tu) and A-site tRNA accommodation, leading to excessive rejection of cognate aminoacylated tRNAs in initial selection and proofreading. Our current and prior findings highlight how chemical modifications of mRNA tune the dynamics of protein synthesis at different steps of translation elongation.

Endothelial cell markers in vascular neoplasms: an immunohistochemical study comparing factor VIII-related antigen, blood group specific antigens, 6-keto-PGF1 alpha, and Ulex europaeus 1 lectin.

Science.gov (United States)

Little, D; Said, J W; Siegel, R J; Fealy, M; Fishbein, M C

1986-06-01

Markers for endothelial cells including Ulex europaeus 1 lectin, blood group A, B, and H, and the prostaglandin metabolite 6-keto-PGF1 alpha were evaluated in paraffin secretions from formalin-fixed benign and malignant vascular neoplasms using a variety of immunohistochemical techniques, and results compared with staining for factor VIII-related antigen. Staining for Ulex appeared more sensitive than factor VIII-related antigen in identifying poorly differentiated neoplasms including haemangiosarcomas and spindle cell proliferations in Kaposi's sarcoma. Staining for blood group related antigens correlated with blood group in all cases. Ulex europaeus 1 lectin was the only marker for endothelial cells in lymphangiomas.

Expression and autoregulation of transforming growth factor beta receptor mRNA in small-cell lung cancer cell lines

DEFF Research Database (Denmark)

Nørgaard, P; Spang-Thomsen, M; Poulsen, H S

1996-01-01

In small-cell lung cancer cell lines resistance to growth inhibition by transforming growth factor (TGF)-beta 1, was previously shown to correlate with lack of TGF-beta receptor I (RI) and II (RII) proteins. To further investigate the role of these receptors, the expression of mRNA for RI, RII...... and beta-glycan (RIII) was examined. The results showed that loss of RII mRNA correlated with TGF-beta 1 resistance. In contrast, RI-and beta-glycan mRNA was expressed by all cell lines, including those lacking expression of these proteins. According to Southern blot analysis, the loss of type II m......RNA was not due to gross structural changes in the gene. The effect of TGF-beta 1 on expression of TGF-beta receptor mRNA (receptor autoregulation) was examined by quantitative Northern blotting in four cell lines with different expression of TGF-beta receptor proteins. In two cell lines expressing all three TGF...

A Killer Immunoglobulin - Like Receptor Gene - Content Haplotype and A Cognate Human Leukocyte Antigen Ligand are Associated with Autism

OpenAIRE

Torres, Anthony; Westover, Jonna; Benson, Michael; Johnson, Randall; Dykes, Annelise

2016-01-01

The killing activity of natural killer cells is largely regulated by the binding of class I human leukocyte antigen cognate ligands to killer cell immunoglobulin - like receptor proteins. The killer cell immunoglobulin - like receptor gene - complex contains genes that activate and others that inhibit the killing state of natural killer cells depending on the binding of specific human leukocyte antigen cognate ligands. It has been suggested in previous publications that activating human leuko...

Evaluation of variants of melanoma-associated antigen genes and mRNA transcripts in melanomas of dogs.

Science.gov (United States)

Stell, Anneliese J; Dobson, Jane M; Scase, Timothy J; Catchpole, Brian

2009-12-01

OBJECTIVE-To characterize variability in melanoma-associated antigen (MAA) genes and gene expression in melanomas of dogs. ANIMALS-18 dogs with malignant melanomas and 8 healthy control dogs. PROCEDURES-cDNA was prepared from malignant melanoma biopsy specimens and from pigmented oral mucocutaneous tissues of healthy control dogs. Genomic DNA was extracted from poorly pigmented melanomas. A PCR assay was performed by use of Melan-A, SILV, or tyrosinase-specific primers. RESULTS-Splice variants of Melan-A and SILV were identified in malignant melanomas and also in healthy pigmented tissues, whereas a tyrosinase splice variant was detected in melanoma tissues only. A short interspersed nuclear element (SINE) insertion mutation was identified in the SILV gene in 1 of 10 poorly pigmented melanomas. Six novel exonic single nucleotide polymorphisms (SNPs; 3 synonymous and 3 nonsynonymous) were detected in the tyrosinase gene, and 1 nonsynonymous exonic SNP was detected in the SILV gene. CONCLUSIONS AND CLINICAL RELEVANCE-Variants of MAA mRNA were detected in malignant melanoma tissues of dogs. The importance of MAA alternative transcripts expressed in melanomas and normal pigmented tissues was unclear, but they may have represented a means of regulating melanin synthesis. The tyrosinase splice variant was detected only in melanomas and could potentially be a tumor-specific target for immunotherapy. A SILV SINE insertion mutation was identified in a melanoma from a Great Dane, a breed known to carry this mutation (associated with merle coat color). The nonsynonymous SNPs detected in tyrosinase and SILV transcripts did not appear to affect tumor pigmentation.

Intake of branched-chain amino acids influences the levels of MAFbx mRNA and MuRF-1 total protein in resting and exercising human muscle.

Science.gov (United States)

Borgenvik, Marcus; Apró, William; Blomstrand, Eva

2012-03-01

Resistance exercise and amino acids are two major factors that influence muscle protein turnover. Here, we examined the effects of resistance exercise and branched-chain amino acids (BCAA), individually and in combination, on the expression of anabolic and catabolic genes in human skeletal muscle. Seven subjects performed two sessions of unilateral leg press exercise with randomized supplementation with BCAA or flavored water. Biopsies were collected from the vastus lateralis muscle of both the resting and exercising legs before and repeatedly after exercise to determine levels of mRNA, protein phosphorylation, and amino acid concentrations. Intake of BCAA reduced (P exercising legs, respectively. The level of MuRF-1 mRNA was elevated (P exercising leg two- and threefold under the placebo and BCAA conditions, respectively, whereas MuRF-1 total protein increased by 20% (P exercising muscle. In conclusion, BCAA ingestion reduced MAFbx mRNA and prevented the exercise-induced increase in MuRF-1 total protein in both resting and exercising leg. Further-more, resistance exercise differently influenced MAFbx and MuRF-1 mRNA expression, suggesting both common and divergent regulation of these two ubiquitin ligases.

Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

Science.gov (United States)

Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

2018-03-29

The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

Increasing vaccine potency through exosome antigen targeting.

Science.gov (United States)

Hartman, Zachary C; Wei, Junping; Glass, Oliver K; Guo, Hongtao; Lei, Gangjun; Yang, Xiao-Yi; Osada, Takuya; Hobeika, Amy; Delcayre, Alain; Le Pecq, Jean-Bernard; Morse, Michael A; Clay, Timothy M; Lyerly, Herbert K

2011-11-21

While many tumor associated antigens (TAAs) have been identified in human cancers, efforts to develop efficient TAA "cancer vaccines" using classical vaccine approaches have been largely ineffective. Recently, a process to specifically target proteins to exosomes has been established which takes advantage of the ability of the factor V like C1C2 domain of lactadherin to specifically address proteins to exosomes. Using this approach, we hypothesized that TAAs could be targeted to exosomes to potentially increase their immunogenicity, as exosomes have been demonstrated to traffic to antigen presenting cells (APC). To investigate this possibility, we created adenoviral vectors expressing the extracellular domain (ECD) of two non-mutated TAAs often found in tumors of cancer patients, carcinoembryonic antigen (CEA) and HER2, and coupled them to the C1C2 domain of lactadherin. We found that these C1C2 fusion proteins had enhanced expression in exosomes in vitro. We saw significant improvement in antigen specific immune responses to each of these antigens in naïve and tolerant transgenic animal models and could further demonstrate significantly enhanced therapeutic anti-tumor effects in a human HER2+ transgenic animal model. These findings demonstrate that the mode of secretion and trafficking can influence the immunogenicity of different human TAAs, and may explain the lack of immunogenicity of non-mutated TAAs found in cancer patients. They suggest that exosomal targeting could enhance future anti-tumor vaccination protocols. This targeting exosome process could also be adapted for the development of more potent vaccines in some viral and parasitic diseases where the classical vaccine approach has demonstrated limitations. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Escherichia coli antiterminator protein BglG stabilizes the 5 ...

Indian Academy of Sciences (India)

Unknown

Keywords. Antitermination; mRNA stability; RNA binding protein ... factor, Rho, and the pBR322 copy number protein, Rop, have been .... Transcription analysis using the oligo- ..... Retarded RNA turnover in Escherichia coli a means of main-.

The human factor in the nuclear industry

International Nuclear Information System (INIS)

Colas, Armand

1998-01-01

After having evoked the progressive reduction and stabilization of significant incidents occurring every year in French nuclear power plants, and the challenges faced by nuclear energy (loss of public confidence, loss of competitiveness), and then outlined the importance of safety to overcome these challenges, the author comments EDF's approach to the human factor. He first highlights the importance of information and communication towards the population. He briefly discusses the meaning of human factors for the nuclear industry, sometimes perceived as the contribution people to the company's safety and performance. He comments the evolution observed in the perception of human error in different industrial or technical environments and situations, and outlines what is at stake to reduce the production of faults and organize a 'hunt for latent defects'

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

Energy Technology Data Exchange (ETDEWEB)

Kleinhenz, M.E.; Ellner, J.J.

1985-07-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the (/sup 3/H)thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced (/sup 3/H)thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% (SD)) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition.

Immunoregulatory adherent cells in human tuberculosis: radiation-sensitive antigen-specific suppression by monocytes

International Nuclear Information System (INIS)

Kleinhenz, M.E.; Ellner, J.J.

1985-01-01

In human tuberculosis, adherent mononuclear cells (AMC) selectively depress in vitro responses to the mycobacterial antigen tuberculin purified protein derivative (PPD). The phenotype of this antigen-specific adherent suppressor cell was characterized by examining the functional activity of adherent cells after selective depletion of sheep erythrocyte-rosetting T cells or OKM1-reactive monocytes. Adherent cell suppression was studied in the [ 3 H]thymidine-incorporation microculture assay by using T cells rigorously depleted of T cells with surface receptors for the Fc portion of IgG (T gamma cells) as antigen-responsive cells. PPD-induced [ 3 H]thymidine incorporation by these non gamma T cells was uniformly reduced (mean, 42% +/- 10% [SD]) when autologous AMC were added to non gamma T cells at a ratio of 1:2. Antigen-specific suppression by AMC was not altered by depletion of sheep erythrocyte-rosetting T cells or treatment with indomethacin. However, AMC treated with OKM1 and complement or gamma irradiation (1,500 rads) no longer suppressed tuberculin responses in vitro. These studies identify the antigen-specific adherent suppressor cell in tuberculosis as an OKM1-reactive, non-erythrocyte-rosetting monocyte. The radiosensitivity of this monocyte immunoregulatory function may facilitate its further definition

Classification and analysis of factors that affect stability of oil and gas enterprise staff

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Zelinska Haluna Olexiivna

2016-12-01

Full Text Available The relevance of human resources as a strategic goal of sustainable development of oil and gas companies is determined. It is shown that the stability of staff, as the main component of the social components of sustainable enterprise development, research and evaluation needs in terms of an integrated system of factors influence the behavior of staff. Addressing issues related to the management personnel can be based classification study the factors affecting its stability in the formation of high quality human resources strategy. In particular noted that the needs of each employee should become an integral part of the concept of work and life balance. Analysis of the results of the study showed that in areas of oil and gas industry has a number of factors that negatively affect its operation and development, which are caused not only technical, technological and natural factors, but also due to neglect behavioral characteristics personnel. It is found that without understanding of the behavioral characteristics of staff and its values can`t implement a quality model of human resource management and provide optimal scenarios of oil companies in general.

Human leukocyte antigen-G within the male reproductive system

DEFF Research Database (Denmark)

Hviid, Thomas Vauvert F

2015-01-01

by “priming” the woman’s immune system before conception and at conception. Recent studies have demonstrated the presence of the immunoregulatory and tolerance-inducible human leukocyte antigen (HLA)-G in the male reproductive organs. The expression of HLA-G in the blastocyst and by extravillous trophoblast......In sexual reproduction in humans, a man has a clear interest in ensuring that the immune system of his female partner accepts the semi-allogenic fetus. Increasing attention has been given to soluble immunomodulatory molecules in the seminal fluid as one mechanism of ensuring this, possibly...... plasma may even be associated with the chance of pregnancy in couples, where the male partner has reduced semen quality. More studies are needed to verify these preliminary findings....

Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

Science.gov (United States)

Schumacher, Jens; Bacic, Tijana; Staritzbichler, René; Daneschdar, Matin; Klamp, Thorsten; Arnold, Philipp; Jägle, Sabrina; Türeci, Özlem; Markl, Jürgen; Sahin, Ugur

2018-04-13

Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element

Hypoxic stress simultaneously stimulates vascular endothelial growth factor via hypoxia-inducible factor-1α and inhibits stromal cell-derived factor-1 in human endometrial stromal cells.

Science.gov (United States)

Tsuzuki, Tomoko; Okada, Hidetaka; Cho, Hisayuu; Tsuji, Shoko; Nishigaki, Akemi; Yasuda, Katsuhiko; Kanzaki, Hideharu

2012-02-01

Hypoxia of the human endometrium is a physiologic event occurring during the perimenstrual period and the local stimulus for angiogenesis. The aim of this study was to investigate the effects of hypoxic stress on the regulation of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1/CXCL12), and the potential role of hypoxia-inducible factor-1α (HIF-1α) in the endometrium. Human endometrial stromal cells (ESCs, n= 22 samples) were studied in vitro. ESCs were cultured under hypoxic and normoxic conditions and treated with cobalt chloride (CoCl₂; a hypoxia-mimicking agent) and/or echinomycin, a small-molecule inhibitor of HIF-1α activity. The mRNA levels and production of VEGF and SDF-1 were assessed by real-time PCR and ELISA, respectively. The HIF-1α protein levels were measured using western blot analysis. Hypoxia simultaneously induced the expression of mRNA and production of VEGF and attenuated the expression and production of SDF-1 from ESCs in a time-dependent manner. Similar changes were observed in the ESCs after stimulation with CoCl₂ in a dose-dependent manner. CoCl₂ significantly induced the expression of HIF-1α protein, and its highest expression was observed at 6 h. Echinomycin inhibited hypoxia-induced VEGF production without affecting the HIF-1α protein level and cell toxicity and had no effect on SDF-1 secretion (P hypoxic conditions that could influence angiogenesis in the human endometrium.

Heterosubtypic protection against pathogenic human and avian influenza viruses via in vivo electroporation of synthetic consensus DNA antigens.

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Dominick J Laddy

Full Text Available BACKGROUND: The persistent evolution of highly pathogenic avian influenza (HPAI highlights the need for novel vaccination techniques that can quickly and effectively respond to emerging viral threats. We evaluated the use of optimized consensus influenza antigens to provide broad protection against divergent strains of H5N1 influenza in three animal models of mice, ferrets, and non-human primates. We also evaluated the use of in vivo electroporation to deliver these vaccines to overcome the immunogenicity barrier encountered in larger animal models of vaccination. METHODS AND FINDINGS: Mice, ferrets and non-human primates were immunized with consensus plasmids expressing H5 hemagglutinin (pH5HA, N1 neuraminidase (pN1NA, and nucleoprotein antigen (pNP. Dramatic IFN-gamma-based cellular immune responses to both H5 and NP, largely dependent upon CD8+ T cells were seen in mice. Hemaggutination inhibition titers classically associated with protection (>1:40 were seen in all species. Responses in both ferrets and macaques demonstrate the ability of synthetic consensus antigens to induce antibodies capable of inhibiting divergent strains of the H5N1 subtype, and studies in the mouse and ferret demonstrate the ability of synthetic consensus vaccines to induce protection even in the absence of such neutralizing antibodies. After challenge, protection from morbidity and mortality was seen in mice and ferrets, with significant reductions in viral shedding and disease progression seen in vaccinated animals. CONCLUSIONS: By combining several consensus influenza antigens with in vivo electroporation, we demonstrate that these antigens induce both protective cellular and humoral immune responses in mice, ferrets and non-human primates. We also demonstrate the ability of these antigens to protect from both morbidity and mortality in a ferret model of HPAI, in both the presence and absence of neutralizing antibody, which will be critical in responding to the

Synthetic mRNA devices that detect endogenous proteins and distinguish mammalian cells.

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Kawasaki, Shunsuke; Fujita, Yoshihiko; Nagaike, Takashi; Tomita, Kozo; Saito, Hirohide

2017-07-07

Synthetic biology has great potential for future therapeutic applications including autonomous cell programming through the detection of protein signals and the production of desired outputs. Synthetic RNA devices are promising for this purpose. However, the number of available devices is limited due to the difficulty in the detection of endogenous proteins within a cell. Here, we show a strategy to construct synthetic mRNA devices that detect endogenous proteins in living cells, control translation and distinguish cell types. We engineered protein-binding aptamers that have increased stability in the secondary structures of their active conformation. The designed devices can efficiently respond to target proteins including human LIN28A and U1A proteins, while the original aptamers failed to do so. Moreover, mRNA delivery of an LIN28A-responsive device into human induced pluripotent stem cells (hiPSCs) revealed that we can distinguish living hiPSCs and differentiated cells by quantifying endogenous LIN28A protein expression level. Thus, our endogenous protein-driven RNA devices determine live-cell states and program mammalian cells based on intracellular protein information. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Migration of human antigen-presenting cells in a human skin graft onto nude mice model after contact sensitization

NARCIS (Netherlands)

Hoefakker, S.; Balk, H.P.; Boersma, W.J.A.; Joost, T. van; Notten, W.R.F.; Claassen, E.

1995-01-01

Fluorescent contact chemical allergens provoke sensitization after application on both syngeneic and allogeneic skin grafts in mice. We attempted to determine whether the functional activity in a contact sensitization response of human skin graft was affected at the level of antigen uptake and

The Leishmania promastigote surface antigen-2 (PSA-2) is specifically recognised by Th1 cells in humans with naturally acquired immunity to L. major

DEFF Research Database (Denmark)

Kemp, M; Handman, E; Kemp, K

1998-01-01

The promastigote surface antigen-2 (PSA-2) is a Leishmania parasite antigen, which can induce Th1-mediated protection against murine leishmaniasis when used as a vaccine. To evaluate PSA-2 as a human vaccine candidate the specific T-cell response to PSA-2 was characterised in individuals immune...... to cutaneous leishmaniasis. Peripheral blood mononuclear cells from Sudanese individuals with a past history of self-healing cutaneous leishmaniasis proliferated vigorously in response to PSA-2 isolated from Leishmania major, whereas the antigen did not activate cells from presumably unexposed Danes....... Peripheral blood mononuclear cells from individuals with previous L. major infection had varying proliferative responses to PSA-2 derived from L. donovani promastigotes. Peripheral blood mononuclear cells activated by PSA-2 from L. major produced high amounts of interferon-gamma and tumour necrosis factor...

FcepsilonRI-alpha siRNA inhibits the antigen-induced activation of mast cells.

Science.gov (United States)

Safaralizadeh, Reza; Soheili, Zahra-Soheila; Deezagi, Abdolkhaleg; Pourpak, Zahra; Samiei, Shahram; Moin, Mostafa

2009-12-01

FcepsilonRI, The high affinity receptor for IgE plays a critical role in triggering the allergic reactions. It is responsible for inducing mast cell degranulation and deliberation of allergy mediators when it is aggregated by allergen and IgE complexes. FcepsilonRI on the mast cells consists of three subunits; alpha chain directly binds IgE, beta chain and dimmer of gamma chains together mediate intracellular signaling. Cross-linking of IgE-bound FcepsilonRI on the surface of mast cells and basophils by the multivalent antigen induces release of chemical mediators. The present in vitro study was designed to investigate the effect of synthetic FcepsilonRI-alpha siRNA on the antigen-induced activation of MC/9 cells. MC/9 cells which are murine mast cells were transfected by FcepsilonRI-alpha siRNA and negative control siRNA. After 6 h, anti-DNP (Dinitrophenyl) IgE was used for the cells sensitization. Then the cells were challenged with Dinitrophenyl-Human Serum Albumin (DNP-HSA) for mast cell degranulation induction before collection of supernatants. The amount of mRNA and protein expression was measured by Real Time PCR and western blot analysis, respectively. Determination of the expression rate of FcepsilonRI-alpha on cell surface was achieved by flow cytometry. ELISA and spectrophotometry methods were used subsequently for measuring the effects of FcepsilonRI-alpha siRNA on antigen-induced histamine and beta-hexosaminidase release. FcepsilonRI-alpha siRNA treated cells showed significant decrease in FcepsilonRI-alpha mRNA and protein expression in comparison to control cells. FcepsilonRI-mediated mast cell release of beta-hexosaminidase and histamine were also inhibited. In this study it was shown that FcepsilonRI-alpha siRNA could suppress FcepsilonRI-alpha expression and inhibited degranulation and histamine release in antigen-stimulated MC/9 cells. In conclusion, knock-down of FcepsilonRI-alpha by siRNA could be a promising method for inhibition of the mast

Effect of low-dose irradiation on expression of mRNA and protein. Pt.1. Induction of thioredoxin as radioprotective protein in human lymphocytes

International Nuclear Information System (INIS)

Hoshi, Yuko; Tanooka, Hiroshi; Wakasugi, Hiro; Miyasaki, Kunihisa

1997-01-01

To elucidate the mechanism of hormetic effect by low-dose ionizing radiation, we studied the expression of the thioredoxin (TRX) gene in human lymphocytes after irradiation. TRX is a radioprotector and a key protein regulating cellular functions through redox reaction. The major results obtained were as follows; (1) The peaks of TRX mRNA expression and protein synthesis in human lymphocytes appeared 6-8 hr after irradiation with 25cGy. (2) At 6 hr after irradiation, the optimum dose for induction of TRX mRNA and TRX protein in human lymphocytes appeared to be 25-50cGy. (3) Induction of expression TRX mRNA had individual variations about twice. (4) Lymphocytes prepared from fresh venous blood showed the lowest TRX mRNA level in other cells such a Jurkat cells, lymphocytes stimulated for now with IL-2 and CD3 and the immortalized cell line 1G8. (5) The optimal dose and time course of induction of TRX by low-dose radiation suggest that TRX is related to the radio-adaptive response. (author)

Bioinspired nanocomplex for spatiotemporal imaging of sequential mRNA expression in differentiating neural stem cells.

Science.gov (United States)

Wang, Zhe; Zhang, Ruili; Wang, Zhongliang; Wang, He-Fang; Wang, Yu; Zhao, Jun; Wang, Fu; Li, Weitao; Niu, Gang; Kiesewetter, Dale O; Chen, Xiaoyuan

2014-12-23

Messenger RNA plays a pivotal role in regulating cellular activities. The expression dynamics of specific mRNA contains substantial information on the intracellular milieu. Unlike the imaging of stationary mRNAs, real-time intracellular imaging of the dynamics of mRNA expression is of great value for investigating mRNA biology and exploring specific cellular cascades. In addition to advanced imaging methods, timely extracellular stimulation is another key factor in regulating the mRNA expression repertoire. The integration of effective stimulation and imaging into a single robust system would significantly improve stimulation efficiency and imaging accuracy, producing fewer unwanted artifacts. In this study, we developed a multifunctional nanocomplex to enable self-activating and spatiotemporal imaging of the dynamics of mRNA sequential expression during the neural stem cell differentiation process. This nanocomplex showed improved enzymatic stability, fast recognition kinetics, and high specificity. With a mechanism regulated by endogenous cell machinery, this nanocomplex realized the successive stimulating motif release and the dynamic imaging of chronological mRNA expression during neural stem cell differentiation without the use of transgenetic manipulation. The dynamic imaging montage of mRNA expression ultimately facilitated genetic heterogeneity analysis. In vivo lateral ventricle injection of this nanocomplex enabled endogenous neural stem cell activation and labeling at their specific differentiation stages. This nanocomplex is highly amenable as an alternative tool to explore the dynamics of intricate mRNA activities in various physiological and pathological conditions.

Epidemiology of human leukocyte antigens among omani population.

Science.gov (United States)

Al Salmi, Issa; Metry, Abdul Massiah; Al Ismaili, Faisal; Hola, Alan; Shaheen, Faissal; Fakhoury, Hana; Hannawi, Suad

2017-01-01

Oman is located on the Southeastern coast of the Arabian Peninsula, and its population has high levels of consanguinity. Human leukocytic antigen (HLA) typing analysis in human population holds unexploited potential for elucidating the genetic causes of human disease and possibly leads to personalized medicine. This is a retrospective, descriptive study evaluating HLA frequencies of Omani individuals who underwent workup for kidney transplantation at the Royal Hospital (RH) from 2005 to 2016. Data on 870 subjects were collected from the Oman kidney transplant registry at RH as well from electronic medical record system. The mean age (standard deviation) years for the cohort were 33.2 (13.0). Males constituted 56.3% (490) while females constituted 43.7% (380). Seven HLA-A alleles accounted for more than 70% of the total alleles. Of which, HLA-A2 contributed the highest frequency (24%), followed by HLA A11 (9.4%), and A32 (8.1%). Ten alleles accounted for 70% of HLA-B alleles. Of which, HLA-B51 was the most common (18.9%), followed by HLA-B-35 (13.6%), and HLA-B8 (7.9%). Seven HLA-DRB1 alleles accounted for more than 70% of the total HLA DRB1 alleles, of which HLA- DRB1*16 contributed the highest frequency (29.56%). This was followed by HLA-DRB1*03 (14.57%) and HLA-DRB1*11 (9.48%). While three alleles accounted for more than 75% of the total HLA DQB1alleles. Of which, HLA-DQB1*05 contributed the highest frequency (37.56%). This was followed by allele HLA-DQB1*02 (26.48%) and HLA-DQB1*03 (17.18%). This study showed considerable heterogeneity in both HLA Class I and Class II antigens, which reflects admixture of our population with rest of old world countries. Despite the high levels of consanguinity, this population is genetically highly heterogeneous. These findings may be useful for transplantation programs, noncommunicable diseases, epidemiology of HLA linked diseases, pharmacogenomics, and anthropology.

Epidemiology of human leukocyte antigens among omani population

Directory of Open Access Journals (Sweden)

Issa Al Salmi

2017-01-01

Full Text Available Oman is located on the Southeastern coast of the Arabian Peninsula, and its population has high levels of consanguinity. Human leukocytic antigen (HLA typing analysis in human population holds unexploited potential for elucidating the genetic causes of human disease and possibly leads to personalized medicine. This is a retrospective, descriptive study evaluating HLA frequencies of Omani individuals who underwent workup for kidney transplantation at the Royal Hospital (RH from 2005 to 2016. Data on 870 subjects were collected from the Oman kidney transplant registry at RH as well from electronic medical record system. The mean age (standard deviation years for the cohort were 33.2 (13.0. Males constituted 56.3% (490 while females constituted 43.7% (380. Seven HLA-A alleles accounted for more than 70% of the total alleles. Of which, HLA-A2 contributed the highest frequency (24%, followed by HLA A11 (9.4%, and A32 (8.1%. Ten alleles accounted for 70% of HLA-B alleles. Of which, HLA-B51 was the most common (18.9%, followed by HLA-B-35 (13.6%, and HLA-B8 (7.9%. Seven HLA-DRB1 alleles accounted for more than 70% of the total HLA DRB1 alleles, of which HLA- DRB1*16 contributed the highest frequency (29.56%. This was followed by HLA-DRB1*03 (14.57% and HLA-DRB1*11 (9.48%. While three alleles accounted for more than 75% of the total HLA DQB1alleles. Of which, HLA-DQB1*05 contributed the highest frequency (37.56%. This was followed by allele HLA-DQB1*02 (26.48% and HLA-DQB1*03 (17.18%. This study showed considerable heterogeneity in both HLA Class I and Class II antigens, which reflects admixture of our population with rest of old world countries. Despite the high levels of consanguinity, this population is genetically highly heterogeneous. These findings may be useful for transplantation programs, noncommunicable diseases, epidemiology of HLA linked diseases, pharmacogenomics, and anthropology.

Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA

International Nuclear Information System (INIS)

Indik, Z.; Yeh, H.; Ornstein-goldstein, N.; Sheppard, P.; Anderson, N.; Rosenbloom, J.C.; Peltonen, L.; Rosenbloom, J.

1987-01-01

Poly(A) + RNA, isolated from a single 7-mo fetal human aorta, was used to synthesize cDNA by the RNase H method, and the cDNA was inserted into λgt10. Recombinant phage containing elastin sequences were identified by hybridization with cloned, exon-containing fragments of the human elastin gene. Three clones containing inserts of 3.3, 2.7, and 2.3 kilobases were selected for further analysis. Three overlapping clones containing 17.8 kilobases of the human elastin gene were also isolated from genomic libraries. Complete sequence analysis of the six clones demonstrated that: (i) the cDNA encompassed the entire translated portion of the mRNA encoding 786 amino acids, including several unusual hydrophilic amino acid sequences not previously identified in porcine tropoelastin, (ii) exons encoding either hydrophobic or crosslinking domains in the protein alternated in the gene, and (iii) a great abundance of Alu repetitive sequences occurred throughout the introns. The data also indicated substantial alternative splicing of the mRNA. These results suggest the potential for significant variation in the precise molecular structure of the elastic fiber in the human population

Human leukocyte antigen (HLA)-G during pregnancy part I

DEFF Research Database (Denmark)

Klitkou, Louise; Dahl, Mette; Hviid, Thomas Vauvert F

2015-01-01

Human leukocyte antigen (HLA)-G is a class Ib molecule with restricted tissue distribution expressed on trophoblast cells and has been proposed to have immunomodulatory functions during pregnancy. Soluble HLA-G1 (sHLA-G1) can be generated by the shedding of membrane-bound HLA-G molecules; however...... of importance for production of sHLA-G in the mother and child, or it may support the theory that sHLA-G in the pregnant woman and the fetus is partly derived from a "shared organ", the placenta....

Recognition of Histo-Blood Group Antigen-Like Carbohydrates in Lettuce by Human GII.4 Norovirus.

Science.gov (United States)

Gao, Xiang; Esseili, Malak A; Lu, Zhongyan; Saif, Linda J; Wang, Qiuhong

2016-05-15

Human norovirus (HuNoV) genogroup II genotype 4 (GII.4) strains account for about 80% of the gastroenteritis outbreaks in the United States. Contaminated food is a major transmission vehicle for this virus. In humans, pigs, and oysters, histo-blood group antigens (HBGAs) act as attachment factors for HuNoVs. In lettuce, although the virus-like particles (VLPs) of a GII.4 HuNoV were found to bind to cell wall carbohydrates, the exact binding site has not been investigated. Here, we show the presence of HBGA-like carbohydrates in the cell wall of lettuce. The digestion of lettuce leaves with cell wall-degrading enzymes exposed more binding sites and significantly increased the level of binding of GII.4 HuNoV VLPs. Competition assays showed that both the HBGA monoclonal antibody, recognizing the H type, and plant lectins, recognizing α-l-fucose in the H type, effectively inhibited VLP binding to lettuce tissues. Lettuce cell wall components were isolated and their NoV VLP binding characteristics were tested by enzyme-linked immunosorbent assays. The binding was inhibited by pretreatment of the lettuce cell wall materials with α-1,2-fucosidase. Collectively, our results indicate that H-type HBGA-like carbohydrates exist in lettuce tissues and that GII.4 HuNoV VLPs can bind the exposed fucose moiety, possibly in the hemicellulose component of the cell wall. Salad crops and fruits are increasingly recognized as vehicles for human norovirus (HuNoV) transmission. A recent study showed that HuNoVs specifically bind to the carbohydrates of the lettuce cell wall. Histo-blood group antigens (HBGAs) are carbohydrates and are known as the attachment factors for HuNoV infection in humans. In this study, we show the presence of HBGA-like carbohydrates in lettuce, to which HuNoVs specifically bind. These results suggest that specifically bound HuNoVs cannot be removed by simple washing, which may allow viral transmission to consumers. Our findings provide new information needed

Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

Science.gov (United States)

Rasmussen, Martin Krøyer; Bertholdt, Lærke; Gudiksen, Anders; Pilegaard, Henriette; Knudsen, Jakob G

2018-01-05

The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification. Copyright © 2017 Elsevier B.V. All rights reserved.

Global regulation of mRNA translation and stability in the early Drosophila embryo by the Smaug RNA-binding protein.

Science.gov (United States)

Chen, Linan; Dumelie, Jason G; Li, Xiao; Cheng, Matthew Hk; Yang, Zhiyong; Laver, John D; Siddiqui, Najeeb U; Westwood, J Timothy; Morris, Quaid; Lipshitz, Howard D; Smibert, Craig A

2014-01-07

Smaug is an RNA-binding protein that induces the degradation and represses the translation of mRNAs in the early Drosophila embryo. Smaug has two identified direct target mRNAs that it differentially regulates: nanos and Hsp83. Smaug represses the translation of nanos mRNA but has only a modest effect on its stability, whereas it destabilizes Hsp83 mRNA but has no detectable effect on Hsp83 translation. Smaug is required to destabilize more than one thousand mRNAs in the early embryo, but whether these transcripts represent direct targets of Smaug is unclear and the extent of Smaug-mediated translational repression is unknown. To gain a panoramic view of Smaug function in the early embryo, we identified mRNAs that are bound to Smaug using RNA co-immunoprecipitation followed by hybridization to DNA microarrays. We also identified mRNAs that are translationally repressed by Smaug using polysome gradients and microarrays. Comparison of the bound mRNAs to those that are translationally repressed by Smaug and those that require Smaug for their degradation suggests that a large fraction of Smaug's target mRNAs are both translationally repressed and degraded by Smaug. Smaug directly regulates components of the TRiC/CCT chaperonin, the proteasome regulatory particle and lipid droplets, as well as many metabolic enzymes, including several glycolytic enzymes. Smaug plays a direct and global role in regulating the translation and stability of a large fraction of the mRNAs in the early Drosophila embryo, and has unanticipated functions in control of protein folding and degradation, lipid droplet function and metabolism.

Characterization of Antigen-Specific B Cells Using Nominal Antigen-Coated Flow-Beads

Science.gov (United States)

Akl, Ahmed; Lepetit, Maud; Crochette, Romain; Giral, Magali; Lepourry, Julie; Pallier, Annaick; Castagnet, Stéphanie; Dugast, Emilie; Guillot-Gueguen, Cécile; Jacq-Foucher, Marylène; Saulquin, Xavier; Cesbron, Anne; Laplaud, David; Nicot, Arnaud; Brouard, Sophie; Soulillou, Jean-Paul

2013-01-01

In order to characterize the reactivity of B cells against nominal antigens, a method based on the coupling of antigens onto the surface of fluorescent core polystyrene beads was developed. We first demonstrate that murine B cells with a human MOG-specific BCR are able to interact with MOG-coated beads and do not recognize beads coated with human albumin or pp65. B cells purified from human healthy volunteer blood or immunized individuals were tested for their ability to interact with various nominal antigens, including viral, vaccine, self and alloantigens, chosen for their usefulness in studying a variety of pathological processes. A substantial amount of B cells binding self-antigen MOG-coated beads can be detected in normal blood. Furthermore, greater frequencies of B cell against anti-Tetanic Toxin or anti-EBNA1 were observed in primed individuals. This method can reveal increased frequencies of anti-HLA committed B cells in patients with circulating anti-HLA antibodies compared to unsensitized patients and normal individuals. Of interest, those specific CD19 cells were preferentially identified within CD27−IgD+ (i-e naïve) subset. These observations suggest that a broad range of medical situations could benefit from a tool that allows the detection, the quantification and the characterization of antigen-specific blood B cells. PMID:24386360

Isorhapontigenin (ISO) inhibited cell transformation by inducing G0/G1 phase arrest via increasing MKP-1 mRNA Stability.

Science.gov (United States)

Gao, Guangxun; Chen, Liang; Li, Jingxia; Zhang, Dongyun; Fang, Yong; Huang, Haishan; Chen, Xiequn; Huang, Chuanshu

2014-05-15

The cancer chemopreventive property of Chinese herb new isolate isorhapontigenin (ISO) and mechanisms underlying its activity have never been explored. Here we demonstrated that ISO treatment with various concentrations for 3 weeks could dramatically inhibit TPA/EGF-induced cell transformation of Cl41 cells in Soft Agar assay, whereas co-incubation of cells with ISO at the same concentrations could elicit G0/G1 cell-cycle arrest without redundant cytotoxic effects on non-transformed cells. Further studies showed that ISO treatment resulted in cyclin D1 downregulation in dose- and time-dependent manner. Our results indicated that ISO regulated cyclin D1 at transcription level via targeting JNK/C-Jun/AP-1 activation. Moreover, we found that ISO-inhibited JNK/C-Jun/AP-1 activation was mediated by both upregulation of MKP-1 expression through increasing its mRNA stability and deactivating MKK7. Most importantly, MKP-1 knockdown could attenuate ISO-mediated suppression of JNK/C-Jun activation and cyclin D1 expression, as well as G0/G1 cell cycle arrest and cell transformation inhibition, while ectopic expression of FLAG-cyclin D1 T286A mutant also reversed ISO-induced G0/G1 cell-cycle arrest and inhibition of cell transformation. Our results demonstrated that ISO is a promising chemopreventive agent via upregulating mkp-1 mRNA stability, which is distinct from its cancer therapeutic effect with downregulation of XIAP and cyclin D1 expression.

Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

Directory of Open Access Journals (Sweden)

Chiou-Yueh Yeh

Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

Conservation of myeloid surface antigens on primate granulocytes.

Science.gov (United States)

Letvin, N L; Todd, R F; Palley, L S; Schlossman, S F; Griffin, J D

1983-02-01

Monoclonal antibodies reactive with myeloid cell surface antigens were used to study evolutionary changes in granulocyte surface antigens from primate species. Certain of these granulocyte membrane antigens are conserved in phylogenetically distant species, indicating the potential functional importance of these structures. The degree of conservation of these antigens reflects the phylogenetic relationship between primate species. Furthermore, species of the same genus show similar patterns of binding to this panel of anti-human myeloid antibodies. This finding of conserved granulocyte surface antigens suggests that non-human primates may provide a model system for exploring uses of monoclonal antibodies in the treatment of human myeloid disorders.

Altered expression of asparagine synthetase mRNA in human leukemic and carcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Goodwin, L.O.; Guzowski, D.E.; Millan, C.A. [North Shore Univ. Hospital/Cornell Univ. Medical College, Manhasset, NY (United States)] [and others

1994-09-01

Asparagine synthetase (AS) is the enzyme responsible for the ATP-dependant conversion of aspartic acid to asparagine. The AS gene is expressed constitutively in most mammalian cells, including cells of the lymphoid lineage, as a 2 kb mRNA. In some leukemic phenotypes, AS expression is abrogated, resulting in no detectable enzyme activity. These cells are rendered sensitive to killing by L-asparaginase, which destroys extracellular asparagine. Prolonged treatment of leukemic cells with this agent can lead to resistance and the reappearance of AS activity, suggesting derepression of the AS gene, which has been shown to be regulated by intracellular levels of asparagine. Modulation of AS expression by asparagine employs cis and trans-acting elements involved in transcriptional and translational regulation. We have cloned and sequenced the human AS gene and surrounding sequence elements as well as the full-length cDNA. Using probes specific to the third and fourth exons of AS, we have identified an additional higher molecular weight mRNA (2.7 kb) in Northern blots derived from a chronic myelogenous leukemia and a colon carcinoma but not in normal lymphocytic or other human cell lines. We speculate that elements present in the cancer-derived mRNAs may be involved in the derepression of AS activity. This hypothesis is being evaluated by RNase protection assays using RNA isolated from a variety of human cell lines to characterize and elucidate the nature of this additional AS encoded message.

Differential expression of the human thymosin-β4 gene in lymphocytes, macrophages, and granulocytes

International Nuclear Information System (INIS)

Gondo, H.; Kudo, J.; White, J.W.; Barr, C.; Selvanayagam, P.; Saunders, G.F.

1987-01-01

A cDNA clone encoding human thymosin-β 4 was isolated from a cDNA library prepared from peripheral blood leukocytes of a patient with acute lymphocytic leukemia. This clone contained the entire coding sequence of 43 amino acid residues of thymosin-β 4 and had an initiation codon and two termination codons. The amino acid and nucleotide sequences in the coding region were well conserved between rat and human. No signal peptide was found in the deduced protein sequence. Human thymosin-β 4 mRNA, approximately 830 nucleotides in length, was about 30 nucleotides larger than rat thymosin-β 4 mRNA. Expression of the human thymosin-β 4 gene in various primary myeloid and lymphoid malignant cells and in a few human hemopoietic cell lines was studied. Northern blot analyses of different neoplastic B lymphocytes revealed that steady state levels of thymosin-β 4 mRNA varied as a function of differentiation stage. Thymosin-β 4 mRNA levels were decreased in myeloma cells as are class II human leukocyte antigen, Fc receptor, and complement receptor, suggesting a relationship between thymosin-β 4 and the immune response. Treatment of THP-1 cells, a human monocytic cell line, with recombinant human interferon-γ reduced the levels of thymosin-β 4 mRNA. The pattern of thymosin-β 4 gene expression suggests that it may play a fundamental role in the host defense mechanism

Protective Effect of Human Leukocyte Antigen (HLA Allele DRB1*13:02 on Age-Related Brain Gray Matter Volume Reduction in Healthy Women

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Lisa M. James

2018-03-01

Full Text Available Background: Reduction of brain volume (brain atrophy during healthy brain aging is well documented and dependent on genetic, lifestyle and environmental factors. Here we investigated the possible dependence of brain gray matter volume reduction in the absence of the Human Leukocyte Antigen (HLA allele DRB1*13:02 which prevents brain atrophy in Gulf War Illness (James et al., 2017. Methods: Seventy-one cognitively healthy women (32–69 years old underwent a structural Magnetic Resonance Imaging (sMRI scan to measure the volumes of total gray matter, cerebrocortical gray matter, and subcortical gray matter. Participants were assigned to two groups, depending on whether they lacked the DRB1*13:02 allele (No DRB1*13:02 group, N = 60 or carried the DRB1*13:02 allele (N = 11. We assessed the change of brain gray matter volume with age in each group by performing a linear regression where the brain volume (adjusted for total intracranial volume was the dependent variable and age was the independent variable. Findings: In the No DRB1*13:02 group, the volumes of total gray matter, cerebrocortical gray matter, and subcortical gray matter were reduced highly significantly. In contrast, none of these volumes showed a statistically significant reduction with age in the DRB1*13:02 group. Interpretation: These findings document the protective effect of DRB1*13:02 on age-dependent reduction of brain gray matter in healthy individuals. Since the role of this allele is to connect to matching epitopes of external antigens for the subsequent production of antibodies and elimination of the offending antigen, we hypothesize that its protective effect may be due to the successful elimination of such antigens to which we are exposed during the lifespan, antigens that otherwise would persist causing gradual brain atrophy. In addition, we consider a possible beneficial role of DRB1*13:02 attributed to its binding to cathepsin S, a known harmful substance in brain

Insulin-like growth factor I enhances collagen synthesis in engineered human tendon tissue

DEFF Research Database (Denmark)

Herchenhan, Andreas; Bayer, Monika L.; Eliasson, Pernilla

2015-01-01

OBJECTIVE: Isolated human tendon cells form 3D tendon constructs that demonstrate collagen fibrillogenesis and feature structural similarities to tendon when cultured under tensile load. The exact role of circulating growth factors for collagen formation in tendon is sparsely examined. We...... investigated the influence of insulin-like growth factor I (IGF-I) on tendon construct formation in 3D cell culture. DESIGN: Tendon constructs were grown in 0.5 or 10% FBS with or without IGF-I (250 mg/ml) supplementation. Collagen content (fluorometric), mRNA levels (PCR) and fibril diameter (transmission...... electron microscopy) were determined at 7, 10, 14, 21 and 28 days. RESULTS: IGF-I revealed a stimulating effect on fibril diameter (up to day 21), mRNA for collagen (to day 28), tenomodulin (to day 28) and scleraxis (at days 10 and 14), and on overall collagen content. 10% FBS diminished the development...

IMP3 RNP Safe Houses Prevent miRNA-Directed HMGA2 mRNA Decay in Cancer and Development

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Lars Jønson

2014-04-01

Full Text Available The IMP3 RNA-binding protein is associated with metastasis and poor outcome in human cancer. Using solid cancer transcriptome data, we found that IMP3 correlates with HMGA2 mRNA expression. Cytoplasmic IMP3 granules contain HMGA2, and IMP3 dose-dependently increases HMGA2 mRNA. HMGA2 is regulated by let-7, and let-7 antagomiRs make HMGA2 refractory to IMP3. Removal of let-7 target sites eliminates IMP3-dependent stabilization, and IMP3-containing bodies are depleted of Ago1-4 and miRNAs. The relationship between Hmga2 mRNA and IMPs also exists in the developing limb bud, where IMP1-deficient embryos show dose-dependent Hmga2 mRNA downregulation. Finally, IMP3 ribonucleoproteins (RNPs contain other let-7 target mRNAs, including LIN28B, and a global gene set enrichment analysis demonstrates that miRNA-regulated transcripts in general are upregulated following IMP3 induction. We conclude that IMP3 RNPs may function as cytoplasmic safe houses and prevent miRNA-directed mRNA decay of oncogenes during tumor progression.

Molecular cloning of a human gene that is a member of the nerve growth factor family

Energy Technology Data Exchange (ETDEWEB)

Jones, K.R.; Reichardt, L.F. (Howard Hughes Medical Institute, San Francisco, CA (USA))

1990-10-01

Cell death within the developing vertebrate nervous system is regulated in part by interactions between neurons and their innervation targets that are mediated by neurotrophic factors. These factors also appear to have a role in the maintenance of the adult nervous system. Two neurotrophic factors, nerve growth factor and brain-derived neurotrophic factor, share substantial amino acid sequence identity. The authors have used a screen that combines polymerase chain reaction amplification of genomic DNA and low-stringency hybridization with degenerate oligonucleotides to isolate human BDNF and a human gene, neurotrophin-3, that is closely related to both nerve growth factor and brain-derived neurotrophic factor. mRNA products of the brain-derived neurotrophic factor and neurotrophin-3 genes were detected in the adult human brain, suggesting that these proteins are involved in the maintenance of the adult nervous system. Neurotrophin-3 is also expected to function in embryonic neural development.

RNA-Based Vaccines in Cancer Immunotherapy

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Megan A. McNamara

2015-01-01

Full Text Available RNA vaccines traditionally consist of messenger RNA synthesized by in vitro transcription using a bacteriophage RNA polymerase and template DNA that encodes the antigen(s of interest. Once administered and internalized by host cells, the mRNA transcripts are translated directly in the cytoplasm and then the resulting antigens are presented to antigen presenting cells to stimulate an immune response. Alternatively, dendritic cells can be loaded with either tumor associated antigen mRNA or total tumor RNA and delivered to the host to elicit a specific immune response. In this review, we will explain why RNA vaccines represent an attractive platform for cancer immunotherapy, discuss modifications to RNA structure that have been developed to optimize mRNA vaccine stability and translational efficiency, and describe strategies for nonviral delivery of mRNA vaccines, highlighting key preclinical and clinical data related to cancer immunotherapy.

Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic β-cells

DEFF Research Database (Denmark)

Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel

2011-01-01

of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic β-cells, and to examine this in relation to β-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP...

Suppression of FAT/CD36 mRNA by human growth hormone in pancreatic ß-cells

DEFF Research Database (Denmark)

Dalgaard, Louise Torp; Thams, Peter Grevsen; Gaarn, Louise Winkel

2011-01-01

of this study was to examine the effect of human growth hormone (hGH) on mRNAs of fatty acid transport and binding proteins expressed in pancreatic ß-cells, and to examine this in relation to ß-cell survival after exposure to fatty acids. hGH decreased mRNA levels of FAT/CD36, whereas mRNAs of GPR40, FASN, FABP...

Effects of exogenous ATM gene on mRNA expression of human telomerase reverse transcriptase in AT cells induced by irradiation

International Nuclear Information System (INIS)

Sheng Fangjun; Cao Jianping; Luo Jialin; Zhu Wei; Liu Fenju; Feng Shuang; Song Jianyuan; Li Chong

2005-01-01

The study is to observe effects of exogenous ATM gene on mRNA expression of hTERT (human telomerase reverse transcriptase) in fibroblast cells (AT5BIVA cells) from skin of Ataxia-telangiectasia (AT) patients and to study the regulation of ATM to hTERT. Using reverse transcription polymerase chain reaction (RT-PCR), mRNA expression of hTERT in AT, PEBS7-AT, ATM + -AT and GM cells irradiated with 0 and 3 Gy of 60 Co γ-rays were examined respectively. The difference of the mRNA expression of hTERT among AT, PEBS7-AT, ATM + -AT and GM cells were analyzed. Difference of the mRNA expression of hTERT between 0 Gy and 3 Gy groups was analyzed, too. The results showed that the mRNA expression of hTERT in GM cells was negative, but positive mRNA expression of hTERT in AT cells. The mRNA expression of hTERT in ATM + -AT cells decreased significantly (p 60 Co γ-rays, the mRNA expression of hTERT in GM cells was positive, and that in AT, PEBS7-AT, ATM + -AT cells was increased (p + -AT cells was lower than that in AT and PEBS7-AT cells respectively (p<0.05). It is postulated that exogenous ATM is able to downregulate the mRNA expression of hTERT in AT cells, ionizing radiation can induce the mRNA expression of hTERT in cells and telomerase anticipates the repair of damaged DNA. (authors)

MHC Class II and Non-MHC Class II Genes Differentially Influence Humoral Immunity to Bacillus anthracis Lethal Factor and Protective Antigen

OpenAIRE

Garman, Lori; Dumas, Eric K.; Kurella, Sridevi; Hunt, Jonathan J.; Crowe, Sherry R.; Nguyen, Melissa L.; Cox, Philip M.; James, Judith A.; Farris, A. Darise

2012-01-01

Anthrax Lethal Toxin consists of Protective Antigen (PA) and Lethal Factor (LF), and current vaccination strategies focus on eliciting antibodies to PA. In human vaccination, the response to PA can vary greatly, and the response is often directed toward non-neutralizing epitopes. Variable vaccine responses have been shown to be due in part to genetic differences in individuals, with both MHC class II and other genes playing roles. Here, we investigated the relative contribution of MHC class I...

Red Maca (Lepidium meyenii) did not affect cell viability despite increased androgen receptor and prostate-specific antigen gene expression in the human prostate cancer cell line LNCaP.

Science.gov (United States)

Díaz, P; Cardenas, H; Orihuela, P A

2016-10-01

We examined whether aqueous extract of Lepidium meyenii (red Maca) could inhibit growth, potentiate apoptotic activity of two anticancer drugs Taxol and 2-methoxyestradiol (2ME) or change mRNA expression for the androgen target genes, androgen receptor (Ar) and prostate-specific antigen (Psa) in the human prostate cancer cell line LNCaP. Red Maca aqueous extract at 0, 10, 20, 40 or 80 μg/ml was added to LNCaP cells, and viability was evaluated by the MTS assay at 24 or 48 hr after treatment. Furthermore, LNCaP cells were treated with 80 μg/ml of red Maca plus Taxol or 2ME 5 μM and viability was assessed 48 hr later. Finally, LNCaP cells were treated with red Maca 0, 20, 40 or 80 μg/ml, and 12 hr later, mRNA level for Ar or Psa was assessed by real-time PCR. Treatment with red Maca did not affect viability of LNCaP cells. Apoptotic activity induced by Taxol and 2ME in LNCaP cells was not altered with red Maca treatment. Relative expression of the mRNA for Ar and Psa increased with red Maca 20 and 40 μg/ml, but not at 80 μg/ml. We conclude that red Maca aqueous extract does not have toxic effects, but stimulates androgen signalling in LNCaP cells. © 2016 Blackwell Verlag GmbH.

Effects of roasting, blanching, autoclaving, and microwave heating on antigenicity of almond (Prunus dulcis L.) proteins.

Science.gov (United States)

Venkatachalam, M; Teuber, S S; Roux, K H; Sathe, S K

2002-06-05

Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated ras oncogene and SV40 T-antigen.

Science.gov (United States)

Su, L N; Little, J B

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. Cells transfected with EJ ras alone showed no morphological alterations nor significant changes in radiosensitivity. Cell clones expressing ras and/or SV40 T-antigen showed a reduced requirement for serum supplements, an increase in aneuploidy and chromosomal aberrations, and enhanced growth in soft agar as an early cellular response to SV40 T-antigen expression. The sequential order of transfection with SV40 T-antigen and ras influenced radio-sensitivity but not the induction of morphological changes. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself.

Topical stabilized retinol treatment induces the expression of HAS genes and HA production in human skin in vitro and in vivo.

Science.gov (United States)

Li, Wen-Hwa; Wong, Heng-Kuan; Serrano, José; Randhawa, Manpreet; Kaur, Simarna; Southall, Michael D; Parsa, Ramine

2017-05-01

Skin Aging manifests primarily with wrinkles, dyspigmentations, texture changes, and loss of elasticity. During the skin aging process, there is a loss of moisture and elasticity in skin resulting in loss of firmness finally leading to skin sagging. The key molecule involved in skin moisture is hyaluronic acid (HA), which has a significant water-binding capacity. HA levels in skin decline with age resulting in decrease in skin moisture, which may contribute to loss of firmness. Clinical trials have shown that topically applied ROL effectively reduces wrinkles and helps retain youthful appearance. In the current study, ROL was shown to induce HA production and stimulates the gene expression of all three forms of hyaluronic acid synthases (HAS) in normal human epidermal keratinocytes monolayer cultures. Moreover, in human skin equivalent tissues and in human skin explants, topical treatment of tissues with a stabilized-ROL formulation significantly induced the gene expression of HAS mRNA concomitant with an increased HA production. Finally, in a vehicle-controlled human clinical study, histochemical analysis confirmed increased HA accumulation in the epidermis in ROL-treated human skin as compared to vehicle. These results show that ROL increases skin expression of HA, a significant contributing factor responsible for wrinkle formation and skin moisture, which decrease during aging. Taken together with the activity to increase collagen, elastin, and cell proliferation, these studies establish that retinol provides multi-functional activity for photodamaged skin.

Combinatorial Control of mRNA Fates by RNA-Binding Proteins and Non-Coding RNAs

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Valentina Iadevaia

2015-09-01

Full Text Available Post-transcriptional control of gene expression is mediated by RNA-binding proteins (RBPs and small non-coding RNAs (e.g., microRNAs that bind to distinct elements in their mRNA targets. Here, we review recent examples describing the synergistic and/or antagonistic effects mediated by RBPs and miRNAs to determine the localisation, stability and translation of mRNAs in mammalian cells. From these studies, it is becoming increasingly apparent that dynamic rearrangements of RNA-protein complexes could have profound implications in human cancer, in synaptic plasticity, and in cellular differentiation.

Purification and characterization of a major human Pneumocystis carinii surface antigen

DEFF Research Database (Denmark)

Lundgren, B; Lipschik, G Y; Kovacs, J A

1991-01-01

. To evaluate humoral immune responses to the human P. carinii protein, an enzyme-linked immunosorbent assay using purified protein was developed. Some, but not all, patients who subsequently developed P. carinii pneumonia demonstrated a serum antibody response to the surface antigen. Nearly all subjects...... without a history of P. carinii pneumonia had no detectable antibodies. Purified P. carinii proteins will greatly facilitate the investigation of host-P. carinii interactions....

Determining the binding affinity of therapeutic monoclonal antibodies towards their native unpurified antigens in human serum.

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Christine Bee

Full Text Available Monoclonal antibodies (mAbs are a growing segment of therapeutics, yet their in vitro characterization remains challenging. While it is essential that a therapeutic mAb recognizes the native, physiologically occurring epitope, the generation and selection of mAbs often rely on the use of purified recombinant versions of the antigen that may display non-native epitopes. Here, we present a method to measure both, the binding affinity of a therapeutic mAb towards its native unpurified antigen in human serum, and the antigen's endogenous concentration, by combining the kinetic exclusion assay and Biacore's calibration free concentration analysis. To illustrate the broad utility of our method, we studied a panel of mAbs raised against three disparate soluble antigens that are abundant in the serum of healthy donors: proprotein convertase subtilisin/kexin type 9 (PCSK9, progranulin (PGRN, and fatty acid binding protein (FABP4. We also determined the affinity of each mAb towards its purified recombinant antigen and assessed whether the interactions were pH-dependent. Of the six mAbs studied, three did not appear to discriminate between the serum and recombinant forms of the antigen; one mAb bound serum antigen with a higher affinity than recombinant antigen; and two mAbs displayed a different affinity for serum antigen that could be explained by a pH-dependent interaction. Our results highlight the importance of taking pH into account when measuring the affinities of mAbs towards their serum antigens, since the pH of serum samples becomes increasingly alkaline upon aerobic handling.

Multiple correlation analyses revealed complex relationship between DNA methylation and mRNA expression in human peripheral blood mononuclear cells.

Science.gov (United States)

Xie, Fang-Fei; Deng, Fei-Yan; Wu, Long-Fei; Mo, Xing-Bo; Zhu, Hong; Wu, Jian; Guo, Yu-Fan; Zeng, Ke-Qin; Wang, Ming-Jun; Zhu, Xiao-Wei; Xia, Wei; Wang, Lan; He, Pei; Bing, Peng-Fei; Lu, Xin; Zhang, Yong-Hong; Lei, Shu-Feng

2018-01-01

DNA methylation is an important regulator on the mRNA expression. However, a genome-wide correlation pattern between DNA methylation and mRNA expression in human peripheral blood mononuclear cells (PBMCs) is largely unknown. The comprehensive relationship between mRNA and DNA methylation was explored by using four types of correlation analyses and a genome-wide methylation-mRNA expression quantitative trait locus (eQTL) analysis in PBMCs in 46 unrelated female subjects. An enrichment analysis was performed to detect biological function for the detected genes. Single pair correlation coefficient (r T1 ) between methylation level and mRNA is moderate (-0.63-0.62) in intensity, and the negative and positive correlations are nearly equal in quantity. Correlation analysis on each gene (T4) found 60.1% genes showed correlations between mRNA and gene-based methylation at P correlation (R T4  > 0.8). Methylation sites have regulation effects on mRNA expression in eQTL analysis, with more often observations in region of transcription start site (TSS). The genes under significant methylation regulation both in correlation analysis and eQTL analysis tend to cluster to the categories (e.g., transcription, translation, regulation of transcription) that are essential for maintaining the basic life activities of cells. Our findings indicated that DNA methylation has predictive regulation effect on mRNA with a very complex pattern in PBMCs. The results increased our understanding on correlation of methylation and mRNA and also provided useful clues for future epigenetic studies in exploring biological and disease-related regulatory mechanisms in PBMC.

Development of a new in vivo kit for detection of prostate specific antigen in human serum using immunoradiometric assay method

International Nuclear Information System (INIS)

Babaei, M. H.; Behradkia, P.; Shafii, M.; Movla, M.; Forutan, H.; Najafi, R.

2006-01-01

Prostate is a leading site for the cancer incidence, accounted for 31.0% of new cancer cases in men. Prostate-specific antigen is widely used in the detection and monitoring of the prostate cancer. Currently, immunoassay is used to detect Prostate-specific antigen in human serum. This technique is based on the interaction between antibody and antigen. The varied immunoassay formats and equipment to run the assays allow the users to measure the analytes rapidly, with the flexibility to run a small or a large number of samples. Among different immunoassay methods, immunoradiometric assay is a more sensitive and valuable detection approach. This study has been made in 4 parts: (1) purification of Prostate-specific antigen from seminal fluid; (2) preparation of hybridoma cells which secrete monoclonal antibody (mAb) against Prostate-specific antigen , (3) selection of pair monoclonal antibody among those antibodies, and finally (4) design of an immunoradiometric assay kit and it's quality control . The results of this study were: (1) obtaining a huge amount of Prostate-specific antigen as semi-purified and purified, that is a valuable material for preparation of standard kits; (2) preparation of 8 kinds of monoclonal antibodies; (3) finding 4 pairs of monoclonal antibodies which react with different epitopes on Prostate-specific antigen molecule; and (4) preparation of immunoradiometric assay kit for measuring Prostate-specific antigen concentration in human serum

Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

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Ji-Hyun Yeom

Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

Cloning of cDNA sequences of a progestin-regulated mRNA from MCF7 human breast cancer cells

Energy Technology Data Exchange (ETDEWEB)

Chalbos, D; Westley, B; Alibert, C; Rochefort, H

1986-01-24

A cDNA clone corresponding to an mRNA regulated by the progestin R5020, has been isolated by differential screening of a cDNA library from the MCF7 breast cancer cell line, which contains estrogen and progesterone receptors. This probe hybridized with a single species of poly A + RNA of 8-kb molecular weight as shown by Northern blot analysis and could also be used to total RNA preparation. This recombinant cone hybridized specifically to an mRNA coding for a 250,000 daltons protein when translated in vitro. This protein was identical to the 250 kDa progestin-regulated protein that the authors previously described as shown by immunoprecipitation with specific rabbit polyclonal antibodies. Dose-response curve and specificity studies show that the accumulation of the Pg8 mRNA and that of the 250-kDa protein was increased by 5 to 30-fold following progestin treatment and that this effect was mediated by the progesterone receptor. Time course of induction indicated that the accumulation of mRNA was rapid and preceded that of the protein. This is the first report on a cloned cDNA probe of progestin-regulated mRNA in human cell lines.

Sequence, 'subtle' alternative splicing and expression of the CYYR1 (cysteine/tyrosine-rich 1) mRNA in human neuroendocrine tumors

International Nuclear Information System (INIS)

Vitale, Lorenza; Coppola, Domenico; Strippoli, Pierluigi; Frabetti, Flavia; Huntsman, Shane A; Canaider, Silvia; Casadei, Raffaella; Lenzi, Luca; Facchin, Federica; Carinci, Paolo; Zannotti, Maria

2007-01-01

CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG - isoform, the first described mRNA for CYYR1 locus) or the presence (CAG + isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG - or CAG + isoform expression compared to control tissues. CYYR1 CAG - isoform was significantly more expressed than CAG + isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. A new 'subtle' splicing isoform (CAG + ) of CYYR1 mRNA, the sequence and

Heritability in the efficiency of nonsense-mediated mRNA decay in humans.

LENUS (Irish Health Repository)

Seoighe, Cathal

2010-01-01

BACKGROUND: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. PRINCIPAL FINDINGS: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. CONCLUSIONS: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3.

Heritability in the efficiency of nonsense-mediated mRNA decay in humans

KAUST Repository

Seoighe, Cathal

2010-07-21

Background: In eukaryotes mRNA transcripts of protein-coding genes in which an intron has been retained in the coding region normally result in premature stop codons and are therefore degraded through the nonsense-mediated mRNA decay (NMD) pathway. There is evidence in the form of selective pressure for in-frame stop codons in introns and a depletion of length three introns that this is an important and conserved quality-control mechanism. Yet recent reports have revealed that the efficiency of NMD varies across tissues and between individuals, with important clinical consequences. Principal Findings: Using previously published Affymetrix exon microarray data from cell lines genotyped as part of the International HapMap project, we investigated whether there are heritable, inter-individual differences in the abundance of intron-containing transcripts, potentially reflecting differences in the efficiency of NMD. We identified intronic probesets using EST data and report evidence of heritability in the extent of intron expression in 56 HapMap trios. We also used a genome-wide association approach to identify genetic markers associated with intron expression. Among the top candidates was a SNP in the DCP1A gene, which forms part of the decapping complex, involved in NMD. Conclusions: While we caution that some of the apparent inter-individual difference in intron expression may be attributable to different handling or treatments of cell lines, we hypothesize that there is significant polymorphism in the process of NMD, resulting in heritable differences in the abundance of intronic mRNA. Part of this phenotype is likely to be due to a polymorphism in a decapping enzyme on human chromosome 3. © 2010 Seoighe, Gehring.

Impact of adrenaline and metabolic stress on exercise-induced intracellular signaling and PGC-1α mRNA response in human skeletal muscle

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Brandt, Nina; Gunnarsson, Thomas Gunnar Petursson; Hostrup, Morten

2016-01-01

This study tested the hypothesis that elevated plasma adrenaline or metabolic stress enhances exercise-induced PGC-1α mRNA and intracellular signaling in human muscle. Trained (VO2-max: 53.8 ± 1.8 mL min(-1) kg(-1)) male subjects completed four different exercise protocols (work load of the legs...... exercise than at rest in all protocols, and higher (P adrenaline nor muscle metabolic stress determines the magnitude of PGC-1α mRNA response in human muscle. Furthermore, higher exercise-induced changes in AMPK, p38, and CREB...

Human papillomavirus mRNA and DNA testing in women with atypical squamous cells of undetermined significance

DEFF Research Database (Denmark)

Thomsen, Louise T; Dehlendorff, Christian; Junge, Jette

2016-01-01

In this prospective cohort study, we compared the performance of human papillomavirus (HPV) mRNA and DNA testing of women with atypical squamous cells of undetermined significance (ASC-US) during cervical cancer screening. Using a nationwide Danish pathology register, we identified women aged 30......-65 years with ASC-US during 2005-2011 who were tested for HPV16/18/31/33/45 mRNA using PreTect HPV-Proofer (n = 3,226) or for high-risk HPV (hrHPV) DNA using Hybrid Capture 2 (HC2) (n = 9,405) or Linear Array HPV-Genotyping test (LA) (n = 1,533). Women with ≥1 subsequent examination in the register (n = 13...... those testing HC2 negative (3.2% [95% CI: 2.2-4.2%] versus 0.5% [95% CI: 0.3-0.7%]). Patterns were similar after 18 months and 5 years'; follow-up; for CIN2+ and cancer as outcomes; across all age groups; and when comparing mRNA testing to hrHPV DNA testing using LA. In conclusion, the HPV16...

Oncogenic p95HER2 regulates Na+-HCO3- cotransporter NBCn1 mRNA stability in breast cancer cells via 3'UTR-dependent processes.

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Gorbatenko, Andrej; Olesen, Christina W; Loebl, Nathalie; Sigurdsson, Haraldur H; Bianchi, Carolina; Pedraz-Cuesta, Elena; Christiansen, Jan; Pedersen, Stine Falsig

2016-11-01

The Na + -HCO 3 - cotransporter NBCn1 (SLC4A7) is up-regulated in breast cancer, important for tumor growth, and a single nucleotide polymorphism (SNP), rs4973768, in its 3' untranslated region (3'UTR) correlates with increased breast cancer risk. We previously demonstrated that NBCn1 expression and promoter activity are strongly increased in breast cancer cells expressing a constitutively active oncogenic human epidermal growth factor receptor 2 (HER2) (p95HER2). Here, we address the roles of p95HER2 in regulating NBCn1 expression via post-transcriptional mechanisms. p95HER2 expression in MCF-7 cells reduced the rate of NBCn1 mRNA degradation. The NBCn1 3'UTR down-regulated luciferase reporter expression in control cells, and this was reversed by p95HER2, suggesting that p95HER2 counteracts 3'UTR-mediated suppression of NBCn1 expression. Truncation analyses identified three NBCn1 3'UTR regions of regulatory importance. Mutation of putative miRNA-binding sites (miR-374a/b, miR-200b/c, miR-29a/b/c, miR-488) in these regions did not have significant impact on 3'UTR activity. The NBCn1 3'UTR interacted directly with the RNA-binding protein human antigen R (HuR), and HuR knockdown reduced NBCn1 expression. Conversely, ablation of a distal AU-rich element increased 3'UTR-driven reporter activity, suggesting complex regulatory roles of these sites. The cancer-associated SNP variant decreased reporter expression in T-47D breast cancer cells, yet not in MCF-7, MDA-MB-231 and SK-BR-3 cells, arguing against a general role in regulating NBCn1 expression. Finally, p95HER2 expression increased total and plasma membrane NBCn1 protein levels and decreased the rate of NBCn1 protein degradation. Collectively, this is the first work to demonstrate 3'UTR-mediated NBCn1 regulation, shows that p95HER2 regulates NBCn1 expression at multiple levels, and substantiates the central position of p95HER2-NBCn1 signaling in breast cancer. © 2016 The Author(s); published by Portland Press

Mesoporous Silica Nanoparticle-Coated Microneedle Arrays for Intradermal Antigen Delivery.

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Tu, Jing; Du, Guangsheng; Reza Nejadnik, M; Mönkäre, Juha; van der Maaden, Koen; Bomans, Paul H H; Sommerdijk, Nico A J M; Slütter, Bram; Jiskoot, Wim; Bouwstra, Joke A; Kros, Alexander

2017-08-01

To develop a new intradermal antigen delivery system by coating microneedle arrays with lipid bilayer-coated, antigen-loaded mesoporous silica nanoparticles (LB-MSN-OVA). Synthesis of MSNs with 10-nm pores was performed and the nanoparticles were loaded with the model antigen ovalbumin (OVA), and coated with a lipid bilayer (LB-MSN-OVA). The uptake of LB-MSN-OVA by bone marrow-derived dendritic cells (BDMCs) was studied by flow cytometry. The designed LB-MSN-OVA were coated onto pH-sensitive pyridine-modified microneedle arrays and the delivery of LB-MSN-OVA into ex vivo human skin was studied. The synthesized MSNs demonstrated efficient loading of OVA with a maximum loading capacity of about 34% and the lipid bilayer enhanced the colloidal stability of the MSNs. Uptake of OVA loaded in LB-MSN-OVA by BMDCs was higher than that of free OVA, suggesting effective targeting of LB-MSN-OVA to antigen-presenting cells. Microneedles were readily coated with LB-MSN-OVA at pH 5.8, yielding 1.5 μg of encapsulated OVA per microneedle array. Finally, as a result of the pyridine modification, LB-MSN-OVA were effectively released from the microneedles upon piercing the skin. Microneedle arrays coated with LB-MSN-OVA were successfully developed and shown to be suitable for intradermal delivery of the encapsulated protein antigen.

The potential usefulness of human leukocyte antigen typing for celiac disease screening: a systematic review and meta-analysis

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Alicia Díaz-Redondo

2015-07-01

Full Text Available Background and aim: The presence of specific human leukocyte antigen-DQ2 and DQ8 seems to be necessary for celiac disease development, but the real contribution of its typing for screening is still uncertain. We aim to conduct a systematic review and meta-analysis of the diagnostic performance of human leukocyte antigen typing tests for celiac disease screening. Methods: Systematic review of published studies assessing accuracy of human leukocyte antigen DQ2 and DQ8 typing for the detection of celiac disease were selected. MEDLINE and EMBASE were searched from 1st January 2004 until 31st December 2013. Two independent researchers carried out selection and classification of studies, data extraction and analysis. Meta-analysis combining sensitivities, specificities and likelihood ratios of HLA-DQ2 and DQ8 for the diagnosis of celiac disease were carried out. Results: 6 studies including 1303 individuals were finally evaluated. Pooled sensitivity was 98% (95% confidence interval: 97-99. Overall specificity was 45% (95% confidence interval: 41-48. Regarding specificity, studies were heterogeneous and a subgroup analysis was done according to the type of population included. Overall negative likelihood ratio was 0.05 (0.03-0.09. Conclusions: Due to its great sensitivity and low negative likelihood ratio, human leukocyte antigen-DQ2/DQ8 typing would be an appropriate test for ruling out celiac disease in the general population suffering related symptoms, and even more in at risk population.

Rational design of protamine nanocapsules as antigen delivery carriers.

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González-Aramundiz, José Vicente; Presas, Elena; Dalmau-Mena, Inmaculada; Martínez-Pulgarín, Susana; Alonso, Covadonga; Escribano, José M; Alonso, María J; Csaba, Noemi Stefánia

2017-01-10

Current challenges in global immunization indicate the demand for new delivery strategies, which could be applied to the development of new vaccines against emerging diseases, as well as to improve safety and efficacy of currently existing vaccine formulations. Here, we report a novel antigen nanocarrier consisting of an oily core and a protamine shell, further stabilized with pegylated surfactants. These nanocarriers, named protamine nanocapsules, were rationally designed to promote the intracellular delivery of antigens to immunocompetent cells and to trigger an efficient and long-lasting immune response. Protamine nanocapsules have nanometric size, positive zeta potential and high association capacity for H1N1 influenza hemagglutinin, a protein that was used here as a model antigen. The new formulation shows an attractive stability profile both, as an aqueous suspension or a freeze-dried powder formulation. In vitro studies showed that protamine nanocapsules were efficiently internalized by macrophages without eliciting significant toxicity. In vivo studies indicate that antigen-loaded nanocapsules trigger immune responses comparable to those achieved with alum, even when using significantly lower antigen doses, thus indicating their adjuvant properties. These promising in vivo data, alongside with their versatility for the loading of different antigens and oily immunomodulators and their excellent stability profile, make these nanocapsules a promising platform for the delivery of antigens. Protamine sulphate (PubChem SID: 7849283), Sodium Cholate (PubChem CID: 23668194), Miglyol (PubChem CID: 53471835), α tocopherol (PubChem CID: 14985), Tween® 20(PubChem CID: 443314), Tween® 80(PubChem CID: 5281955), TPGS (PubChem CID: 71406). Copyright © 2016 Elsevier B.V. All rights reserved.

Lewis antigen mediated adhesion of freshly removed human bladder tumors to E-selectin

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Skorsteensgaard, Karna; Vestergaard, Else Marie; Langkilde, Niels

1999-01-01

PURPOSE: Twenty fresh surgical specimens of human bladder tumors were tested for their ability to adhere to recombinant P and E-selectin. The adhesion was correlated to immunological detection of carbohydrate structures. MATERIALS AND METHODS: A static titertray assay with immobilized selectins.......003), whereas no correlation was found to secretor and Lewis genotypes. CONCLUSIONS: These data on clinical specimens indicate that Lewis antigen mediated E-selectin adhesion may play a role in the human bladder cancer disease....

Exonuclease hDIS3L2 specifies an exosome-independent 3'-5' degradation pathway of human cytoplasmic mRNA

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Lubas, Michal Szymon; Damgaard, Christian Kroun; Tomecki, Rafal

2013-01-01

Turnover of mRNA in the cytoplasm of human cells is thought to be redundantly conducted by the monomeric 5'-3' exoribonuclease hXRN1 and the 3'-5' exoribonucleolytic RNA exosome complex. However, in addition to the exosome-associated 3'-5' exonucleases hDIS3 and hDIS3L, the human genome encodes...

Autocrine secretion of tumor necrosis factor under the influence of interferon-γ amplifies HLA-DR gene induction in human monocytes

International Nuclear Information System (INIS)

Arenzana-Seisdedos, F.; Mogensen, S.C.; Vuillier, F.; Fiers, W.; Virelizier, J.L.

1988-01-01

Recombinant interferon-γ (IFN-γ) induced HLA-DR gene expression in both U937 and THP-1 human monocytic cell lines, although the former was only very weakly inducible. Combination of recombinant tumor necrosis factor (TNF) and IFN-γ resulted in a synergistic enhancement of DR mRNA and protein induction in both cell lines. TNF alone increased the constitutive expression of the DR gene in THP-1 cells. In the HLA class II-negative U937 cells, TNF used alone was not able to induce DR gene expression. Such a negative result was not due to a lack of TNF receptor expression in U937 cells, since TNF clearly induced HLA class I and TNF gene expression in this cell line. THP-1, but not U937, cells secreted TNF under the influence of IFN-γ. Neutralization of TNF by a specific antibody decreased IFN-γ-induced DR antigen expression in THP-1 cultures. These observations indicate that TNF is not able to directly induce DR gene expression, but rather amplifies ongoing expression of this gene, whether constitutive or induced by IFN-γ. In the two cell lines tested, the level of DR inducibility under the influence of IFN-γ used alone depended on a different inducibility of TNF secretion by IFN-γ. Altogether, the observations indicate that TNF, whether exogenous or endogenously produced under the influence of IFN-γ, amplifies DR gene expression in monocytes, a phenomenon that may provide to such antigen-presenting cells a selective sensitivity to the DR-inducing effects of IFN-γ

Expression of insulin-like growth factor I, insulin-like growth factor binding proteins, and collagen mRNA in mechanically loaded plantaris tendon

DEFF Research Database (Denmark)

Olesen, Jens L; Heinemeier, Katja M; Haddad, Fadia

2006-01-01

Insulin-like growth factor I (IGF-I) is known to exert an anabolic effect on tendon fibroblast production of collagen. IGF-I's regulation is complex and involves six different IGF binding proteins (IGFBPs). Of these, IGFBP-4 and -5 could potentially influence the effect of IGF-I in the tendon...... because they both are produced in fibroblast; however, the response of IGFBP-4 and -5 to mechanical loading and their role in IGF-I regulation in tendinous tissue are unknown. A splice variant of IGF-I, mechano-growth factor (MGF) is upregulated and known to be important for adaptation in loaded muscle....... However, it is not known whether MGF is expressed and upregulated in mechanically loaded tendon. This study examined the effect of mechanical load on tendon collagen mRNA in relation to changes in the IGF-I systems mRNA expression. Data were collected at 2, 4, 8 and 16 days after surgical removal...

Wig1 prevents cellular senescence by regulating p21 mRNA decay through control of RISC recruitment.

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Kim, Bong Cho; Lee, Hyung Chul; Lee, Je-Jung; Choi, Chang-Min; Kim, Dong-Kwan; Lee, Jae Cheol; Ko, Young-Gyu; Lee, Jae-Seon

2012-11-14

Premature senescence, a key strategy used to suppress carcinogenesis, can be driven by p53/p21 proteins in response to various stresses. Here, we demonstrate that Wig1 plays a critical role in this process through regulation of p21 mRNA stability. Wig1 controls the association of Argonaute2 (Ago2), a central component of the RNA-induced silencing complex (RISC), with target p21 mRNA via binding of the stem-loop structure near the microRNA (miRNA) target site. Depletion of Wig1 prohibited miRNA-mediated p21 mRNA decay and resulted in premature senescence. Wig1 plays an essential role in cell proliferation, as demonstrated in tumour xenografts in mice, and Wig1 and p21 mRNA levels are inversely correlated in human normal and cancer tissues. Together, our data indicate a novel role of Wig1 in RISC target accessibility, which is a key step in RNA-mediated gene silencing. In addition, these findings indicate that fine-tuning of p21 levels by Wig1 is essential for the prevention of cellular senescence.

Induction of Robust B Cell Responses after Influenza mRNA Vaccination Is Accompanied by Circulating Hemagglutinin-Specific ICOS+ PD-1+ CXCR3+ T Follicular Helper Cells

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Gustaf Lindgren

2017-11-01

Full Text Available Modified mRNA vaccines have developed into an effective and well-tolerated vaccine platform that offers scalable and precise antigen production. Nevertheless, the immunological events leading to strong antibody responses elicited by mRNA vaccines are largely unknown. In this study, we demonstrate that protective levels of antibodies to hemagglutinin were induced after two immunizations of modified non-replicating mRNA encoding influenza H10 encapsulated in lipid nanoparticles (LNP in non-human primates. While both intradermal (ID and intramuscular (IM administration induced protective titers, ID delivery generated this response more rapidly. Circulating H10-specific memory B cells expanded after each immunization, along with a transient appearance of plasmablasts. The memory B cell pool waned over time but remained detectable throughout the 25-week study. Following prime immunization, H10-specific plasma cells were found in the bone marrow and persisted over time. Germinal centers were formed in vaccine-draining lymph nodes along with an increase in circulating H10-specific ICOS+ PD-1+ CXCR3+ T follicular helper cells, a population shown to correlate with high avidity antibody responses after seasonal influenza vaccination in humans. Collectively, this study demonstrates that mRNA/LNP vaccines potently induce an immunological repertoire associated with the generation of high magnitude and quality antibodies.

Molecular mimics of the tumour antigen MUC1.

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Tharappel C James

Full Text Available A key requirement for the development of cancer immunotherapy is the identification of tumour-associated antigens that are differentially or exclusively expressed on the tumour and recognized by the host immune system. However, immune responses to such antigens are often muted or lacking due to the antigens being recognized as "self", and further complicated by the tumour environment and regulation of immune cells within. In an effort to circumvent the lack of immune responses to tumour antigens, we have devised a strategy to develop potential synthetic immunogens. The strategy, termed mirror image phage display, is based on the concept of molecular mimicry as demonstrated by the idiotype/anti-idiotype paradigm in the immune system. Here as 'proof of principle' we have selected molecular mimics of the well-characterised tumour associated antigen, the human mucin1 protein (MUC1 from two different peptide phage display libraries. The putative mimics were compared in structure and function to that of the native antigen. Our results demonstrate that several of the mimic peptides display T-cell stimulation activity in vitro when presented by matured dendritic cells. The mimic peptides and the native MUC1 antigenic epitopes can cross-stimulate T-cells. The data also indicate that sequence homology and/or chemical properties to the original epitope are not the sole determining factors for the observed immunostimulatory activity of the mimic peptides.

Recruitment of PfSET2 by RNA polymerase II to variant antigen encoding loci contributes to antigenic variation in P. falciparum.

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Uchechi E Ukaegbu

2014-01-01

Full Text Available Histone modifications are important regulators of gene expression in all eukaryotes. In Plasmodium falciparum, these epigenetic marks regulate expression of genes involved in several aspects of host-parasite interactions, including antigenic variation. While the identities and genomic positions of many histone modifications have now been cataloged, how they are targeted to defined genomic regions remains poorly understood. For example, how variant antigen encoding loci (var are targeted for deposition of unique histone marks is a mystery that continues to perplex the field. Here we describe the recruitment of an ortholog of the histone modifier SET2 to var genes through direct interactions with the C-terminal domain (CTD of RNA polymerase II. In higher eukaryotes, SET2 is a histone methyltransferase recruited by RNA pol II during mRNA transcription; however, the ortholog in P. falciparum (PfSET2 has an atypical architecture and its role in regulating transcription is unknown. Here we show that PfSET2 binds to the unphosphorylated form of the CTD, a property inconsistent with its recruitment during mRNA synthesis. Further, we show that H3K36me3, the epigenetic mark deposited by PfSET2, is enriched at both active and silent var gene loci, providing additional evidence that its recruitment is not associated with mRNA production. Over-expression of a dominant negative form of PfSET2 designed to disrupt binding to RNA pol II induced rapid var gene expression switching, confirming both the importance of PfSET2 in var gene regulation and a role for RNA pol II in its recruitment. RNA pol II is known to transcribe non-coding RNAs from both active and silent var genes, providing a possible mechanism by which it could recruit PfSET2 to var loci. This work unifies previous reports of histone modifications, the production of ncRNAs, and the promoter activity of var introns into a mechanism that contributes to antigenic variation by malaria parasites.

Analysis of MDM2 and MDM4 single nucleotide polymorphisms, mRNA splicing and protein expression in retinoblastoma.

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Justina McEvoy

Full Text Available Retinoblastoma is a childhood cancer of the developing retina that begins in utero and is diagnosed in the first years of life. Biallelic RB1 gene inactivation is the initiating genetic lesion in retinoblastoma. The p53 gene is intact in human retinoblastoma but the pathway is believed to be suppressed by increased expression of MDM4 (MDMX and MDM2. Here we quantify the expression of MDM4 and MDM2 mRNA and protein in human fetal retinae, primary retinoblastomas, retinoblastoma cell lines and several independent orthotopic retinoblastoma xenografts. We found that MDM4 is the major p53 antagonist expressed in retinoblastoma and in the developing human retina. We also discovered that MDM4 protein steady state levels are much higher in retinoblastoma than in human fetal retinae. This increase would not have been predicted based on the mRNA levels. We explored several possible post-transcriptional mechanisms that may contribute to the elevated levels of MDM4 protein. A proportion of MDM4 transcripts are alternatively spliced to produce protein products that are reported to be more stable and oncogenic. We also discovered that a microRNA predicted to target MDM4 (miR191 was downregulated in retinoblastoma relative to human fetal retinae and a subset of samples had somatic mutations that eliminated the miR-191 binding site in the MDM4 mRNA. Taken together, these data suggest that post-transcriptional mechanisms may contribute to stabilization of the MDM4 protein in retinoblastoma.

Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse

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Green Carla B

2001-05-01

Full Text Available Abstract Background Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. Results cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. Conclusion The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector.

Identification and Functional Characterization of Human Cd4+Cd25+ T Cells with Regulatory Properties Isolated from Peripheral Blood

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Jonuleit, Helmut; Schmitt, Edgar; Stassen, Michael; Tuettenberg, Andrea; Knop, Jurgen; Enk, Alexander H.

2001-01-01

A subpopulation of peripheral human CD4+CD25+ T cells that expresses CD45RO, histocompatibility leukocyte antigen DR, and intracellular cytotoxic T lymphocyte–associated antigen (CTLA) 4 does not expand after stimulation and markedly suppresses the expansion of conventional T cells in a contact-dependent manner. After activation, CD4+CD25+ T cells express CTLA-4 on the surface detectable for several weeks. These cells show a G1/G0 cell cycle arrest and no production of interleukin (IL)-2, IL-4, or interferon (IFN)-γ on either protein or mRNA levels. The anergic state of CD4+CD25+ T cells is not reversible by the addition of anti-CD28, anti–CTLA-4, anti–transforming growth factor β, or anti–IL-10 antibody. However, the refractory state of CD4+CD25+ T cells was partially reversible by the addition of IL-2 or IL-4. These data demonstrate that human blood contains a resident T cell population with potent regulatory properties. PMID:11390435

Detection of specific antibody producing cells in porcine colostrum by in ovo translation of their mRNA

International Nuclear Information System (INIS)

Kortbeek-Jacobs, N.; Donk, H. van der

1978-01-01

An improved method is described for the determination of antibody producing cells in sows colostrum. The test system comprises in ovo translation of mRNA from swine colostral cells and analysis of the translation products by radioimmunoassay with specific antibodies and antigen. (C.F.)

Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s).

Science.gov (United States)

Mckelvie, J; Foster, A P; Hamblin, A S; Cunningham, F M

2001-04-01

Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active phase of disease; 4 microg protein ml(-1)CAgX; 168 h]. A heat labile factor(s) which caused eosinophil adherence was also released [sweet itch ponies: 6.0 (1.6) per cent adherence versus 1.3 (0.4) per cent; normal ponies: 6.6 (0.5) per cent adherence versus 0.9 (0.1) per cent for supernatants from CAgX (4 microg protein ml(-1); 48 hours) stimulated versus unstimulated BMN cells, respectively]. These results suggest that soluble proteins released from T lymphocytes could affect eosinophil function in the lesional skin of sweet itch horses. Copyright 2001 Harcourt Publishers Ltd.

Transcription factor Brn-3α mRNA in cancers, relationship with AR, ER receptors and AKT/m-TOR pathway components

Science.gov (United States)

Spirina, L. V.; Gorbunov, A. K.; Chigevskaya, S. Y.; Usynin, Y. A.; Kondakova, I. V.; Slonimskaya, E. M.; Usynin, E. A.; Choinzonov, E. L.; Zaitseva, O. S.

2017-09-01

Transcription factors POU4F1 (neurogenic factor Brn-3α) play a pivotal role in cancers development. The aim of the study was to reveal the Brn-3α expression, AR, ER expression in cancers development, association with AKT/mTOR pathway activation. 30 patients with locally advanced prostate cancer, 20 patients with papillary thyroid cancer, T2-3N0-1M0 stages and 40 patients with renal cell cancer T2-3N0M0-1 were involved into the study. The expressions of Brn-3α, AR, ERα, components of AKT/m-TOR signaling pathway genes were performed by real-time PCR. The dependence of Brn-3α expression on mRNA levels of steroid hormone receptors and components of AKT/m-TOR signaling pathway in studied cancers were shown. High levels of mRNA of nuclear factor, steroid hormone receptors were found followed by the activation of this signaling pathway in prostate cancer tissue. The reduction of transcription factor Brn-3α was accompanied with tumor invasive growth with increasing rates of AR, ER and 4E-BP1 mRNA. Thyroid cancer development happened in a case of a Brn-3α and steroid hormone receptors decrease. The activation of AKT/m-TOR signaling pathway was established in the metastatic renal cancers, accompanied with the increase of ER mRNA. But there was no correlation between the steroid receptor and Brn-3α. One-direction changes of Brn-3α were observed in the development of prostate and thyroid cancer due to its effect on the steroid hormone receptors and the activation of AKT/m-TOR signaling pathway components. The influence of this factor on the development of the kidney cancer was mediated through m-TOR activity modifications, the key enzyme of oncogenesis.

Hepatocyte and keratinocyte growth factors and their receptors in human lung emphysema

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Marchal Joëlle

2005-10-01

Full Text Available Abstract Background Hepatocyte and keratinocyte growth factors are key growth factors in the process of alveolar repair. We hypothesized that excessive alveolar destruction observed in lung emphysema involves impaired expression of hepatocyte and keratinocyte growth factors or their respective receptors, c-met and keratinocyte growth factor receptor. The aim of our study was to compare the expression of hepatocyte and keratinocyte growth factors and their receptors in lung samples from 3 groups of patients: emphysema; smokers without emphysema and non-smokers without emphysema. Methods Hepatocyte and keratinocyte growth factor proteins were analysed by immunoassay and western blot; mRNA expression was measured by real time quantitative polymerase chain reaction. Results Hepatocyte and keratinocyte growth factors, c-met and keratinocyte growth factor receptor mRNA levels were similar in emphysema and non-emphysema patients. Hepatocyte growth factor mRNA correlated negatively with FEV1 and the FEV1/FVC ratio both in emphysema patients and in smokers with or without emphysema. Hepatocyte and keratinocyte growth factor protein concentrations were similar in all patients' groups. Conclusion The expression of hepatocyte and keratinocyte growth factors and their receptors is preserved in patients with lung emphysema as compared to patients without emphysema. Hepatocyte growth factor mRNA correlates with the severity of airflow obstruction in smokers.

IL-4 mRNA Is Downregulated in the Liver of Pancreatic Cancer Patients Suffering from Cachexia.

Science.gov (United States)

Prokopchuk, Olga; Steinacker, Jürgen M; Nitsche, Ulrich; Otto, Stephanie; Bachmann, Jeannine; Schubert, Elaine C; Friess, Helmut; Martignoni, Marc E

2017-01-01

Interleukin-4 (IL-4) together with interleukin-13 (IL-13) play an important role in inflammation and wound repair, and are known to be upregulated in human skeletal muscle after strenuous physical exercise. Additionally, these cytokines may act as autocrine growth factors in pancreatic cancer cells. We hypothesize that IL-4, IL-13, and their corresponding receptors are involved in mechanism of cancer cachexia. Tissue samples from human skeletal muscle, white fat, liver, healthy pancreas, and pancreatic ductal adenocarcinoma were analyzed by quantitative real-time polymerase chain reaction for mRNA expression levels of IL-4, IL-13, IL-4 receptor α, and IL-13 receptor α1. We demonstrate for the first time that liver IL-4 mRNA is downregulated in vivo in patients with pancreatic cancer and cachexia. Additionally, IL-4 mRNA in the liver inversely correlated with musculus psoas thickness. We speculate that suppression of IL-4 is involved in cancer cachexia, although the exact mechanisms have to be further elucidated.

Fas ligand expression in human and mouse cancer cell lines; a caveat on over-reliance on mRNA data

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Ryan Aideen E

2006-02-01

Full Text Available Abstract Background During carcinogenesis, tumors develop multiple mechanisms for evading the immune response, including upregulation of Fas ligand (FasL/CD95L expression. Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of anti-tumor immune effector cells. Recently this idea has been challenged by studies reporting that tumor cells of varying origin do not express FasL. In the present study, we aimed to comprehensively characterize FasL expression in tumors of both murine and human origin over a 72 hour time period. Methods RNA and protein was extracted from six human (SW620, HT29, SW480, KM12SM, HCT116, Jurkat and three mouse (CMT93, CT26, B16F10 cancer cell lines at regular time intervals over a 72 hour time period. FasL expression was detected at the mRNA level by RT-PCR, using intron spanning primers, and at the protein level by Western Blotting and immunofluorescence, using a polyclonal FasL- specific antibody. Results Expression of FasL mRNA and protein was observed in all cell lines analysed. However, expression of FasL mRNA varied dramatically over time, with cells negative for FasL mRNA at many time points. In contrast, 8 of the 9 cell lines constitutively expressed FasL protein. Thus, cells can abundantly express FasL protein at times when FasL mRNA is absent. Conclusion These findings demonstrate the importance of complete analysis of FasL expression by tumor cells in order to fully characterize its biological function and may help to resolve the discrepancies present in the literature regarding FasL expression and tumor immune privilege.

Evaluation of an mRNA lipofection procedure for human dendritic cells and induction of cytotoxic T lymphocytes against enhanced green fluorescence protein.

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Okano, Kozue; Fukui, Mikiko; Suehiro, Yutaka; Hamanaka, Yuichiro; Imai, Kohzoh; Hinoda, Yuji

2003-01-01

We utilized an mRNA lipofection procedure in human dendritic cells (DCs) and attempted to induce cytotoxic T lymphocytes (CTLs) against enhanced green fluorescence protein (EGFP). EGFP mRNA was transfected into phytohemagglutinin (PHA)-stimulated lymphocytes or adherent peripheral blood mononuclear cell-derived DCs using a liposomal reagent. Lipofection efficiency was measured by flow cytometry. In PHA-stimulated lymphocytes, increasing concentrations of liposome or mRNA increased EGFP expression levels by up to 64.4%, but caused a decrease in cell viability. A similar trend was also observed in DCs. For 70% DC viability, the concentration of liposomes was 24 microl/ml, and the mRNA concentration was 6 microg/ml. Under these conditions, ELISPOT and (51)Cr release assays were performed on CD8+ T cells stimulated twice with EGFP mRNA-transfected DCs. The number of interferon-gamma-producing cells was increased when the CD8+ T cells were cocultured for 24 h with PHA-stimulated lymphocytes transfected with EGFP mRNA. The level of specific lysis of EGFP mRNA-transfected DCs also increased to approximately 80%, with an effector to target ratio of 40:1. These data suggest that EGFP is immunogenic for human T cells, confirming that our lipofection procedure may be of use for inducing specific CTLs. Copyright 2003 S. Karger AG, Basel

Selection of antigenic markers on a GFP-Cκ fusion scaffold with high sensitivity by eukaryotic ribosome display

International Nuclear Information System (INIS)

Yang Yongmin; Barankiewicz, Teresa J.; He Mingyue; Taussig, Michael J.; Chen, Swey-Shen

2007-01-01

Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (Cκ) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5') or Cκ (3') were selected by anti-GFP or anti-Cκ antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins

Human leukocyte antigen-G polymorphism in relation to expression, function, and disease

DEFF Research Database (Denmark)

Larsen, Margit Hørup; Hviid, Thomas

2009-01-01

Human leukocyte antigen-G (HLA-G) is a nonclassical class Ib molecule belonging to the major histocompatibility complex. HLA-G appears to play a role in the suppression of immune responses and contribute to long-term immune escape or tolerance. The focus of this review is polymorphism in the HLA......-G gene and protein and its possible importance in expression, function, and disease associations....

Crimean-Congo Hemorrhagic Fever Virus Nucleocapsid Protein Augments mRNA Translation.

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Jeeva, Subbiah; Cheng, Erdong; Ganaie, Safder S; Mir, Mohammad A

2017-08-01

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne Nairovirus of the Bunyaviridae family, causing severe illness with high mortality rates in humans. Here, we demonstrate that CCHFV nucleocapsid protein (CCHFV-NP) augments mRNA translation. CCHFV-NP binds to the viral mRNA 5' untranslated region (UTR) with high affinity. It facilitates the translation of reporter mRNA both in vivo and in vitro with the assistance of the viral mRNA 5' UTR. CCHFV-NP equally favors the translation of both capped and uncapped mRNAs, demonstrating the independence of this translation strategy on the 5' cap. Unlike the canonical host translation machinery, inhibition of eIF4F complex, an amalgam of three initiation factors, eIF4A, eIF4G, and eIF4E, by the chemical inhibitor 4E1RCat did not impact the CCHFV-NP-mediated translation mechanism. However, the proteolytic degradation of eIF4G alone by the human rhinovirus 2A protease abrogated this translation strategy. Our results demonstrate that eIF4F complex formation is not required but eIF4G plays a critical role in this translation mechanism. Our results suggest that CCHFV has adopted a unique translation mechanism to facilitate the translation of viral mRNAs in the host cell cytoplasm where cellular transcripts are competing for the same translation apparatus. IMPORTANCE Crimean-Congo hemorrhagic fever, a highly contagious viral disease endemic to more than 30 countries, has limited treatment options. Our results demonstrate that NP favors the translation of a reporter mRNA harboring the viral mRNA 5' UTR. It is highly likely that CCHFV uses an NP-mediated translation strategy for the rapid synthesis of viral proteins during the course of infection. Shutdown of this translation mechanism might selectively impact viral protein synthesis, suggesting that an NP-mediated translation strategy is a target for therapeutic intervention against this viral disease. Copyright © 2017 American Society for Microbiology.

Donor-recipient human leukocyte antigen matching practices in vascularized composite tissue allotransplantation: a survey of major transplantation centers.

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Ashvetiya, Tamara; Mundinger, Gerhard S; Kukuruga, Debra; Bojovic, Branko; Christy, Michael R; Dorafshar, Amir H; Rodriguez, Eduardo D

2014-07-01

Vascularized composite tissue allotransplant recipients are often highly sensitized to human leukocyte antigens because of multiple prior blood transfusions and other reconstructive operations. The use of peripheral blood obtained from dead donors for crossmatching may be insufficient because of life support measures taken for the donor before donation. No study has been published investigating human leukocyte antigen matching practices in this field. A survey addressing human leukocyte antigen crossmatching methods was generated and sent to 22 vascularized composite tissue allotransplantation centers with active protocols worldwide. Results were compiled by center and compared using two-tailed t tests. Twenty of 22 centers (91 percent) responded to the survey. Peripheral blood was the most commonly reported donor sample for vascularized composite tissue allotransplant crossmatching [78 percent of centers (n=14)], with only 22 percent (n=4) using lymph nodes. However, 56 percent of the 18 centers (n=10) that had performed vascularized composite tissue allotransplantation reported that they harvested lymph nodes for crossmatching. Of responding individuals, 62.5 percent (10 of 16 individuals) felt that lymph nodes were the best donor sample for crossmatching. A slight majority of vascularized composite tissue allotransplant centers that have performed clinical transplants have used lymph nodes for human leukocyte antigen matching, and centers appear to be divided on the utility of lymph node harvest. The use of lymph nodes may offer a number of potential benefits. This study highlights the need for institutional review board-approved crossmatching protocols specific to vascularized composite tissue allotransplantation, and the need for global databases for sharing of vascularized composite tissue allotransplantation experiences.

Natural selection and algorithmic design of mRNA.

Science.gov (United States)

Cohen, Barry; Skiena, Steven

2003-01-01

Messenger RNA (mRNA) sequences serve as templates for proteins according to the triplet code, in which each of the 4(3) = 64 different codons (sequences of three consecutive nucleotide bases) in RNA either terminate transcription or map to one of the 20 different amino acids (or residues) which build up proteins. Because there are more codons than residues, there is inherent redundancy in the coding. Certain residues (e.g., tryptophan) have only a single corresponding codon, while other residues (e.g., arginine) have as many as six corresponding codons. This freedom implies that the number of possible RNA sequences coding for a given protein grows exponentially in the length of the protein. Thus nature has wide latitude to select among mRNA sequences which are informationally equivalent, but structurally and energetically divergent. In this paper, we explore how nature takes advantage of this freedom and how to algorithmically design structures more energetically favorable than have been built through natural selection. In particular: (1) Natural Selection--we perform the first large-scale computational experiment comparing the stability of mRNA sequences from a variety of organisms to random synonymous sequences which respect the codon preferences of the organism. This experiment was conducted on over 27,000 sequences from 34 microbial species with 36 genomic structures. We provide evidence that in all genomic structures highly stable sequences are disproportionately abundant, and in 19 of 36 cases highly unstable sequences are disproportionately abundant. This suggests that the stability of mRNA sequences is subject to natural selection. (2) Artificial Selection--motivated by these biological results, we examine the algorithmic problem of designing the most stable and unstable mRNA sequences which code for a target protein. We give a polynomial-time dynamic programming solution to the most stable sequence problem (MSSP), which is asymptotically no more complex

Human Leucocyte Antigen-G (HLA-G and Its Murine Functional Homolog Qa2 in the Trypanosoma cruzi Infection

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Fabrício C. Dias

2015-01-01

Full Text Available Genetic susceptibility factors, parasite strain, and an adequate modulation of the immune system seem to be crucial for disease progression after Trypanosoma cruzi infection. HLA-G and its murine functional homolog Qa2 have well-recognized immunomodulatory properties. We evaluated the HLA-G 3′ untranslated region (3′UTR polymorphic sites (associated with mRNA stability and target for microRNA binding and HLA-G tissue expression (heart, colon, and esophagus in patients presenting Chagas disease, stratified according to the major clinical variants. Further, we investigated the transcriptional levels of Qa2 and other pro- and anti-inflammatory genes in affected mouse tissues during T. cruzi experimental acute and early chronic infection induced by the CL strain. Chagas disease patients exhibited differential HLA-G 3′UTR susceptibility allele/genotype/haplotype patterns, according to the major clinical variant (digestive/cardiac/mixed/indeterminate. HLA-G constitutive expression on cardiac muscle and colonic cells was decreased in Chagasic tissues; however, no difference was observed for Chagasic and non-Chagasic esophagus tissues. The transcriptional levels of Qa2 and other anti and proinflammatory (CTLA-4, PDCD1, IL-10, INF-γ, and NOS-2 genes were induced only during the acute T. cruzi infection in BALB/c and C57BL/6 mice. We present several lines of evidence indicating the role of immunomodulatory genes and molecules in human and experimental T. cruzi infection.

Clinical significance of serum circulating insulin-like growth factor-1 (IGF-1) mRNA in hepatocellular carcinoma.

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Karabulut, S; Duranyıldız, D; Tas, F; Gezer, U; Akyüz, F; Serilmez, M; Ozgür, E; Yasasever, C T; Vatansever, S; Aykan, N F

2014-03-01

The principal aim of our study was to investigate the usefulness of serum protein and circulating mRNA of insulin-like growth factor-1 (IGF-1) as a diagnostic and prognostic tool in hepatocellular carcinoma (HCC). Fifty-four HCC patients and age- and sex-matched 20 healthy controls were enrolled into this study. Pretreatment serum IGF-1 and IGF-1 mRNA were determined by the solid-phase sandwich ELISA and quantitative RT-PCR method, respectively. The median age at diagnosis was 60 years, range 36-77 years; where majority of group were male (n = 48, 88.8%). All patients had cirrhotic history. Forty-six percent (n = 25) of patients had Child-Pugh score A, 30% (n = 16) had score B or C. All of the patients were treated with local therapies and none of them received sorafenib. The baseline serum IGF-1 mRNA levels were significantly higher in HCC patients than in the control group (p = 0.04), whereas no significant difference was observed for IGF-1 protein levels between the two group (p = 0.18). Patients with history of HBV infection, who were not treated, and who received multiple palliative treatment for HCC had higher serum IGF-1 mRNA levels (p = 0.03, 0.03, and 0.05, respectively). Poor performance status (p IGF-1 nor serum IGF-1 mRNA had significantly adverse effect on survival (p = 0.53 and 0.42, respectively).

Invasive breast cancer in Argentine women: association between risk and prognostic factors with antigens of a peptidic and carbohydrate nature

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Croce MV

2011-12-01

Full Text Available Sandra O Demichelis, Marina T Isla-Larrain, Luciano Cermignani, Cecilio G Alberdi, Amada Segal-Eiras, María Virginia CroceCentre of Basic and Applied Immunological Research, Faculty of Medical Sciences, National University of La Plata, La Plata, ArgentinaObjective: In breast cancer, several tumor markers have been identified. The marker most extensively associated with breast cancer is MUC1. The objective of the study was to analyze prognostic and risk factors in relation to tumor markers in order to clarify breast cancer biology. A total of 349 primary tumor samples and lymph nodes from breast cancer patients were studied. Risk and prognostic factors were considered. An immunohistochemical approach was applied and an extensive statistical analysis was performed, including frequency analysis and analysis of variance. Correlation among variables was performed with principal component analysis.Results: All the antigens showed an increased expression according to tumor size increment; moreover, sialyl Lewis x expression showed a significant increase in relation to disease stage, whereas Tn and TF presented a positive tendency. Vascular invasion was related to sialyl Lewis x expression and number of metastatic lymph nodes. Taking into account risk factors, when a patient had at least one child, Lewis antigens diminished their expression. In relation to breastfeeding, sialyl Lewis x expression diminished, although its apical expression increased.Conclusion: Associations between MUC1 and carbohydrate antigens and risk and prognostic factors show the complexity of the cellular biological behavior that these antigens modulate in breast cancer.Keywords: breast cancer, Argentine women, risk factors, prognostic factors, antigenic expression

Quality Improvement to Demonstrate the Lack of Reliability of the Human Papillomavirus mRNA Assay to Identify Women With Latent Human Papillomavirus Infections.

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Cotton, Sarah; Brown, Robert E; Nugent, Elizabeth K; Robazetti, Sonia C; Berens, Pamela D; Smith, Judith A

2018-04-01

To assess the consistency between human papillomavirus (HPV) mRNA testing in women with a history of previous HPV infections diagnosed by HPV DNA assay and the potential effects on follow-up HPV screening. This was a quality improvement study that used data from a pathology laboratory software database reviewed from November 2014 to June 2016 to identify female patients aged 30 years or older with greater than one HPV-positive result, including one or more HPV mRNA assay results and one or more documented HPV DNA assay results for comparison. Previous correlative cytology and colposcopic histopathology were also documented. American College of Obstetricians and Gynecologists' cervical cancer screening guidelines were used to compare potential differences in follow-up recommendations. Four hundred twenty-five charts for female patients 30 years of age or older were identified with one or more prior high-risk HPV infections by DNA assay. There was a 69.3% difference in HPV mRNA results compared with previous HPV DNA-positive results. There was a potential change in follow-up for 71.7% of patients with one prior high-risk-HPV-positive result and 60.0% of patients with two or more prior high-risk HPV-positive results. There were 231 colposcopy reports evaluated in this study. Of these, 62 (26.8%) were abnormal colposcopy reports, including 45 low-grade squamous intraepithelial lesions, 15 high-grade squamous intraepithelial lesions, and two cancers. Twenty-five (40.3%) abnormal colposcopy findings were in patients with a history of at least than two prior HPV DNA-positive results and a report of currently being HPV-negative with the mRNA assay. The HPV mRNA assays are less sensitive for detection of latent HPV infections compared with HPV DNA assays. Based on these data and the potential change in follow-up care, the HPV mRNA assay should not be used for a primary screening tool for cervical cancer. Many pathology laboratories have shifted to using the HPV mRNA assay

Differential expression of THOC1 and ALY mRNP biogenesis/export factors in human cancers

International Nuclear Information System (INIS)

Domínguez-Sánchez, María S; Sáez, Carmen; Japón, Miguel A; Aguilera, Andrés; Luna, Rosa

2011-01-01

One key step in gene expression is the biogenesis of mRNA ribonucleoparticle complexes (mRNPs). Formation of the mRNP requires the participation of a number of conserved factors such as the THO complex. THO interacts physically and functionally with the Sub2/UAP56 RNA-dependent ATPase, and the Yra1/REF1/ALY RNA-binding protein linking transcription, mRNA export and genome integrity. Given the link between genome instability and cancer, we have performed a comparative analysis of the expression patterns of THOC1, a THO complex subunit, and ALY in tumor samples. The mRNA levels were measured by quantitative real-time PCR and hybridization of a tumor tissue cDNA array; and the protein levels and distribution by immunostaining of a custom tissue array containing a set of paraffin-embedded samples of different tumor and normal tissues followed by statistical analysis. We show that the expression of two mRNP factors, THOC1 and ALY are altered in several tumor tissues. THOC1 mRNA and protein levels are up-regulated in ovarian and lung tumors and down-regulated in those of testis and skin, whereas ALY is altered in a wide variety of tumors. In contrast to THOC1, ALY protein is highly detected in normal proliferative cells, but poorly in high-grade cancers. These results suggest a differential connection between tumorogenesis and the expression levels of human THO and ALY. This study opens the possibility of defining mRNP biogenesis factors as putative players in cell proliferation that could contribute to tumor development

Squamous Cell Carcinoma Antigen-encoding Genes SERPINB3/B4 as Potentially Useful Markers for the Stratification of HNSCC Tumours.

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Saidak, Zuzana; Morisse, Mony Chenda; Chatelain, Denis; Sauzay, Chloé; Houessinon, Aline; Guilain, Nelly; Soyez, Marion; Chauffert, Bruno; Dakpé, Stéphanie; Galmiche, Antoine

2018-03-01

The squamous cell carcinoma antigen (SCCA), encoded by the genes SERPINB3/B4, is a tumour marker produced by head and neck squamous cell carcinoma (HNSCC). We aimed to examine SERPINB3/B4 mRNA levels and its clinical significance in the therapeutic context. We retrieved mRNA expression levels, clinical, pathological and genomic data for 520 HNSCC from The Cancer Genome Atlas (TCGA). HNSCC tumours express high levels of SERPINB3/B4 mRNA. SERPINB3 expression differs depending on Human papillomavirus (HPV) infection status, primary tumour location, grade and differentiation, extension to lymph nodes and extracapsular spread. Interestingly, we observed an association between SERPINB3/B4 and the presence of tumour immune infiltrate as well as the expression of the immune checkpoint regulators PD-L1/PD-L2 that depended on HPV status. Our findings point to potential interest of SERPINB3/B4 for the stratification of HNSCC patients in the therapeutic context. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Regulation of human histone gene expression: transcriptional and posttranscriptional control in the coupling of histone messenger RNA stability with DNA replication

International Nuclear Information System (INIS)

Baumbach, L.L.; Stein, G.S.; Stein, J.L.

1987-01-01

The extent to which transcriptional and posttranscriptional regulation contributes to the coupling of histone gene expression and DNA replication was examined during the cell cycle in synchronized HeLa S3 cells. Rates of transcription were determined in vitro in isolated nuclei. A 3-5-fold increase in cell cycle dependent histone gene transcription was observed in early S phase, prior to the peak of DNA synthesis. This result is consistent with a previous determination of histone mRNA synthesis in intact cells. The transcription of these genes did not change appreciably after inhibition of DNA replication by hydroxyurea treatment, although Northern blot analysis indicated that cellular levels of histone mRNA decreased rapidly in the presence of the drug. Total cellular levels of histone mRNA closely parallel the rate of DNA synthesis as a function of cell cycle progression, reaching a maximal 20-fold increase as compared with non S phase levels. This DNA synthesis dependent accumulation of histone mRNA occurs predominantly in the cytoplasm and appears to be mediated primarily by control of histone mRNA stability. Changes in nuclear histone mRNA levels were less pronounced. These combined observations suggest that both transcriptional regulation and posttranscriptional regulation contribute toward control of the cell cycle dependent accumulation of histone mRNA during S phase, while the stability of histone mRNA throughout S phase and the selective turnover of histone mRNAs, either at the natural termination of S phase or following inhibition of DNA synthesis, are posttranscriptionally regulated

Myogenic, matrix and growth factor mRNA expression in human skeletal muscle: effect of contraction intensity and feeding

DEFF Research Database (Denmark)

Agergaard, Jakob; Reitelseder, Søren; Pedersen, T.G.

2013-01-01

. RESULTS: Relative muscle activity differed between HL and LL resistance exercise, whereas median power frequency was even, suggesting an equal muscle-fiber-type recruitment distribution. mRNA expression of Myf6, myogenin, and p21 was mostly increased, and myostatin was mostly depressed by HL resistance...

Impact of a single bout of high-intensity interval exercise and short-term interval training on interleukin-6, FNDC5, and METRNL mRNA expression in human skeletal muscle

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Malcolm Eaton

2018-04-01

Full Text Available Background: Exercise promotes numerous phenotypic adaptations in skeletal muscle that contribute to improved function and metabolic capacity. An emerging body of evidence suggests that skeletal muscle also releases a myriad of factors during exercise, termed “myokines”. The purpose of this study was to examine the effects of high-intensity interval training (HIIT on the acute regulation of the mRNA expression of several myokines, including the prototypical myokine interleukin-6 (IL-6, and recently identified myokines fibronectin type III domain-containing protein 5 (FNDC5 (irisin and meteorin-like protein (METRNL. Methods: Both before and after a 20-day period of twice-daily high-volume HIIT, 9 healthy males (20.5 ± 1.5 years performed a standardized bout of high-intensity interval exercise (HIIE; 5 × 4 min at ~80% pretraining peak power output with skeletal muscle biopsy samples (vastus lateralis obtained at rest, immediately following exercise, and at 3 h recovery. Results: Before training, a single bout of HIIE increased IL-6 (p < 0.05 and METRNL (p < 0.05 mRNA expression measured at 3 h recovery when compared to rest. Following 20 days of HIIT, IL-6 and FNDC5 mRNA were increased at 3 h recovery from the standardized HIIE bout when compared to rest (both p < 0.05. Resting METRNL and FNDC5 mRNA expression were higher following training (p < 0.05, and there was an overall increase in FNDC5 mRNA post-training (main effect of training, p < 0.05. Conclusion: In human skeletal muscle (1 an acute bout of HIIE can induce upregulation of skeletal muscle IL-6 mRNA both before and after a period of intensified HIIT; (2 Resting and overall FNDC5 mRNA expression is increased by 20 days of HIIT; and (3 METRNL mRNA expression is responsive to both acute HIIE and short-term intense HIIT. Future studies are needed to confirm these findings at the protein and secretion level in humans. Keywords: Brown adipose tissue

Enzymatic Activity of Free-Prostate-Specific Antigen (f-PSA) Is Not Required for Some of its Physiological Activities

Science.gov (United States)

Chadha, Kailash C.; Nair, Bindukumar B.; Chakravarthi, Srikant; Zhou, Rita; Godoy, Alejandro; Mohler, James L.; Aalinkeel, Ravikumar; Schwartz, Stanley A.; Smith, Gary J.

2015-01-01

BACKGROUND Prostate specific antigen (PSA) is a well known biomarker for early diagnosis and management of prostate cancer. Furthermore, PSA has been documented to have anti-angiogenic and anti-tumorigenic activities in both in vitro and in vivo studies. However, little is known about the molecular mechanism(s) involved in regulation of these processes, in particular the role of the serine-protease enzymatic activity of PSA. METHODS Enzymatic activity of PSA isolated directly from seminal plasma was inhibited specifically (>95%) by incubation with zinc2+. Human umbilical vein endothelial cells (HUVEC) were utilized to compare/contrast the physiological effects of enzymatically active versus inactive PSA. RESULTS Equimolar concentrations of enzymatically active PSA and PSA enzymatically inactivated by incubation with Zn2+ had similar physiological effects on HUVEC, including inhibiting the gene expression of pro-angiogenic growth factors, like VEGF and bFGF, and up-regulation of expression of the anti-angiogenic growth factor IFN-γ; suppression of mRNA expression for markers of blood vessel development, like FAK, FLT, KDR, TWIST-1; P-38; inhibition of endothelial tube formation in the in vitro Matrigel Tube Formation Assay; and inhibition of endothelial cell invasion and migration properties. DISCUSSION Our data provides compelling evidence that the transcriptional regulatory and the anti-angiogenic activities of human PSA are independent of the innate enzymatic activity PMID:21446007

Progestins inhibit estradiol-induced vascular endothelial growth factor and stromal cell-derived factor 1 in human endometrial stromal cells.

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Okada, Hidetaka; Okamoto, Rika; Tsuzuki, Tomoko; Tsuji, Shoko; Yasuda, Katsuhiko; Kanzaki, Hideharu

2011-09-01

To investigate whether 17β-estradiol (E(2)) and progestins exert direct effects on vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1/CXCL12) in human endometrial stromal cells (ESCs) and thereby to clarify the regulatory function of these local angiogenic factors in the endometrium. In vitro experiment. Research laboratory at Kansai Medical University. Fourteen patients undergoing hysterectomy for benign reasons. ESCs were cultured with E(2) and/or various clinically relevant progestins (medroxyprogesterone acetate [MPA], norethisterone [NET], levonorgestrel [LNG], dienogest [DNG], and progesterone [P]). The mRNA levels and production of VEGF and SDF-1 were assessed by real-time reverse-transcription polymerase chain reaction and ELISA, respectively. E(2) significantly induced the mRNA levels and protein production of VEGF and SDF-1 in ESCs. MPA could antagonize the E(2)-stimulated effects in a time- and dose-dependent manner, and this effect could be reversed by RU-486 (P receptor antagonist). All of the progestins (MPA, NET, LNG, and DNG; 10(-9) to 10(-7) mol/L) attenuated E(2)-induced VEGF and SDF-1 production, whereas P showed these inhibitory effects only when present in a high concentration (10(-7) mol/L). Progestins have inhibitory effects on E(2)-induced VEGF and SDF-1 in ESCs. These results may indicate a potential mechanism for action of the female sex steroids in the human endometrium that can be helpful for various clinical applications. Copyright © 2011 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

Science.gov (United States)

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

Combined sequencing of mRNA and DNA from human embryonic stem cells

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Florian Mertes

2016-06-01

Full Text Available Combined transcriptome and whole genome sequencing of the same ultra-low input sample down to single cells is a rapidly evolving approach for the analysis of rare cells. Besides stem cells, rare cells originating from tissues like tumor or biopsies, circulating tumor cells and cells from early embryonic development are under investigation. Herein we describe a universal method applicable for the analysis of minute amounts of sample material (150 to 200 cells derived from sub-colony structures from human embryonic stem cells. The protocol comprises the combined isolation and separate amplification of poly(A mRNA and whole genome DNA followed by next generation sequencing. Here we present a detailed description of the method developed and an overview of the results obtained for RNA and whole genome sequencing of human embryonic stem cells, sequencing data is available in the Gene Expression Omnibus (GEO database under accession number GSE69471.

Humoral and cell-mediated immune response against human retinal antigens in relation to ocular onchocerciasis

NARCIS (Netherlands)

van der Lelij, A.; Rothova, A.; Stilma, J. S.; Vetter, J. C.; Hoekzema, R.; Kijlstra, A.

1990-01-01

Autoimmune mechanisms are thought to be involved in the pathogenesis of the chorioretinal changes in ocular onchocerciasis. The humoral autoimmune response was determined by measuring serum levels of autoantibodies, directed against human S-antigen and interphotoreceptor retinoid binding protein

Tumor necrosis factor-alpha regulates the Hypocretin system via mRNA degradation and ubiquitination.

Science.gov (United States)

Zhan, Shuqin; Cai, Guo-Qiang; Zheng, Anni; Wang, Yuping; Jia, Jianping; Fang, Haotian; Yang, Youfeng; Hu, Meng; Ding, Qiang

2011-04-01

Recent studies recognize that Hypocretin system (also known as Orexin) plays a critical role in sleep/wake disorders and feeding behaviors. However, little is known about the regulation of the Hypocretin system. It is also known that tumor necrosis factor alpha (TNF-α) is involved in the regulation of sleep/wake cycle. Here, we test our hypothesis that the Hypocretin system is regulated by TNF-α. Prepro-Hypocretin and Hypocretin receptor 2 (HcrtR2) can be detected at a very low level in rat B35 neuroblastoma cells. In response to TNF-α, Prepro-Hypocretin mRNA and protein levels are down-regulated, and also HcrtR2 protein level is down-regulated in B35 cells. To investigate the mechanism, exogenous rat Prepro-Hypocretin and rat HcrtR2 were overexpressed in B35 cells. In response to TNF-α, protein and mRNA of Prepro-Hypocretin are significantly decreased (by 93% and 94%, respectively), and the half-life of Prepro-Hypocretin mRNA is decreased in a time- and dose-dependent manner. The level of HcrtR2 mRNA level is not affected by TNF-α treatment; however, HcrtR2 protein level is significantly decreased (by 86%) through ubiquitination in B35 cells treated with TNF-α. Downregulation of cellular inhibitor of apoptosis protein-1 and -2 (cIAP-1 and -2) abrogates the HcrtR2 ubiquitination induced by TNF-α. The control green fluorescent protein (GFP) expression is not affected by TNF-α treatment. These studies demonstrate that TNF-α can impair the function of the Hypocretin system by reducing the levels of both Prepro-Hypocretin and HcrtR2. Copyright © 2010 Elsevier B.V. All rights reserved.

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

Energy Technology Data Exchange (ETDEWEB)

Su, L.-N.; Little, J.B. (Harvard School of Public Health, Boston, MA (United States))

1992-08-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author).

Transformation and radiosensitivity of human diploid skin fibroblasts transfected with activated RAS oncogene and SV40 T-antigen

International Nuclear Information System (INIS)

Su, L.-N.; Little, J.B.

1992-01-01

Three normal human diploid cell strains were transfected with an activated Ha-ras oncogene (EJ ras) or SV40 T-antigen. Multiple clones were examined for morphological alterations, growth requirements, ability to grow under anchorage independent conditions, immortality and tumorigenicity in nude mice. Clones expressing SV40 T-antigen alone or in combination with ras protein p21 were significantly radioresistant as compared with their parent cells or clones transfected with the neo gene only. This radioresistant phenotype persisted in post-crisis, immortalized cell lines. These data suggest that expression of the SV40 T-antigen but not activated Ha-ras plays an important role in the radiosensitivity of human diploid cells. The radioresistant phenotype in SV40 T transfected cells was not related to the enhanced level of genetic instability seen in pre-crisis and newly immortalized cells, nor to the process of immortalization itself. (author)

A study of a steering system algorithm for pleasure boats based on stability analysis of a human-machine system model

International Nuclear Information System (INIS)

Ikeda, Fujio; Toyama, Shigehiro; Ishiduki, Souta; Seta, Hiroaki

2016-01-01

Maritime accidents of small ships continue to increase in number. One of the major factors is poor manoeuvrability of the Manual Hydraulic Steering Mechanism (MHSM) in common use. The manoeuvrability can be improved by using the Electronic Control Steering Mechanism (ECSM). This paper conducts stability analyses of a pleasure boat controlled by human models in view of path following on a target course, in order to establish design guidelines for the ECSM. First, to analyse the stability region, the research derives the linear approximated model in a planar global coordinate system. Then, several human models are assumed to develop closed-loop human-machine controlled systems. These human models include basic proportional, derivative, integral and time-delay actions. The stability analysis simulations for those human-machine systems are carried out. The results show that the stability region tends to spread as a ship's velocity increases in the case of the basic proportional human model. The derivative action and time-delay action of human models are effective in spreading the stability region in their respective ranges of frontal gazing points. (paper)

A study of a steering system algorithm for pleasure boats based on stability analysis of a human-machine system model

Science.gov (United States)

Ikeda, Fujio; Toyama, Shigehiro; Ishiduki, Souta; Seta, Hiroaki

2016-09-01

Maritime accidents of small ships continue to increase in number. One of the major factors is poor manoeuvrability of the Manual Hydraulic Steering Mechanism (MHSM) in common use. The manoeuvrability can be improved by using the Electronic Control Steering Mechanism (ECSM). This paper conducts stability analyses of a pleasure boat controlled by human models in view of path following on a target course, in order to establish design guidelines for the ECSM. First, to analyse the stability region, the research derives the linear approximated model in a planar global coordinate system. Then, several human models are assumed to develop closed-loop human-machine controlled systems. These human models include basic proportional, derivative, integral and time-delay actions. The stability analysis simulations for those human-machine systems are carried out. The results show that the stability region tends to spread as a ship's velocity increases in the case of the basic proportional human model. The derivative action and time-delay action of human models are effective in spreading the stability region in their respective ranges of frontal gazing points.

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement*

Science.gov (United States)

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-01-01

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. PMID:26887951

A Point Mutation in the Exon Junction Complex Factor Y14 Disrupts Its Function in mRNA Cap Binding and Translation Enhancement.

Science.gov (United States)

Chuang, Tzu-Wei; Lee, Kuo-Ming; Lou, Yuan-Chao; Lu, Chia-Chen; Tarn, Woan-Yuh

2016-04-15

Eukaryotic mRNA biogenesis involves a series of interconnected steps mediated by RNA-binding proteins. The exon junction complex core protein Y14 is required for nonsense-mediated mRNA decay (NMD) and promotes translation. Moreover, Y14 binds the cap structure of mRNAs and inhibits the activity of the decapping enzyme Dcp2. In this report, we show that an evolutionarily conserved tryptophan residue (Trp-73) of Y14 is critical for its binding to the mRNA cap structure. A Trp-73 mutant (W73V) bound weakly to mRNAs and failed to protect them from degradation. However, this mutant could still interact with the NMD and mRNA degradation factors and retained partial NMD activity. In addition, we found that the W73V mutant could not interact with translation initiation factors. Overexpression of W73V suppressed reporter mRNA translation in vitro and in vivo and reduced the level of a set of nascent proteins. These results reveal a residue of Y14 that confers cap-binding activity and is essential for Y14-mediated enhancement of translation. Finally, we demonstrated that Y14 may selectively and differentially modulate protein biosynthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunodetection of Fasciola gigantica circulating antigen in sera of infected individuals for laboratory diagnosis of human fascioliasis.

Science.gov (United States)

Attallah, Abdelfattah M; Bughdadi, Faisal A; El-Shazly, Atef M; Ismail, Hisham

2013-10-01

Currently, the laboratory diagnosis of human fascioliasis is based on the parasitological examination of parasite eggs in stool specimens and serological detection of specific antibodies in serum samples, which are often unreliable diagnostic approaches. Ideally, a sensitive and specific diagnostic test for Fasciola infection should be based on the detection of circulating Fasciola antigen, which implies active infection. Here, a 27-kDa-molecular-mass antigen was identified in a Fasciola gigantica adult worm antigen preparation, excretory-secretory products, and sera from F. gigantica-infected individuals, and it was not detected in antigenic extracts of other parasites and sera from noninfected individuals. The target antigen was isolated and partially characterized as a protein. Immunoperoxidase staining located the target epitope within teguments and guts of F. gigantica adult worms. The performance characteristics of a newly developed enzyme-linked immunosorbent assay (ELISA) based on F. gigantica circulating antigen detection in serum (FgCA-27 ELISA) were investigated using sera of 120 parasitologically diagnosed F. gigantica-infected individuals and 80 noninfected individuals. The area under the receiving operating characteristic (ROC) curve (AUC) for ELISA was significantly high (AUC = 0.961, P 93%), and a significant correlation (r = 0.715, P fascioliasis.

The raccoon polyomavirus genome and tumor antigen transcription are stable and abundant in neuroglial tumors.

Science.gov (United States)

Brostoff, Terza; Dela Cruz, Florante N; Church, Molly E; Woolard, Kevin D; Pesavento, Patricia A

2014-11-01

Raccoon polyomavirus (RacPyV) is associated with 100% of neuroglial tumors in free-ranging raccoons. Other tumor-associated polyomaviruses (PyVs), including simian virus 40 (SV40), murine PyV, and Merkel cell PyV, are found integrated in the host genome in neoplastic cells, where they constitutively express splice variants of the tumor antigen (TAg) gene. We have previously reported that RacPyV exists only as an episome (nonintegrated) in neuroglial tumors. Here, we have investigated TAg transcription in primary tumor tissue by transcriptome analysis, and we identified the alternatively spliced TAg transcripts for RacPyV. We also determined that TAg was highly transcribed relative to host cellular genes. We further colocalized TAg DNA and mRNA by in situ hybridization and found that the majority of tumor cells showed positive staining. Lastly, we examined the stability of the viral genome and TAg transcription by quantitative reverse transcriptase PCR in cultured tumor cells in vitro and in a mouse xenograft model. When tumor cells were cultured in vitro, TAg transcription increased nearly 2 log-fold over that of parental tumor tissue by passage 17. Both episomal viral genome and TAg transcription were faithfully maintained in culture and in tumors arising from xenotransplantation of cultured cells in mice. This study represents a minimal criterion for RacPyV's association with neuroglial tumors and a novel mechanism of stability for a polyomavirus in cancer. The natural cycle of polyomaviruses in mammals is to persist in the host without causing disease, but they can cause cancer in humans or in other animals. Because this is an unpredictable and rare event, the oncogenic potential of polyomavirus is primarily evaluated in laboratory animal models. Recently, raccoon polyomavirus (RacPyV) was identified in neuroglial tumors of free-ranging raccoons. Viral copy number was consistently high in these tumors but was low or undetectable in nontumor tissue or in

The role of the human leukocyte antigen system in retinopathy of prematurity: a pilot study.

Science.gov (United States)

Flor-de-Lima, Filipa; Rocha, Gustavo; Proença, Elisa; Tafulo, Sandra; Freitas, Fátima; Guimarães, Hercília

2013-12-01

To assess the association between the human leukocyte antigen system and retinopathy of prematurity. Neonates of prematurity. A large-scale population-based study should be performed to clarify this association. ©2013 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

Antigen Cross-Presentation of Immune Complexes

Science.gov (United States)

Platzer, Barbara; Stout, Madeleine; Fiebiger, Edda

2014-01-01

The ability of dendritic cells (DCs) to cross-present tumor antigens has long been a focus of interest to physicians, as well as basic scientists, that aim to establish efficient cell-based cancer immune therapy. A prerequisite for exploiting this pathway for therapeutic purposes is a better understanding of the mechanisms that underlie the induction of tumor-specific cytotoxic T-lymphocyte (CTL) responses when initiated by DCs via cross-presentation. The ability of humans DC to perform cross-presentation is of utmost interest, as this cell type is a main target for cell-based immunotherapy in humans. The outcome of a cross-presentation event is guided by the nature of the antigen, the form of antigen uptake, and the subpopulation of DCs that performs presentation. Generally, CD8α+ DCs are considered to be the most potent cross-presenting DCs. This paradigm, however, only applies to soluble antigens. During adaptive immune responses, immune complexes form when antibodies interact with their specific epitopes on soluble antigens. Immunoglobulin G (IgG) immune complexes target Fc-gamma receptors on DCs to shuttle exogenous antigens efficiently into the cross-presentation pathway. This receptor-mediated cross-presentation pathway is a well-described route for the induction of strong CD8+ T cell responses. IgG-mediated cross-presentation is intriguing because it permits the CD8− DCs, which are commonly considered to be weak cross-presenters, to efficiently cross-present. Engaging multiple DC subtypes for cross-presentation might be a superior strategy to boost CTL responses in vivo. We here summarize our current understanding of how DCs use IgG-complexed antigens for the efficient induction of CTL responses. Because of its importance for human cell therapy, we also review the recent advances in the characterization of cross-presentation properties of human DC subsets. PMID:24744762

CD Nomenclature 2015: Human Leukocyte Differentiation Antigen Workshops as a Driving Force in Immunology

Czech Academy of Sciences Publication Activity Database

Engel, P.; Boumsell, L.; Balderas, R.; Gattei, V.; Hořejší, Václav; Jin, B.Q.; Malavasi, F.; Mortari, F.; Schwartz-Albiez, R.; Stockinger, H.; van Zelm, M.C.; Zola, H.; Clark, G.

2015-01-01

Roč. 165, č. 10 (2015), s. 4555-4563 ISSN 0022-1767 Institutional support: RVO:68378050 Keywords : CD nomenclature, , * leukocyte antigens * HLDA workshop Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.985, year: 2015

Cloning of human tumor necrosis factor (TNF) receptor cDNA and expression of recombinant soluble TNF-binding protein

International Nuclear Information System (INIS)

Gray, P.W.; Barrett, K.; Chantry, D.; Turner, M.; Feldmann, M.

1990-01-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extracellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10 -9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ)

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

Science.gov (United States)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Human sensitization to Prosopis juliflora antigen in Saudi Arabia.

Science.gov (United States)

Al-Frayh, A; Hasnain, S M; Gad-El-Rab, M O; Al-Turki, T; Al-Mobeireek, K; Al-Sedairy, S T

1999-01-01

Allergenicity to Prosopis juliflora pollen antigen has been reported from only a few countries, including the US, South Africa, India and Kuwait. In some parts of Saudi Arabia, species of Prosopis have been introduced by the millions as roadside ornamentation. There appear to be four flowering seasons during which pollen grains float in all directions. However, the role of Prosopis pollen as the sensitizing and/or triggering agent of allergic asthma and/or rhinitis in the Kingdom has never been evaluated. A total of 473 allergic patients suffering from bronchial asthma in four different geographical regions (Abha, Qassim, Hofuf and Gizan), and attending allergy clinics and chest disease centers of university and Ministry of Health hospitals in the region were tested for immediate hypersensitivity reaction to Prosopis juliflora allergens. Airborne pollen grains at one center were also studied for one full year, using volumetric sampling techniques. A total of 76.1% patients in Qassim, 37.5% in Gizan, 29% in Abha and 11% in Hofuf reacted positively to Prosopis antigen. Multiple sensitivities to other pollen antigens were detected in all patients. The level of airborne Prosopis pollen detected in Gizan exceeded 90 grains m -3 of air. In view of the documented evidence of Prosopis-involved allergenicity, the role of Prosopis pollen as a sensitizing factor in Saudi Arabia has been confirmed. However, the cause of elicitation of symptoms in many multiple sensitive patients, together with the question of cross-reactivities, needs thorough and detailed investigation. In vitro confirmation of all positive results is also required to incriminate Prosopis as one of the major allergens in parts of Saudi Arabia.

Hydrogen sulfide upregulated mRNA expressions of sodium bicarbonate cotransporter1, trefoil factor1 and trefoil factor2 in gastric mucosa in rats.

Science.gov (United States)

Cheraghi, Parisa; Mard, Seyyed Ali; Nagi, Tahereh

2016-01-01

Hydrogen sulfide (H 2 S) has been shown to protect the gastric mucosa through several protective mechanisms but till now its effect on mRNA expression of sodium bicarbonate cotransporter 1 (NBC1), trefoil factor1 (TFF1) and trefoil factor2 (TFF2) was not investigated. This study was aimed to evaluate the effect of H 2 S on mRNA expression of NBC1, TFF1 and TFF2 in rat gastric mucosa in response to gastric distention. Thirty two rats were randomly assigned into four equal groups. They were control (C), distention (D), propargylglycine (PAG)-, and NaHS-treated groups. To evaluate the effect of exogenous and endogenous H 2 S on gene expression of NBC1, TFF1 and TFF2, two groups of rats were received H 2 S donor, intra-peritoneal NaHS (80 µg Kg -1 ), and PAG (50 mg kg -1 ), accompanied to stimulate the gastric acid secretion, respectively. Under general anesthesia and laparotomy, a catheter was inserted into the stomach through duodenum for instillation of isotonic saline for gastric distention. Ninety min after beginning the experiment, animals were sacrificed and the gastric mucosa was collected to determine total acid content of gastric effluents and to quantify the mRNA expression of studied genes by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that A) gastric distention increased the level of mRNA expressions of NBC1, TFF1 and TFF2; B) these levels in NaHS-treated rats were significantly higher than those in Distention group; and C) PAG decreased the expression levels of NBC1 and TFF1. The Findings showed H 2 S upregulated gene expression of NBC1, TFF1 and TFF2 in gastric mucosa.

Human papillomavirus-exposed Langerhans cells are activated by stabilized Poly-I:C.

Science.gov (United States)

Da Silva, Diane M; Woodham, Andrew W; Rijkee, Laurie K; Skeate, Joseph G; Taylor, Julia R; Koopman, Maaike E; Brand, Heike E; Wong, Michael K; McKee, Greg M; Salazar, Andres M; Kast, W Martin

2015-12-01

Human papillomaviruses (HPV) establish persistent infections because of evolved immune evasion mechanisms, particularly HPV-mediated suppression of the immune functions of Langerhans cells (LC), the antigen presenting cells of the epithelium. Polyinosinic-polycytidilic acid (Poly-I:C) is broadly immunostimulatory with the ability to enhance APC expression of costimulatory molecules and inflammatory cytokines resulting in T cell activation. Here we investigated the activation of primary human LC derived from peripheral blood monocytes after exposure to HPV16 virus like particles followed by treatment with stabilized Poly-I:C compounds (s-Poly-I:C), and their subsequent induction of HPV16-specific T cells. Our results indicate that HPV16 particles alone were incapable of inducing LC activation as demonstrated by the lack of costimulatory molecules, inflammatory cytokines, chemokine-directed migration, and HPV16-specific CD8 + T cells in vitro . Conversely, s-Poly-I:C caused significant upregulation of costimulatory molecules and induction of chemokine-directed migration of LC that were pre-exposed to HPV16. In HLA-A*0201-positive donors, s-Poly-I:C treatment was able to induce CD8 + T cell immune responses against HPV16-derived peptides. Thus, s-Poly-I:C compounds are attractive for translation into therapeutics in which they could potentially mediate clearance of persistent HPV infection.

Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigree.

Science.gov (United States)

Yu, T; Wang, X; Ding, Q; Fu, Q; Dai, J; Lu, Y; Xi, X; Wang, H

2009-11-01

Factor VII deficiency which transmitted as an autosomal recessive disorder is a rare haemorrhagic condition. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a Chinese pedigree with FVII deficiency. The proband was diagnosed as inherited coagulation FVII deficiency by reduced plasma levels of FVII activity (4.4%) and antigen (38.5%). All nine exons and their flanking sequence of F7 gene were amplified by polymerase chain reaction (PCR) for the proband and the PCR products were directly sequenced. The compound heterozygous mutations of F7 (NM_000131.3) c.572-1G>A and F7 (NM_000131.3) c.1165T>G; p.Cys389Gly were identified in the proband's F7 gene. To investigate the splicing patterns associated with F7 c.572-1G>A, ectopic transcripts in leucocytes of the proband were analyzed. F7 minigenes, spanning from intron 4 to intron 7 and carrying either an A or a G at position -1 of intron 5, were constructed and transiently transfected into human embryonic kidney (HEK) 293T cells, followed by RT-PCR analysis. The aberrant transcripts from the F7 c.572-1G>A mutant allele were not detected by ectopic transcription study. Sequencing of the RT-PCR products from the mutant transfectant demonstrated the production of an erroneously spliced mRNA with exon 6 skipping, whereas a normal splicing occurred in the wide type transfectant. The aberrant mRNA produced from the F7 c.572-1G>A mutant allele is responsible for the factor VII deficiency in this pedigree.

Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

International Nuclear Information System (INIS)

Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

1987-01-01

The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of 3 H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes

Monoclonal antibodies to antigens on human neutrophils, activated T lymphocytes, and acute leukemia blast cells

Energy Technology Data Exchange (ETDEWEB)

Miterev, G.Yu.; Burova, G.F.; Puzhitskaya, M.S.; Danilevich, S.V.; Bulycheva, T.I.

1987-11-01

The authors describe the production of two mouse hybridomas secreting monoclonal antibodies to antigenic determinants of the surface membranes of human neutrophils, activated T lymphocytes, and acute leukemic blast cells. The degree of lymphocyte stimulation was estimated from incorporation of /sup 3/H-thymidine with parallel microculture. Monoclonal antibodies of supernatants of hybridoma cultures shown here reacted in both immunofluorescence test and cytotoxicity test with surface membrane antigens on the majority of neutrophils and PHA-activated peripheral blood lymphocytes from healthy subjects, but did not give positive reactions with unactivated lymphocytes, adherent monocytes, erythrocytes, and alloantigen-stimulated lymphocytes.

Ta1, a novel 105 KD human T cell activation antigen defined by a monoclonal antibody.

Science.gov (United States)

Fox, D A; Hussey, R E; Fitzgerald, K A; Acuto, O; Poole, C; Palley, L; Daley, J F; Schlossman, S F; Reinherz, E L

1984-09-01

By using a murine monoclonal antibody produced against an IL 2-dependent human T cell line, we defined a T lineage-specific molecule, termed Ta1, that is expressed strongly on activated T lymphocytes of both the T4 and T8 subsets, as well as on T cell lines and clones, but only weakly on a fraction of resting T cells. SDS-PAGE analysis of immunoprecipitates from 125I-labeled, activated T cells demonstrates a single major band of apparent m.w. 105 KD under both reducing and nonreducing conditions. Unlike anti-IL 2 receptor antibodies, anti-Ta1 does not inhibit T cell proliferative responses to mitogen, antigen, or IL 2-containing medium. Moreover, anti-Ta1 has no effect on T cell-mediated cytotoxicity. Ta1 appears to be a novel human T cell-specific activation antigen that may serve as a useful marker of T cell activation in human disease.

Selection of antigenic markers on a GFP-C{kappa} fusion scaffold with high sensitivity by eukaryotic ribosome display

Energy Technology Data Exchange (ETDEWEB)

Yongmin, Yang [Institute of Genetics, San Diego, CA 92121-2233 (United States); IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States); Barankiewicz, Teresa J [Institute of Genetics, San Diego, CA 92121-2233 (United States); IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States); Mingyue, He [Babraham Institute, Cambridge CB2 4AT (United Kingdom); Taussig, Michael J [Babraham Institute, Cambridge CB2 4AT (United Kingdom); Chen, Swey-Shen [Institute of Genetics, San Diego, CA 92121-2233 (United States) and IgE Therapeutics, Inc., San Diego, CA 92121-2233 (United States)

2007-07-27

Ribosome display is a cell-free system permitting gene selection through the physical association of genetic material (mRNA) and its phenotypic (protein) product. While often used to select single-chain antibodies from large libraries by panning against immobilized antigens, we have adapted ribosome display for use in the 'reverse' format in order to select high affinity antigenic determinants against solid-phase antibody. To create an antigenic scaffold, DNA encoding green fluorescent protein (GFP) was fused to a light chain constant domain (C{kappa}) with stop codon deleted, and with 5' signals (T7 promoter, Kozak) enabling coupled transcription/translation in a eukaryotic cell-free system. Epitopes on either GFP (5') or C{kappa} (3') were selected by anti-GFP or anti-C{kappa} antibodies, respectively, coupled to magnetic beads. After selection, mRNA was amplified directly from protein-ribosome-mRNA (PRM) complexes by in situ PCR followed by internal amplification and reassembly PCR. As little as 10 fg of the 1 kb DNA construct, i.e. approximately 7500 molecules, could be recovered following a single round of interaction with solid-phase anti-GFP antibody. This platform is highly specific and sensitive for the antigen-antibody interaction and may permit selection and reshaping of high affinity antigenic variants of scaffold proteins.

The use of /sup 125/I recombinant DNA/sub 125/ derived human erythropoietin (R-HuEPO) as a replacement for /sup 125/I human urinary epo as tracer antigen in a radioimmunoassay for human epo

International Nuclear Information System (INIS)

Cotes, P.M.; Tam, R.C.; GainesDas, R.E.

1987-01-01

This paper represents evidence that in a radioimmunoassay for human erythropoietin, recombinant DNA derived human erythropoietin can replace highly purified human urinary erythropoietin in the preparation of radioiodinated tracer antigen

Rifampin modulation of xeno- and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes.

Science.gov (United States)

Gufford, Brandon T; Robarge, Jason D; Eadon, Michael T; Gao, Hongyu; Lin, Hai; Liu, Yunlong; Desta, Zeruesenay; Skaar, Todd C

2018-04-01

Rifampin is a pleiotropic inducer of multiple drug metabolizing enzymes and transporters. This work utilized a global approach to evaluate rifampin effects on conjugating enzyme gene expression with relevance to human xeno- and endo-biotic metabolism. Primary human hepatocytes from 7 subjects were treated with rifampin (10 μmol/L, 24 hours). Standard methods for RNA-seq library construction, EZBead preparation, and NextGen sequencing were used to measure UDP-glucuronosyl transferase UGT, sulfonyltransferase SULT, N acetyltransferase NAT, and glutathione-S-transferase GST mRNA expression compared to vehicle control (0.01% MeOH). Rifampin-induced (>1.25-fold) mRNA expression of 13 clinically important phase II drug metabolizing genes and repressed (>1.25-fold) the expression of 3 genes ( P  accounting for simultaneous induction of both CYP3A4 and UGT1A4 predicted a ~10-fold decrease in parent midazolam exposure with only a ~2-fold decrease in midazolam N-glucuronide metabolite exposure. These data reveal differential effects of rifampin on the human conjugating enzyme transcriptome and potential associations with miRNAs that form the basis for future mechanistic studies to elucidate the interplay of conjugating enzyme regulatory elements.

p,p'-Dichlorodiphenyltrichloroethane (p,p'-DDT) and p,p'-dichlorodiphenyldichloroethylene (p,p'-DDE) repress prostate specific antigen levels in human prostate cancer cell lines.

Science.gov (United States)

Wong, Lilian I L; Labrecque, Mark P; Ibuki, Naokazu; Cox, Michael E; Elliott, John E; Beischlag, Timothy V

2015-03-25

Despite stringent restrictions on their use by many countries since the 1970s, the endocrine disrupting chemicals, DDT and DDE are still ubiquitous in the environment. However, little attention has been directed to p,p'-DDT and the anti-androgen, p,p'-DDE on androgen receptor (AR) target gene transcription in human cells. Inhibitors of androgenic activity may have a deleterious clinical outcome in prostate cancer screens and progression, therefore we determined whether environmentally relevant concentrations of p,p'-DDT and p,p'-DDE negatively impact AR-regulated expression of prostate-specific antigen (PSA), and other AR target genes in human LNCaP and VCaP prostate cancer cells. Quantitative real-time PCR and immuno-blotting techniques were used to measure intracellular PSA, PSMA and AR mRNA and protein levels. We have shown for the first time that p,p'-DDT and p,p'-DDE repressed R1881-inducible PSA mRNA and protein levels in a dose-dependent manner. Additionally, we used the fully automated COBAS PSA detection system to determine that extracellular PSA levels were also significantly repressed. These chemicals achieve this by blocking the recruitment of AR to the PSA promoter region at 10 μM, as demonstrated by the chromatin immunoprecipitation (ChIP) in LNCaP cells. Both p,p'-DDT and p,p'-DDE repressed R1881-inducible AR protein accumulation at 10 μM. Thus, we conclude that men who have been exposed to either DDT or DDE may produce a false-negative PSA test when screening for prostate cancer, resulting in an inaccurate clinical diagnosis. More importantly, prolonged exposure to these anti-androgens may mimic androgen ablation therapy in individuals with prostate cancer, thus exacerbating the condition by inadvertently forcing adaptation to this stress early in the disease. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Growth differentiation factor 3 is induced by bone morphogenetic protein 6 (BMP-6) and BMP-7 and increases luteinizing hormone receptor messenger RNA expression in human granulosa cells.

Science.gov (United States)

Shi, Jia; Yoshino, Osamu; Osuga, Yutaka; Akiyama, Ikumi; Harada, Miyuki; Koga, Kaori; Fujimoto, Akihisa; Yano, Tetsu; Taketani, Yuji

2012-04-01

To examine the relevance of growth differentiation factor 3 (GDF-3) and bone morphogenetic protein (BMP) cytokines in human ovary. Molecular studies. Research laboratory. Eight women undergoing salpingo-oophorectomy and 30 women undergoing ovarian stimulation for in vitro fertilization. Localizing GDF-3 protein in human ovaries; granulosa cells (GC) cultured with GDF-3, BMP-6, or BMP-7 followed by RNA extraction. The localization of GDF-3 protein in normal human ovaries via immunohistochemical analysis, GDF-3 messenger RNA (mRNA) expression evaluation via quantitative real-time reverse transcription and polymerase chain reaction (RT-PCR), and evaluation of the effect of GDF-3 on leuteinizing hormone (LH) receptor mRNA expression via quantitative real-time RT-PCR. In the ovary, BMP cytokines, of the transforming growth factor beta (TGF-β) superfamily, are known as a luteinization inhibitor by suppressing LH receptor expression in GC. Growth differentiation factor 3, a TGF-β superfamily cytokine, is recognized as an inhibitor of BMP cytokines in other cells. Immunohistochemical analysis showed that GDF-3 was strongly detected in the GC of antral follicles. An in vitro assay revealed that BMP-6 or BMP-7 induced GDF-3 mRNA in GC. Also, GDF-3 increased LH receptor mRNA expression and inhibited the effect of BMP-7, which suppressed the LH receptor mRNA expression in GC. GDF-3, induced with BMP-6 and BMP-7, might play a role in folliculogenesis by inhibiting the effect of BMP cytokines. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii.

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Schmid-Siegert, Emanuel; Richard, Sophie; Luraschi, Amanda; Mühlethaler, Konrad; Pagni, Marco; Hauser, Philippe M

2017-11-07

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different. IMPORTANCE Pneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms

A novel link between Sus1 and the cytoplasmic mRNA decay machinery suggests a broad role in mRNA metabolism

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Llopis Ana

2010-03-01

Full Text Available Abstract Background Gene expression is achieved by the coordinated action of multiple factors to ensure a perfect synchrony from chromatin epigenetic regulation through to mRNA export. Sus1 is a conserved mRNA export/transcription factor and is a key player in coupling transcription initiation, elongation and mRNA export. In the nucleus, Sus1 is associated to the transcriptional co-activator SAGA and to the NPC associated complex termed TREX2/THSC. Through these associations, Sus1 mediates the nuclear dynamics of different gene loci and facilitate the export of the new transcripts. Results In this study, we have investigated whether the yeast Sus1 protein is linked to factors involved in mRNA degradation pathways. We provide evidence for genetic interactions between SUS1 and genes coding for components of P-bodies such as PAT1, LSM1, LSM6 and DHH1. We demonstrate that SUS1 deletion is synthetic lethal with 5'→3' decay machinery components LSM1 and PAT1 and has a strong genetic interaction with LSM6 and DHH1. Interestingly, Sus1 overexpression led to an accumulation of Sus1 in cytoplasmic granules, which can co-localise with components of P-bodies and stress granules. In addition, we have identified novel physical interactions between Sus1 and factors associated to P-bodies/stress granules. Finally, absence of LSM1 and PAT1 slightly promotes the Sus1-TREX2 association. Conclusions In this study, we found genetic and biochemical association between Sus1 and components responsible for cytoplasmic mRNA metabolism. Moreover, Sus1 accumulates in discrete cytoplasmic granules, which partially co-localise with P-bodies and stress granules under specific conditions. These interactions suggest a role for Sus1 in gene expression during cytoplasmic mRNA metabolism in addition to its nuclear function.

Antigen recognition by IgG4 antibodies in human trichinellosis

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Pinelli E.

2001-06-01

Full Text Available The antibody isotype response to Trichinella spiralis excretory/secretory (ES products of muscle larva was examined using sera from patients with confirmed trichinellosis. Using Western blots we identify components of the ES antigen that are recognized by IgM and IgG antibodies. A 45 kDa component was strongly recognized by different antibody classes and subclasses. We observed a 45 kDa-specific lgG4 response that was detected exclusively using sera of patients with trichinellosis and not of patients with echinococcosis, filariasis, cysticercosis, ascariasis, strongyloidiasis or toxocariasis. These results are relevant for the diagnosis of human trichinellosis.

Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

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Chantarawong, Wipa [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Inter Departmental Multidisciplinary Graduate Program in Bioscience, Faculty of Science, Kasetsart University, Bangkok (Thailand); Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan); Pradermwong, Kantimanee [Department of Zoology, Faculty of Science, Kasetsart University, Bangkok (Thailand); Shibahara, Shigeki, E-mail: shibahar@med.tohoku.ac.jp [Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Sendai (Japan)

2014-11-28

Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure.

Microphthalmia-associated transcription factor as the molecular target of cadmium toxicity in human melanocytes

International Nuclear Information System (INIS)

Chantarawong, Wipa; Takeda, Kazuhisa; Sangartit, Weerapon; Yoshizawa, Miki; Pradermwong, Kantimanee; Shibahara, Shigeki

2014-01-01

Highlights: • In human melanocytes, cadmium decreases the expression of MITF-M and tyrosinase and their mRNAs. • In human melanoma cells, cadmium decreases the expression of MITF-M protein and tyrosinase mRNA. • Expression of MITF-H is less sensitive to cadmium toxicity in melanocyte-linage cells. • Cadmium does not decrease the expression of MITF-H in retinal pigment epithelial cells. • MITF-M is the molecular target of cadmium toxicity in melanocytes. - Abstract: Dietary intake of cadmium is inevitable, causing age-related increase in cadmium accumulation in many organs, including hair, choroid and retinal pigment epithelium (RPE). Cadmium has been implicated in the pathogenesis of hearing loss and macular degeneration. The functions of cochlea and retina are maintained by melanocytes and RPE, respectively, and the differentiation of these pigment cells is regulated by microphthalmia-associated transcription factor (MITF). In the present study, we explored the potential toxicity of cadmium in the cochlea and retina by using cultured human melanocytes and human RPE cell lines. MITF consists of multiple isoforms, including melanocyte-specific MITF-M and widely expressed MITF-H. Levels of MITF-M protein and its mRNA in human epidermal melanocytes and HMV-II melanoma cells were decreased significantly by cadmium. In parallel with the MITF reduction, mRNA levels of tyrosinase, the key enzyme of melanin biosynthesis that is regulated by MITF-M, were also decreased. In RPE cells, however, the levels of total MITF protein, constituting mainly MITF-H, were not decreased by cadmium. We thus identify MITF-M as the molecular target of cadmium toxicity in melanocytes, thereby accounting for the increased risk of disability from melanocyte malfunction, such as hearing and vision loss among people with elevated cadmium exposure

Common acute lymphoblastic leukemia antigen: partial characterization by in vivo labeling and isolation of its messenger RNA

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Heinsohn, S.; Kabisch, H.

1987-01-01

Common acute lymphoblastic leukemia (ALL) antigen (CALLA)-like proteins were detected by in vivo labeling experiments carried out with human lymphoblastoid cell line KM3 and also in cell-free translation, directed by CALLA-specific mRNA prepared from immunoadsorbed KM3 polysomes. The CALLA-like structure found in both systems shows an Mr of 95kDa. Additional CALLA-like proteins could be identified in the in vivo experiments with calculated Mrs of 40kDa in the cells and 85 and 38kDa in the culture medium. In the cell-free translation system, an additional product of Mr 80kDa could be detected

Amitriptyline induces brain-derived neurotrophic factor (BDNF) mRNA expression through ERK-dependent modulation of multiple BDNF mRNA variants in primary cultured rat cortical astrocytes and microglia.

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Hisaoka-Nakashima, Kazue; Kajitani, Naoto; Kaneko, Masahiro; Shigetou, Takahiro; Kasai, Miho; Matsumoto, Chie; Yokoe, Toshiki; Azuma, Honami; Takebayashi, Minoru; Morioka, Norimitsu; Nakata, Yoshihiro

2016-03-01

A significant role of brain-derived neurotrophic factor (BDNF) has been previously implicated in the therapeutic effect of antidepressants. To ascertain the contribution of specific cell types in the brain that produce BDNF following antidepressant treatment, the effects of the tricyclic antidepressant amitriptyline on rat primary neuronal, astrocytic and microglial cortical cultures were examined. Amitriptyline increased the expression of BDNF mRNA in astrocytic and microglial cultures but not neuronal cultures. Antidepressants with distinct mechanisms of action, such as clomipramine, duloxetine and fluvoxamine, also increased BDNF mRNA expression in astrocytic and microglial cultures. There are multiple BDNF mRNA variants (exon I, IIA, IV and VI) expressed in astrocytes and microglia and the variant induced by antidepressants has yet to be elaborated. Treatment with antidepressants increased the expression of exon I, IV and VI in astrocyte and microglia. Clomipramine alone significantly upregulated expression of exon IIA. The amitriptyline-induced expression of both total and individual BDNF mRNA variants (exon I, IV and VI) were blocked by MEK inhibitor U0126, indicating MEK/ERK signaling is required in the expression of BDNF. These findings indicate that non-neural cells are a significant target of antidepressants and further support the contention that glial production of BDNF is crucial role in the therapeutic effect of antidepressants. The current data suggest that targeting of glial function could lead to the development of antidepressants with a truly novel mechanism of action. Copyright © 2016 Elsevier B.V. All rights reserved.

Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

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Munisvaradass, Rusheni; Kumar, Suresh; Govindasamy, Chandramohan; Alnumair, Khalid S.; Mok, Pooi Ling

2017-01-01

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours. PMID:28885562

Contribution of enhanced engagement of antigen presentation machinery to the clinical immunogenicity of a human interleukin (IL)-21 receptor-blocking therapeutic antibody.

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Xue, L; Hickling, T; Song, R; Nowak, J; Rup, B

2016-01-01

Reliable risk assessment for biotherapeutics requires accurate evaluation of risk factors associated with immunogenicity. Immunogenicity risk assessment tools were developed and applied to investigate the immunogenicity of a fully human therapeutic monoclonal antibody, ATR-107 [anti-interleukin (IL)-21 receptor] that elicited anti-drug antibodies (ADA) in 76% of healthy subjects in a Phase 1 study. Because the ATR-107 target is expressed on dendritic cells (DCs), the immunogenicity risk related to engagement with DC and antigen presentation pathways was studied. Despite the presence of IL-21R on DCs, ATR-107 did not bind to the DCs more extensively than the control therapeutic antibody (PF-1) that had elicited low clinical ADA incidence. However, ATR-107, but not the control therapeutic antibody, was translocated to the DC late endosomes, co-localized with intracellular antigen-D related (HLA-DR) molecules and presented a dominant T cell epitope overlapping the complementarity determining region 2 (CDR2) of the light chain. ATR-107 induced increased DC activation exemplified by up-regulation of DC surface expression of CD86, CD274 (PD-L1) and CD40, increased expansion of activated DC populations expressing CD86(hi), CD40(hi), CD83(hi), programmed death ligand 1 (PD-L1)(hi), HLA-DR(hi) or CCR7(hi), as well as elevated secretion of tumour necrosis factor (TNF)-α by DCs. DCs exposed to ATR-107 stimulated an autologous T cell proliferative response in human donor cells, in concert with the detection of immunoglobulin (Ig)G-type anti-ATR-107 antibody response in clinical samples. Collectively, the enhanced engagement of antigen presentation machinery by ATR-107 was suggested. The approaches and findings described in this study may be relevant to identifying lower immunogenicity risk targets and therapeutic molecules. © 2015 British Society for Immunology.

mRNA Transcriptomics of Galectins Unveils Heterogeneous Organization in Mouse and Human Brain

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Sebastian John

2016-12-01

Full Text Available Background: Galectins, a family of non-classically secreted, β-galactoside binding proteins is involved in several brain disorders; however no systematic knowledge on the normal neuroanatomical distribution and functions of galectins exits. Hence, the major purpose of this study was to understand spatial distribution and predict functions of galectins in brain and also compare the degree of conservation vs. divergence between mouse and human species. The latter objective was required to determine the relevance and appropriateness of studying galectins in mouse brain which may ultimately enable us to extrapolate the findings to human brain physiology and pathologies.Results: In order to fill this crucial gap in our understanding of brain galectins, we analyzed the in situ hybridization (ISH and microarray data of adult mouse and human brain respectively, from the Allen Brain Atlas, to resolve each galectin-subtype’s spatial distribution across brain distinct cytoarchitecture. Next, transcription factors (TFs that may regulate galectins were identified using TRANSFAC software and the list obtained was further curated to sort TFs on their confirmed transcript expression in the adult brain. Galectin-TF cluster analysis, gene-ontology annotations and co-expression networks were then extrapolated to predict distinct functional relevance of each galectin in the neuronal processes. Data shows that galectins have highly heterogeneous expression within and across brain sub-structures and are predicted to be the crucial targets of brain enriched TFs. Lgals9 had maximal spatial distribution across mouse brain with inferred predominant roles in neurogenesis while LGALS1 was ubiquitously expressed in human. Limbic region associated with learning, memory and emotions and substantia nigra associated with motor movements showed strikingly high expression of LGALS1 and LGALS8 in human vs. mouse brain. The overall expression profile of galectin-8 was most

Chimeric antigen receptor T cells: a novel therapy for solid tumors

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Shengnan Yu

2017-03-01

Full Text Available Abstract The chimeric antigen receptor T (CAR-T cell therapy is a newly developed adoptive antitumor treatment. Theoretically, CAR-T cells can specifically localize and eliminate tumor cells by interacting with the tumor-associated antigens (TAAs expressing on tumor cell surface. Current studies demonstrated that various TAAs could act as target antigens for CAR-T cells, for instance, the type III variant epidermal growth factor receptor (EGFRvIII was considered as an ideal target for its aberrant expression on the cell surface of several tumor types. CAR-T cell therapy has achieved gratifying breakthrough in hematological malignancies and promising outcome in solid tumor as showed in various clinical trials. The third generation of CAR-T demonstrates increased antitumor cytotoxicity and persistence through modification of CAR structure. In this review, we summarized the preclinical and clinical progress of CAR-T cells targeting EGFR, human epidermal growth factor receptor 2 (HER2, and mesothelin (MSLN, as well as the challenges for CAR-T cell therapy.

Coordinate viral induction of tumor necrosis factor α and interferon β in human B cells and monocytes

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Goldfeld, A.E.; Maniatis, T.

1989-01-01

Human tumor necrosis factor α (TNF-α) gene expression can be induced primarily in cells of the monocyte/macrophage lineage by a variety of inducers, including lipopolysaccharide, phorbol esters such as phorbol 12-myristate 13-acetate, and virus or synthetic double-stranded RNA [poly(I)·poly(C)]. In this paper the authors show that the TNF-α gene also responds to virus and phorbol 12-myristate 13-acetate in B lymphocytes and that virus is the most potent inducer of TNF-α mRNA in both monocyte and B-cell lines. In addition, they show that viral infection coinduces the expression of TNF-α and interferon β mRNA and that viral induction of both genes is blocked by the kinase inhibitor 2-aminopurine. Inhibition of protein synthesis with cycloheximide had no effect on mRNA expression of the genes in one of three cell lines tested (U937) but blocked the viral induction of both genes in another (Namalwa). Thus, the regulatory factors required for mRNA induction of both genes are present prior to the addition of virus in U937 but not in Namalwa cells. However, in a third cell line (JY), cycloheximide blocked viral induction of the interferon β gene but not the TNF-α gene. Taken together, these observations suggest that viral induction of TNF-α and interferon β gene expression may involve overlapping pathways with both common and distinct regulatory factors

Analysis of antigen-specific B-cell memory directly ex vivo.

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McHeyzer-Williams, Louise J; McHeyzer-Williams, Michael G

2004-01-01

Helper T-cell-regulated B-cell memory develops in response to initial antigen priming as a cellular product of the germinal center (GC) reaction. On antigen recall, memory response precursors expand rapidly with exaggerated differentiation into plasma cells to produce the high-titer, high-affinity antibody(Ab) that typifies the memory B-cell response in vivo. We have devised a high-resolution flow cytometric strategy to quantify the emergence and maintenance of antigen-specific memory B cells directly ex vivo. Extended cell surface phenotype establishes a level of cellular diversity not previously appreciated for the memory B-cell compartment. Using an "exclusion transfer" strategy, we ascertain the capacity of two distinct memory B-cell populations to transfer antigen-specific memory into naive adoptive hosts. Finally, we sequence expressed messenger ribonucleic acid (mRNA) from single cells within the population to estimate the level of somatic hypermutation as the best molecular indicator of B-cell memory. In this chapter, we describe the methods used in each of these four sections that serve to provide high-resolution quantification of antigen-specific B-cell memory responses directly ex vivo.