Cryopreservation of Human Mucosal Leukocytes.

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Sean M Hughes

Full Text Available Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible.To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension.Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

Chemokine expression by glial cells directs leukocytes to sites of axonal injury in the CNS

DEFF Research Database (Denmark)

Babcock, Alicia A; Kuziel, William A; Rivest, Serge

2003-01-01

Innate responses in the CNS are critical to first line defense against infection and injury. Leukocytes migrate to inflammatory sites in response to chemokines. We studied leukocyte migration and glial chemokine expression within the denervated hippocampus in response to axonal injury caused by e...

Donor exosomes rather than passenger leukocytes initiate alloreactive T cell responses after transplantation

Science.gov (United States)

Marino, Jose; Babiker-Mohamed, Mohamed H.; Crosby-Bertorini, Patrick; Paster, Joshua T.; LeGuern, Christian; Germana, Sharon; Abdi, Reza; Uehara, Mayuko; Kim, James I.; Markmann, James F.; Tocco, Georges; Benichou, Gilles

2016-01-01

Transplantation of allogeneic organs and tissues represents a lifesaving procedure for a variety of patients affected with end-stage diseases. Although current immunosuppressive therapy prevents early acute rejection, it is associated with nephrotoxicity and increased risks for infection and neoplasia. This stresses the need for selective immune-based therapies relying on manipulation of lymphocyte recognition of donor antigens. The passenger leukocyte theory states that allograft rejection is initiated by recipient T cells recognizing donor major histocompatibility complex (MHC) molecules displayed on graft leukocytes migrating to the host’s lymphoid organs. We revisited this concept in mice transplanted with allogeneic skin, heart, or islet grafts using imaging flow cytometry. We observed no donor cells in the lymph nodes and spleen of skin-grafted mice, but we found high numbers of recipient cells displaying allogeneic MHC molecules (cross-dressed) acquired from donor microvesicles (exosomes). After heart or islet transplantation, we observed few donor leukocytes (100 per million) but large numbers of recipient cells cross-dressed with donor MHC (>90,000 per million). Last, we showed that purified allogeneic exosomes induced proinflammatory alloimmune responses by T cells in vitro and in vivo. Collectively, these results suggest that recipient antigen-presenting cells cross-dressed with donor MHC rather than passenger leukocytes trigger T cell responses after allotransplantation. PMID:27942611

Lipofection of plasmid DNA into human mast cell lines using lipid nanoparticles generated by microfluidic mixing.

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Duguay, Brett A; Huang, Kate Wei-Chen; Kulka, Marianna

2018-04-18

Mast cells are important immune cells that have significant roles in mediating allergy and asthma. Therefore, studying the molecular mechanisms regulating these and other processes in mast cells is important to elucidate. Methods such as lipofection, transduction, and electroporation are often employed to dissect these mechanisms by disrupting gene expression in mast cell lines. However, as with other leukocytes, human mast cells (HMCs) are often refractory to the delivery of plasmids by lipofection. In this study, we investigated the utility of lipid nanoparticles (LNPs) containing the ionizable cationic lipids 1,2-dioleoyloxy-3-dimethylaminopropane, 1,2-dioleyloxy-3-dimethylaminopropane, or 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane for the delivery of plasmid DNA into HMC lines. Herein, we demonstrate for the first time the use of LNPs to achieve significant and reproducible levels of plasmid DNA transfection in HMC-1.2 and laboratory of allergic diseases 2 (LAD2) cells. These levels reached 53.2% and 16.0% in HMC-1.2 and LAD2 cells, respectively; and outperformed Lipofectamine 3000 in both cases. Moreover, cell viability in the transfected cells remained above 65% for all LNP conditions tested. Together, these observations illustrate the efficacy of this technique for mast cell researchers and further support the use of LNPs for nucleic acid delivery into leukocytes. ©2018 Society for Leukocyte Biology.

Quantitation of multiple myeloma oncogene 1/interferon-regulatory factor 4 gene expression in malignant B-cell proliferations and normal leukocytes.

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Yamada, M; Asanuma, K; Kobayashi, D; Moriai, R; Yajima, T; Yagihashi, A; Yamamori, S; Watanabe, N

2001-01-01

We studied multiple myeloma oncogene 1/interferon-regulatory factor 4 (MUM1/IRF4) mRNA expression in various malignant human hematopoietic cell lines and normal leukocyte fractions. A quantitative reverse transcription-polymerase chain reaction was used to assess expression and chromosomes were examined for anomalies by fluorescent in situ hybridization. Among 12 cell lines examined, mRNA transcripts were expressed only in B-lymphoblastic and myeloma cell lines. Myeloma cells and malignant cell lines derived from mature B cells expressed more transcript than cell lines derived from immature B cells. Transcript levels, however, showed no association with chromosomal translocations. Expression in B-cell fractions from healthy donors was much less than in the malignant cells. In addition, MUM1/IRF4 mRNA expressed in samples from patients with acute lymphoblastic leukemia derived from B cells but not T cells. Our results suggested that MUM1/IRF4 gene expression is related to stage of differentiation of malignant B cells and they indicated the possibility that the quantitative analysis of MUM1/IRF4 gene is a useful tool for detection of malignant B-cell proliferations in clinical laboratory tests.

Channel catfish (Ictalurus punctatus) leukocytes express estrogen receptor isoforms ERα and ERβ2 and are functionally modulated by estrogens.

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Iwanowicz, Luke R; Stafford, James L; Patiño, Reynaldo; Bengten, Eva; Miller, Norman W; Blazer, Vicki S

2014-09-01

Estrogens are recognized as modulators of immune responses in mammals and teleosts. While it is known that the effects of estrogens are mediated via leukocyte-specific estrogen receptors (ERs) in humans and mice, leucocyte-specific estrogen receptor expression and the effects of estrogens on this cell population is less explored and poorly understood in teleosts. Here in, we verify that channel catfish (Ictalurus punctaus) leukocytes express ERα and ERβ2. Transcripts of these isoforms were detected in tissue-associated leukocyte populations by PCR, but ERβ2 was rarely detected in PBLs. Expression of these receptors was temporally regulated in PBLs following polyclonal activation by concanavalin A, lipopolysaccharide or alloantigen based on evaluation by quantitative and end-point PCR. Examination of long-term leukocyte cell lines demonstrated that these receptors are differentially expressed depending on leukocyte lineage and phenotype. Expression of ERs was also temporally dynamic in some leukocyte lineages and may reflect stage of cell maturity. Estrogens affect the responsiveness of channel catfish peripheral blood leukocytes (PBLs) to mitogens in vitro. Similarly, bactericidal activity and phorbol 12-myristate 13-acetate induced respiratory burst was modulated by 17β-estradiol. These actions were blocked by the pure ER antagonist ICI 182780 indicating that response is, in part, mediated via ERα. In summary, estrogen receptors are expressed in channel catfish leukocytes and participate in the regulation of the immune response. This is the first time leukocyte lineage expression has been reported in teleost cell lines. Published by Elsevier Ltd.

Maternal T-Cell Engraftment Interferes With Human Leukocyte Antigen Typing in Severe Combined Immunodeficiency.

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Liu, Chang; Duffy, Brian; Bednarski, Jeffrey J; Calhoun, Cecelia; Lay, Lindsay; Rundblad, Barrett; Payton, Jacqueline E; Mohanakumar, Thalachallour

2016-02-01

To report the laboratory investigation of a case of severe combined immunodeficiency (SCID) with maternal T-cell engraftment, focusing on the interference of human leukocyte antigen (HLA) typing by blood chimerism. HLA typing was performed with three different methods, including sequence-specific primer (SSP), sequence-specific oligonucleotide, and Sanger sequencing on peripheral blood leukocytes and buccal cells, from a 3-month-old boy and peripheral blood leukocytes from his parents. Short tandem repeat (STR) testing was performed in parallel. HLA typing of the patient's peripheral blood leukocytes using the SSP method demonstrated three different alleles for each of the HLA-B and HLA-C loci, with both maternal alleles present at each locus. Typing results from the patient's buccal cells showed a normal pattern of inheritance for paternal and maternal haplotypes. STR enrichment testing of the patient's CD3+ T lymphocytes and CD15+ myeloid cells confirmed maternal T-cell engraftment, while the myeloid cell profile matched the patient's buccal cells. Maternal T-cell engraftment may interfere with HLA typing in patients with SCID. Selection of the appropriate typing methods and specimens is critical for accurate HLA typing and immunologic assessment before allogeneic hematopoietic stem cell transplantation. © American Society for Clinical Pathology, 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Bacterial reduction by cell salvage washing and leukocyte depletion filtration.

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Waters, Jonathan H; Tuohy, Marion J; Hobson, Donna F; Procop, Gary

2003-09-01

Blood conservation techniques are being increasingly used because of the increased cost and lack of availability of allogeneic blood. Cell salvage offers great blood savings opportunities but is thought to be contraindicated in a number of areas (e.g., blood contaminated with bacteria). Several outcome studies have suggested the safety of this technique in trauma and colorectal surgery, but many practitioners are still hesitant to apply cell salvage in the face of frank bacterial contamination. This study was undertaken to assess the efficacy of bacterial removal when cell salvage was combined with leukocyte depletion filtration. Expired packed erythrocytes were obtained and inoculated with a fixed amount of a stock bacteria (Escherichia coli American Type Culture Collections [ATCC] 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213, or Bacteroides fragilis ATCC 25285) in amounts ranging from 2,000 to 4,000 colony forming units/ml. The blood was processed via a cell salvage machine. The washed blood was then filtered using a leukocyte reduction filter. The results for blood taken during each step of processing were compared using a repeated-measures design. Fifteen units of blood were contaminated with each of the stock bacteria. From the prewash sample to the postfiltration sample, 99.0%, 99.6%, 100%, and 97.6% of E. coli, S. aureus, P. aeruginosa, and B. fragilis were removed, respectively. Significant but not complete removal of contaminating bacteria was seen. An increased level of patient safety may be added to cell salvage by including a leukocyte depletion filter when salvaging blood that might be grossly contaminated with bacteria.

Initial afferent lymphatic vessels controlling outbound leukocyte traffic from skin to lymph nodes.

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Ignacio eMelero

2013-12-01

Full Text Available Tissue drains fluid and macromolecules through lymphatic vessels, which are lined by a specialized endothelium that expresses peculiar differentiation proteins, not found in blood vessels (i.e: LYVE-1, Podoplanin, PROX-1 and VEGFR-3. Lymphatic capillaries are characteristically devoid of a continuous basal membrane and are anchored to the ECM by elastic fibers that act as pulling ropes which open the vessel to avoid oedema if tissue volume increases, as it occurs upon inflammation. Lymphatic vessels are also crucial for the transit of T lymphocytes and antigen presenting cells from tissue to draining lymph nodes. Importantly, cell traffic control across lymphatic endothelium is differently regulated under resting and inflammatory conditions. Under steady-state non-inflammatory conditions, leukocytes enter into the lymphatic capillaries through basal membrane gaps (portals. This entrance is integrin-independent and seems to be mainly guided by CCL21 chemokine gradients acting on leukocytes expressing CCR7. In contrast, inflammatory processes in lymphatic capillaries involve a plethora of cytokines, chemokines, leukocyte integrins and other adhesion molecules. Importantly, under inflammation a role for integrins and their ligands becomes apparent and, as a consequence, the number of leukocytes entering the lymphatic capillaries multiplies several-fold. Enhancing transmigration of dendritic cells en route to lymph nodes is conceivably useful for vaccination and cancer immunotherapy, whereas interference with such key mechanisms may ameliorate autoimmunity or excessive inflammation. Recent findings illustrate how, transient cell-to-cell interactions between lymphatic endothelial cells and leukocytes contribute to shape the subsequent behaviour of leukocytes and condition the lymphatic vessel for subsequent trans-migratory events.

Telomere length of circulating leukocyte subpopulations and buccal cells in patients with ischemic heart failure and their offspring.

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Liza S M Wong

Full Text Available BACKGROUND: We aimed to find support for the hypothesis that telomere length (TL is causally involved in the pathogenesis of ischemic heart failure (IHF. We measured TL in IHF patients and their high-risk offspring and determined whether mean leukocyte TL reflects TL in CD34+ progenitor. We additionally measured TL of offspring of patients and controls to examine heritability throughout different cell types. METHODS AND RESULTS: TL was measured by qPCR in overall leukocytes, CD34+ progenitor cells, mononuclear cells (MNCs, and buccal cells in 27 IHF patients, 24 healthy controls and 60 offspring. TL in IHF patients was shorter than healthy controls in leukocytes (p = 0.002, but not in CD34+ cells (p = 0.39, MNCs (p = 0.31 or buccal cells (p = 0.19. Offspring of IHF patients had shorter TL in leukocytes than offspring of healthy subjects (p = 0.04 but not in other cell types. Controls and offspring showed a good within person correlation between leukocytes and CD34+ cells (r 0.562; p = 0.004 and r 0.602; p = 0.001, respectively. In IHF patients and offspring the correlation among cell types was blunted. Finally, we found strong correlations between parent and offspring TL in all four cell types. CONCLUSIONS: Reduced leukocyte TL in offspring of IHF subjects suggests a potential causal link of TL in ischemic heart disease. However, this causality is unlikely to originate from exhaustion of TL in CD34+ progenitor or MNC cells as their lengths are not well captured by overall leukocyte TL. Additionally, we found strong correlations between parent and offspring TL in all examined cell types, suggesting high heritability of TL among cell types.

Glycocalyx Degradation Induces a Proinflammatory Phenotype and Increased Leukocyte Adhesion in Cultured Endothelial Cells under Flow.

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Karli K McDonald

Full Text Available Leukocyte adhesion to the endothelium is an early step in the pathogenesis of atherosclerosis. Effective adhesion requires the binding of leukocytes to their cognate receptors on the surface of endothelial cells. The glycocalyx covers the surface of endothelial cells and is important in the mechanotransduction of shear stress. This study aimed to identify the molecular mechanisms underlying the role of the glycocalyx in leukocyte adhesion under flow. We performed experiments using 3-D cell culture models, exposing human abdominal aortic endothelial cells to steady laminar shear stress (10 dynes/cm2 for 24 hours. We found that with the enzymatic degradation of the glycocalyx, endothelial cells developed a proinflammatory phenotype when exposed to uniform steady shear stress leading to an increase in leukocyte adhesion. Our results show an up-regulation of ICAM-1 with degradation compared to non-degraded controls (3-fold increase, p<0.05 and we attribute this effect to a de-regulation in NF-κB activity in response to flow. These results suggest that the glycocalyx is not solely a physical barrier to adhesion but rather plays an important role in governing the phenotype of endothelial cells, a key determinant in leukocyte adhesion. We provide evidence for how the destabilization of this structure may be an early and defining feature in the initiation of atherosclerosis.

[Mechanisms of leukocyte formation of endogenous pyrogen].

Science.gov (United States)

Rybakina, E G; Sorokin, A V

1982-06-01

A study was made of the kinetics of endogenous pyrogen production by rabbit blood and exudate leukocytes and possible role played by the products of activated leukocytes in autoregulation of the process. It was established that accumulation of endogenous pyrogen in the cell precedes its release by stimulated cells. Then the processes of active pyrogen formation and release gel interdependent: pyrogen formed releases from the cell; the lowering of pyrogen concentration in the cell is accompanied by the decrease of its content in the medium. No stimulating effect of the products activated during leukocyte inflammation on pyrogen formation by blood leukocytes was discovered.

The human leukocyte antigen G promotes trophoblast fusion and β-hCG production through the Erk1/2 pathway in human choriocarcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Wang, Ji-meng [School of Medicine, Nankai University, Tianjin 300071 (China); State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Zhao, Hong-xi [Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038 (China); Wang, Li [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Gao, Zhi-ying, E-mail: gaozy301@yahoo.com.cn [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China); Yao, Yuan-qing, E-mail: yqyao@126.com [Department of Obstetrics and Gynecology, General Hospital of Chinese People’s Liberation Army, Beijing 100853 (China)

2013-05-10

Highlights: •HLA-G expression promotes BeWo cells fusion and fusogenic gene expression. •HLA-G is capable of inducing β-hCG production in human choriocarcinoma cell lines. •Up-regulation of β-hCG production by HLA-G is mediated via the Erk1/2 pathway. -- Abstract: The human leukocyte antigen G (HLA-G) is expressed on the fetal–maternal interface and plays a role in protecting fetal-derived trophoblasts from the maternal immune response, allowing trophoblasts to invade the uterus. However, HLA-G also possesses immune suppressing-independent functions. We found that HLA-G expressing BeWo choriocarcinoma cells increased cell–cell fusion compared to control BeWo cells under forskolin treatment. Regardless of forskolin treatment, the expression of fusogenic gene mRNAs, including syncytin-1, the transcription factor glial cell missing 1 (Gcm1), and beta human chorionic gonadotropin (β-hCG) were elevated. HLA-G up-regulates β-hCG production in human choriocarcinoma cells because HLA-G knockdown in JEG-3 cells induces a dramatic decrease in β-hCG compared with control cells. The defect in β-hCG production in HLA-G knocked-down cells could not be completely overcome by stimulating hCG production through increasing intracellular cAMP levels. HLA-G expressing cells have increased phosphorylation levels for extracellular signal-regulated kinase1/2 (Erk1/2) in BeWo cells. The Erk1/2 pathway is inactivated after the inhibition of HLA-G expression in JEG-3 cells. Finally, Erk1/2 inhibition was able to suppress the increased hCG production induced by HLA-G expression. Together, these data suggest novel roles for HLA-G in regulating β-hCG production via the modulation of the Erk1/2 pathway and by inducing trophoblast cell fusion.

Synthesis of endogenous pyrogen by rabbit leukocytes.

Science.gov (United States)

Moore, D M; Murphy, P A; Chesney, P J; Wood, W B

1973-05-01

Rabbit ieukocytes from peritoneal exudates and from blood were stimulated to form leukocyte pyrogen in the presence of radiolabeled amino acids. The stimuli used were endotoxin, phagocytosis, and tuberculin. The crude leukocyte pyrogen samples were purified; pyrogen from exudate cells was rendered homogeneous; pyrogen from blood cells was still contaminated with other proteins. All the purified pyrogens were radioactive; and for all it was shown that radioactivity and pyrogenic activity coincided on electrophoresis at pH 3.5 and pH 9 in acrylamide and on isoelectric focusing in acrylamide. Furthermore, pyrogens obtained from exudate cells stimulated in different ways, or from blood cells and exudate cells stimulated with endotoxin, appeared to be identical. These results suggest that leukocyte pyrogen was synthesized de novo from amino acid precursors and that leukocytes made the same pyrogen whatever the stimulus used to activate them.

Human Brain Microvascular Endothelial Cells and Umbilical Vein Endothelial Cells Differentially Facilitate Leukocyte Recruitment and Utilize Chemokines for T Cell Migration

Directory of Open Access Journals (Sweden)

Shumei Man

2008-01-01

Full Text Available Endothelial cells that functionally express blood brain barrier (BBB properties are useful surrogates for studying leukocyte-endothelial cell interactions at the BBB. In this study, we compared two different endothelial cellular models: transfected human brain microvascular endothelial cells (THBMECs and human umbilical vein endothelial cells (HUVECs. With each grow under optimal conditions, confluent THBMEC cultures showed continuous occludin and ZO-1 immunoreactivity, while HUVEC cultures exhibited punctate ZO-1 expression at sites of cell-cell contact only. Confluent THBMEC cultures on 24-well collagen-coated transwell inserts had significantly higher transendothelial electrical resistance (TEER and lower solute permeability than HUVECs. Confluent THBMECs were more restrictive for mononuclear cell migration than HUVECs. Only THBMECs utilized abluminal CCL5 to facilitate T-lymphocyte migration in vitro although both THBMECs and HUVECs employed CCL3 to facilitate T cell migration. These data establish baseline conditions for using THBMECs to develop in vitro BBB models for studying leukocyte-endothelial interactions during neuroinflammation.

Bacteria-induced histamine release from human bronchoalveolar cells and blood leukocytes

DEFF Research Database (Denmark)

Clementsen, P; Milman, N; Struve-Christensen, E

1991-01-01

23187 resulted in histamine release. S. aureus-induced histamine release from basophils was examined in leukocyte suspensions obtained from the same individuals, and in all experiments release was found. The dose-response curves were similar to those obtained with BAL cells. The bacteria...

Genotoxicity of waterpipe smoke in buccal cells and peripheral blood leukocytes as determined by comet assay.

Science.gov (United States)

Al-Amrah, Hadba Jar-Allah; Aboznada, Osama Abdullah; Alam, Mohammad Zubair; ElAssouli, M-Zaki Mustafa; Mujallid, Mohammad Ibrahim; ElAssouli, Sufian Mohamad

2014-12-01

Waterpipe smoke causes DNA damage in peripheral blood leukocytes and in buccal cells of smokers. To determine the exposure effect of waterpipe smoke on buccal cells and peripheral blood leukocytes in regard to DNA damage using comet assay. The waterpipe smoke condensates were analyzed by gas chromatography-mass spectrometry (GC-MS). The study was performed on 20 waterpipe smokers. To perform comet assay on bucaal cells of smokers, 10 µl of cell suspension was mixed with 85 µl of pre-warmed 1% low melting agarose, applied to comet slide and electrophoresed. To analyze the effect of smoke condensate in vitro, 1 ml of peripheral blood was mixed with 10 µl of smoke condensate and subjected for comet assay. The GC-MS analysis revealed the presence of 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4on, nicotine, hydroxymethyl furancarboxaldehyde and 3-ethoxy-4-hydroxybenzaldehyde in the smoke condensates. Waterpipe smoking caused DNA damage in vivo in buccal cells of smokers. The tail moment and tail length in buccal cells of smokers were 186 ± 26 and 456 ± 71, respectively, which are higher than control. The jurak and moassel smoke condensates were found to cause DNA damage in peripheral blood leukocytes. The moassel smoke condensate was more damaging. There is wide misconception that waterpipe smoking is not as harmful as cigarette smoking. This study demonstrated that waterpipe smoke induced DNA damage in exposed cells. Waterpipe smokes cause DNA damage in buccal cells. The smoke condensate of both jurak and moassel caused comet formation suggesting DNA damage in peripheral blood leukocytes.

Studies on the mechanism of endogenous pyrogen production. II. Role of cell products in the regulation of pyrogen release from blood leukocytes.

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Bodel, P

1974-09-01

Some characteristics of the process by which endogenous pyrogen (EP), the mediator of fever, is released from cells were examined by using human blood leukocytes incubated in vitro. Studies were designed to examine a possible role for leukocyte products, including EP, in the induction, augmentation, or suppression of pyrogen release by blood leukocytes. Products of stimulated leukocytes, including a partially purified preparation of EP, did not induce significant activation of nonstimulated cells. Also, no evidence was obtained that stimulated cell products either augment or inhibit pyrogen production by other stimulated cells. A feedback control of EP production was thus not observed. A crude preparation of EP, containing other products of activated cells, maintained its pyrogenicity when incubated at pH 7.4 but not at pH 5.0. These studies thus provide no support for hypothesized control mechanisms regulating production of EP by blood leukocytes. By contrast, local inactivation of EP at inflammatory sites may modify the amount of EP entering the blood, and hence fever.

Cell penetrating peptides to dissect host-pathogen protein-protein interactions in Theileria -transformed leukocytes

KAUST Repository

Haidar, Malak

2017-09-08

One powerful application of cell penetrating peptides is the delivery into cells of molecules that function as specific competitors or inhibitors of protein-protein interactions. Ablating defined protein-protein interactions is a refined way to explore their contribution to a particular cellular phenotype in a given disease context. Cell-penetrating peptides can be synthetically constrained through various chemical modifications that stabilize a given structural fold with the potential to improve competitive binding to specific targets. Theileria-transformed leukocytes display high PKA activity, but PKAis an enzyme that plays key roles in multiple cellular processes; consequently genetic ablation of kinase activity gives rise to a myriad of confounding phenotypes. By contrast, ablation of a specific kinase-substrate interaction has the potential to give more refined information and we illustrate this here by describing how surgically ablating PKA interactions with BAD gives precise information on the type of glycolysis performed by Theileria-transformed leukocytes. In addition, we provide two other examples of how ablating specific protein-protein interactions in Theileria-infected leukocytes leads to precise phenotypes and argue that constrained penetrating peptides have great therapeutic potential to combat infectious diseases in general.

Technique of leukocyte harvesting and labeling: problems and perspectives

International Nuclear Information System (INIS)

McAfee, J.G.; Subramanian, G.; Gagne, G.

1984-01-01

Mixed leukocyte suspensions obtained after gravity sedimentation of red cells and labeled with 111 In lipophilic chelates are now widely used clinically for abscess localization at many medical centers. So far, labeling with 111 In-oxine or tropolone has been more successful than any 99 mTc method. More sophisticated approaches are available for isolation and labeling of specific leukocyte cell types, to study their migration in vivo. The most significant advances in cell harvesting include newer density gradients for isopyknic centrifugation, centrifugal elutriation, and flow cytometry. Unlike current radioactive agents which label many cell types indiscriminately, more selective ligands are being developed which bind to specific cell surface receptors. These will label certain leukocyte populations or subtypes while not reacting with others, thereby avoiding laborious separation techniques. Monoclonal antibodies against leukocyte cell-surface antigens appear particularly promising as agents for selective cell labeling

The effect of varying type and volume of sedimenting agents on leukocyte harvesting and labelling in sickle cell patients

International Nuclear Information System (INIS)

Webber, D.; Nunan, T.O.; O'Doherty, M.J.

1994-01-01

Leukocyte labelling in patients with sickle cell anaemia has been reported as difficult if not impossible due to the slow erythrocyte sedimentation rate (ESR) in these patients. This study investigated standard sedimentation methods in patients with sickle cell disease (n=16) and compared the results obtained with those following changes in the amount and type of sedimenting agent used. Labelling with either 111 In-oxine or 99 Tc m -exametazime was attempted in only five patients. Replacement of the commonly used 6% Hetastarch (Hespan) with Dextran or Haemaccel did not improve leukocyte harvesting, even when the proportions used of these agents were increased. In most cases where standard procedures for leukocyte collection did not lead to harvesting of viable samples, it was possible to collect reasonably pure samples by increasing the proportion of Hespan used. It is possible to obtain adequate leukocyte labelling in the majority of sickle cell patients using a minor modification of standard techniques. In this group of patients a ratio of 8 ml of Hespan to 16 ml of blood should be used for cell separation. If this fails then donor cells, anti-granulocyte antibody labelling or HIG should be considered. (author)

Fundamental studies on ADCC (antibody-dependent cell-mediated cytotoxicity) of human peripheral blood leukocytes using sheep red blood cells as target cells, and the effect of erythrophagocytosis

International Nuclear Information System (INIS)

Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

1979-01-01

We investigated antibody-dependent cell-mediated cytotoxicity (ADCC) of human peripheral blood leukocytes by using 51 Cr-labelled sheep red blood cells (SRBC) as target cells and anti-SRBC rabbit antibody. Lysis of SRBC was mediated by either human peripheral lymphoid cells or phagocytes (Monocytes and granulocytes). SRBC were useful as target cells in ADCC assay against human lymphoid cells, since decreased cytotoxic activity of phagocyte-contaminated crude lymphocyte fraction was recovered by elimination of contaminating phagocytes. The monocytes inhibited ADCC of lymphoid cells through phagocytosis of SRBC. This assay system may be useful for estimating not only Fc receptor-mediated cytotoxicity but also Fc receptor-mediated phagocytic activity of human peripheral blood leukocytes. (author)

LINE1 CpG-DNA Hypomethylation in Granulosa Cells and Blood Leukocytes Is Associated With PCOS and Related Traits.

Science.gov (United States)

Sagvekar, Pooja; Mangoli, Vijay; Desai, Sadhana; Patil, Anushree; Mukherjee, Srabani

2017-04-01

Altered global DNA methylation is indicative of epigenomic instability concerning chronic diseases. Investigating its incidence and association with polycystic ovary syndrome (PCOS) is essential to understand the etiopathogenesis of this disorder. We assessed global DNA methylation differences in peripheral blood leukocytes (PBLs) and cumulus granulosa cells (CGCs) of controls and women with PCOS; and their association with PCOS and its traits. This study included a total of 102 controls and women with PCOS. Forty-one women undergoing controlled ovarian hyperstimulation (COH) and 61 women not undergoing COH were recruited from in vitro fertilization (IVF) and infertility clinics. DNA methylation was measured by ELISA for 5'-methyl-cytosine content and bisulfite sequencing of 5'-untranslated region (5'-UTR) of long interspersed nucleotide element-1 (LINE1/L1). Total 5'-methyl-cytosine and L1 methylation levels in PBLs and CGCs were similar between controls and women with PCOS. Methylation assessed at CpG sites of L1 5'-UTR revealed a single CpG-site (CpG-4) to be consistently hypomethylated in PBLs of both PCOS groups and CGCs of stimulated PCOS group. In unstimulated women, hypomethylation at CpG-4 was strongly associated with PCOS susceptibility, whereas in stimulated group it showed strong associations with PCOS and its hormonal traits. Furthermore, CGCs demonstrated consistent global and CpG-DNA hypomethylation relative to PBLs, irrespective of normal or disease states. Our study revealed strong association of single hypomethylated CpG-site with PCOS. Identification and characterization of more such methyl-CpG signatures in repetitive elements in larger study populations would provide valuable epigenetic insights into PCOS. Copyright © 2017 by the Endocrine Society

A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages.

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Singh, Sarika; Goswami, Poonam; Swarnkar, Supriya; Singh, Sheelendra Pratap; Wahajuddin; Nath, Chandishwar; Sharma, Sharad

2011-11-27

Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages. Copyright © 2011 Elsevier B.V. All rights reserved.

Clumping of labeled leukocyte suspension. A simple measure for avoiding it

International Nuclear Information System (INIS)

Goedemans, W.T.; Hardeman, M.R.; State Univ., Amsterdam

1988-01-01

Leukocytes in mixed suspensions can clump together, resulting in cell clusters which are responsible for false positive hot spots in lungs of patients, in the case of abscess localization studies using 111 In labeled leukocytes. Addition of extra ACD (acid-citrate-dextrose) in those labeled leukocyte suspensions prevented cell clumping and avoided occurrence of focal radioactivity accumulation in lungs. The acidification did not interfere in leukocyte migration under agar. (author)

Cell type-specific variations in the induction of hsp70 in human leukocytes by feverlike whole body hyperthermia.

Science.gov (United States)

Oehler, R; Pusch, E; Zellner, M; Dungel, P; Hergovics, N; Homoncik, M; Eliasen, M M; Brabec, M; Roth, E

2001-10-01

Fever has been associated with shortened duration and improved survival in infectious disease. The mechanism of this beneficial response is still poorly understood. The heat-inducible 70-kDa heat shock protein (Hsp70) has been associated with protection of leukocytes against the cytotoxicity of inflammatory mediators and with improved survival of severe infections. This study characterizes the induction of Hsp70 by feverlike temperatures in human leukocytes in vitro and in vivo. Using flow cytometry, Hsp70 expression was determined in whole blood samples. This approach eliminated cell isolation procedures that would greatly affect the results. Heat treatment of whole blood in vitro for 2 hours at different temperatures revealed that Hsp70 expression depends on temperature and cell type; up to 41 degrees C, Hsp70 increased only slightly in lymphocytes and polymorphonuclear leukocytes. However, in monocytes a strong induction was already seen at 39 degrees C, and Hsp70 levels at 41 degrees C were 10-fold higher than in the 37 degrees C control. To be as close as possible to the physiological situation during fever, we immersed healthy volunteers in a hot water bath, inducing whole body hyperthermia (39 degrees C), and measured leukocyte Hsp70 expression. Hsp70 was induced in all leukocytes with comparable but less pronounced cell type-specific variations as observed in vitro. Thus, a systemic increase of body temperature as triggered by fever stimulates Hsp70 expression in peripheral leukocytes, especially in monocytes. This fever-induced Hsp70 expression may protect monocytes when confronted with cytotoxic inflammatory mediators, thereby improving the course of the disease.

Penetration of equine leukocytes by merozoites of Sarcocystis neurona.

Science.gov (United States)

Lindsay, David S; Mitchell, Sheila M; Yang, Jibing; Dubey, J P; Gogal, Robert M; Witonsky, Sharon G

2006-06-15

Horses are considered accidental hosts for Sarcocystis neurona and they often develop severe neurological disease when infected with this parasite. Schizont stages develop in the central nervous system (CNS) and cause the neurological lesions associated with equine protozoal myeloencephalitis. The present study was done to examine the ability of S. neurona merozoites to penetrate and develop in equine peripheral blood leukocytes. These infected host cells might serve as a possible transport mechanism into the CNS. S. neurona merozoites penetrated equine leukocytes within 5 min of co-culture. Infected leukocytes were usually monocytes. Infected leukocytes were present up to the final day of examination at 3 days. Up to three merozoites were present in an infected monocyte. No development to schizont stages was observed. All stages observed were in the host cell cytoplasm. We postulate that S. neurona merozoites may cross the blood brain barrier hidden inside leukocytes. Once inside the CNS these merozoites can egress and invade additional cells and cause encephalitis.

Decreased UV-induced DNA repair synthesis in peripheral leukocytes from patients with the nevoid basal cell carcinoma syndrome

International Nuclear Information System (INIS)

Ringborg, U.; Lambert, B.; Landergen, J.; Lewensohn, R.

1981-01-01

The uv-induced DNA repair synthesis in peripheral leukocytes from 7 patients with the nevoid basal cell carcinoma syndrome was compared to that in peripheral leukocytes from 5 patients with basal cell carcinomas and 39 healthy subjects. A dose response curve was established for each individual, and maximum DNA repair synthesis was used as a measure of the capacity for DNA repair. The patients with the nevoid basal cell carcinoma syndrome had about 25% lower level of maximum DNA repair synthesis as compared to the patients with basal cell carcinomas and control individuals. The possibility that DNA repair mechanisms may be involved in the etiology to the nevoid basal cell carcinoma syndrome is discussed

The role of regulatory T cells during the attenuation of graft-versus-leukemia activity following donor leukocyte infusion in mice.

Science.gov (United States)

Choi, Mi-Sun; Lim, Ji-Young; Cho, Byung-Sik; Kim, Yoo-Jin; Chung, Nack-Gyun; Jeong, Dae Chul; Youn, Hyewon; Lee, Chulbom; Choi, Eun Young; Min, Chang-Ki

2011-12-01

We investigated how the graft-versus-leukemia (GVL) effect is attenuated in the tumor microenvironment using a murine model of non-myeloablative allo-HSCT (NM-HSCT) plus delayed donor leukocyte infusion (DLI) in a haploidentical B6→F1 strain combination. In-line with aggravated leukemia growth, the proportions of effector T cells expressing IFN-γ (Teffs) in spleen were reduced and attenuated GVL activity was found to be accompanied by a rebound in CD4(+)Foxp3(+) regulatory T cells (Tregs) in tumor-draining lymph nodes and tumor tissues. DLI-derived Tregs and Teffs may be potential indicators of presence of leukemic progression after DLI in this GVL model. Copyright © 2011 Elsevier Ltd. All rights reserved.

[Consequences for labile blood products of leukocyte depletion by whole blood filtration using the Leucoflex LST1 in-line filter. Evaluation of the Leucoflex LST1 filter].

Science.gov (United States)

Janus, G; Samson, T; Faudon, R; Renaudeau, C; Joussemet, M; Fabre, G

1997-12-01

The aim of this study is to evaluate the effects of whole blood filtration after a storage time of 20-24 hours at laboratory temperature using the in line filter Leucoflex LST1. The study concerns 49 blood donations in which we studied leukocyte depletion, proteins (IgG, IgA, IgM, haptoglobin, C3, C4), coagulation factors (fibrinogen, factors XII, XI, IX, VIII, V, proteins S and C, plasminogen, tPA, D-Dimers, PDF) at day 1, the parameters of conservation (ATP, 2-3 DPG, extra cellular potassium, haemolysis, pH) of red blood cell concentrates (RCCs) and bacteriological sterility at day 1 and 42. Despite a correct leukocyte depletion (mean depletion of 3.96 log), a 10 fold higher mean level of residual leukocytes/unit than with buffy coat poor RCC filtration (0.514.10(6) vs 0.051.10(6)) is observed. Moreover a lot of concentrates are not in accordance with French regulations (7/42 with more than 1.10(6) leukocytes/unit). The variation of the rates of IgG, IgA, IgM, haptoglobin, C4 and protein C is not significant. For the others there is a slight decrease with a mean level remaining in a physiological range. No sign of activation is noted. The sterility assays remain negative and the RCC conservation is not altered. In conclusion, even if the quality of the leukocyte depletion is not satisfactory in our study and has to be stated more precisely by multicenter studies, the whole blood filtration does not alter the quality of the derived components and allows us obtain RCC in a bigger volume and containing more haemoglobin than with the classical procedure after removing the buffy-coat [10].

Leukocyte integrins and their ligand interactions

Science.gov (United States)

Hyun, Young-Min; Lefort, Craig T.; Kim, Minsoo

2010-01-01

Although critical for cell adhesion and migration during normal immune-mediated reactions, leukocyte integrins are also involved in the pathogenesis of diverse clinical conditions including autoimmune diseases and chronic inflammation. Leukocyte integrins therefore have been targets for anti-adhesive therapies to treat the inflammatory disorders. Recently, the therapeutic potential of integrin antagonists has been demonstrated in psoriasis and multiple sclerosis. However, current therapeutics broadly affect integrin functions and, thus, yield unfavorable side effects. This review discusses the major leukocyte integrins and the anti-adhesion strategies for treating immune diseases. PMID:19184539

Chemokines in the corpus luteum: Implications of leukocyte chemotaxis

Directory of Open Access Journals (Sweden)

Liptak Amy R

2003-11-01

Full Text Available Abstract Chemokines are small molecular weight peptides responsible for adhesion, activation, and recruitment of leukocytes into tissues. Leukocytes are thought to influence follicular atresia, ovulation, and luteal function. Many studies in recent years have focused attention on the characterization of leukocyte populations within the ovary, the importance of leukocyte-ovarian cell interactions, and more recently, the mechanisms of ovarian leukocyte recruitment. Information about the role of chemokines and leukocyte trafficking (chemotaxis during ovarian function is important to understanding paracrine-autocrine relationships shared between reproductive and immune systems. Recent advances regarding chemokine expression and leukocyte accumulation within the ovulatory follicle and the corpus luteum are the subject of this mini-review.

Kinetics of reversible-sequestration of leukocytes by the isolated perfused rat lung

Energy Technology Data Exchange (ETDEWEB)

Goliaei, B.

1980-08-01

The kinetics and morphology of sequestration and margination of rat leukocytes were studied using an isolated perfused and ventilated rat lung preparation. Whole rat blood, bone marrow suspension, or leukocyte suspensions, were used to perfuse the isolated rat lung. The lung was also perfused with latex particle suspensions and the passage of particles through the lung capillaries was studied. When a leukocyte suspension was perfused through the lung in the single-pass mode, the rate of sequestration decreased as more cells were perfused. In contrast, latex particles of a size comparable to that of leukocytes were totally stopped by the lung. When the leukocyte suspension was recirculated through the lung, cells were rapidly removed from circulation until a steady state was reached, after which no net removal of cells by the lung occurred. These results indicate that leukocytes are reversibly sequestered from circulation. The sequestered cells marginated and attached to the luminal surface of the endothelium of post-capillary venules and veins. A mathematical model was developed based on the assumption that the attachment and detachment of leukocytes to blood vessel walls follows first-order kinetics. The model correctly predicts the following characteristics of the system: (a) the kinetics of the sequestration of leukocytes by the lung; (b) the existence of a steady state when a suspension of leukocytes is recirculated through the lung; and (c) the independence of the fraction of cells remaining in circulation from the starting concentration for all values of starting concentration. (ERB)

West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

Directory of Open Access Journals (Sweden)

Kelsey Roe

Full Text Available Characterizing the mechanisms by which West Nile virus (WNV causes blood-brain barrier (BBB disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE. Infection with WNV (NY99 strain significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1 did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101 strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome.

Tenocytes, pro-inflammatory cytokines and leukocytes: a relationship?

OpenAIRE

Al-Sadi, Onays; Schulze-Tanzil, Gundula; Kohl, Benjamin; Lohan, Anke; Lemke, Marion; Ertel, Wolfgang; John, Thilo

2012-01-01

Leukocyte derived pro-inflammatory mediators could be involved in tendon healing and scar formation. Hence, the effect of autologous leukocytes (PBMCs, peripheral blood mononuclear cells and neutrophils) on primary rabbit Achilles tenocytes gene expression was tested in insert assisted co-cultures.

Leukocyte-associated immunoglobulin-like receptor-1 expressed in epithelial ovarian cancer cells and involved in cell proliferation and invasion

International Nuclear Information System (INIS)

Cao, Qizhi; Fu, Aili; Yang, Shude; He, Xiaoli; Wang, Yue; Zhang, Xiaoshu; Zhou, Jiadi; Luan, Xiying; Yu, Wenzheng; Xue, Jiangnan

2015-01-01

Previous studies have shown that leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is expressed on most types of hamatopoietic cells and negatively regulate immune response, but the roles of LAIR-1 in tumor of the non-hematopoietic lineage have not been determined. Despite advances in therapy of epithelial ovarian cancer (EOC), many questions relating to EOC pathogenesis remain unanswered. The aim of this study was to investigate the clinical significance of LAIR-1 expression in EOC and explore the possible association between LAIR-1 and cancer. In this study, a tissue microarray containing 78 ovarian cancer cases was stained following a standard immunohistochemical protocol for LAIR-1 and the correlation of LAIR-1 expression with clinicopathologic features was assessed. LAIR-1 was detected to express in tumor cells of ovarian cancer tissues (73.1%) and EOC cell lines COC1 and HO8910, not in normal ovarian tissues. In addition, LAIR-1 expression correlates significantly with tumor grade (p = 0.004). Furthermore, down-regulation of LAIR-1 in HO8910 cells increased cell proliferation, colony formation and cell invasion. These data suggest that LAIR-1 has a relevant impact on EOC progression and may be helpful for a better understanding of molecular pathogenesis of cancer. - Highlights: • LAIR-1 is expressed in epithelial ovarian cancer cells. • LAIR-1 expression correlates significantly with tumor grade. • Down-regulation of LAIR-1 expression increased cell proliferation and invasion. • LAIR-1 may be a novel candidate for cancer diagnosis and therapy

Selective suppression of leukocyte recruitment in allergic inflammation

Directory of Open Access Journals (Sweden)

CL Weller

2005-03-01

Full Text Available Allergic diseases result in a considerable socioeconomic burden. The incidence of allergic diseases, notably allergic asthma, has risen to high levels for reasons that are not entirely understood. With an increasing knowledge of underlying mechanisms, there is now more potential to target the inflammatory process rather than the overt symptoms. This focuses attention on the role of leukocytes especially Th2 lymphocytes that regulate allergic inflammation and effector cells where eosinophils have received much attention. Eosinophils are thought to be important based on the high numbers that are recruited to sites of allergic inflammation and the potential of these cells to effect both tissue injury and remodelling. It is hoped that future therapy will be directed towards specific leukocyte types, without overtly compromising essential host defence responses. One obvious target is leukocyte recruitment. This necessitates a detailed understanding of underlying mechanisms, particularly those involving soluble che-moattractants signals and cell-cell adhesion molecules.

Genomic signatures characterize leukocyte infiltration in myositis muscles

Science.gov (United States)

2012-01-01

Background Leukocyte infiltration plays an important role in the pathogenesis and progression of myositis, and is highly associated with disease severity. Currently, there is a lack of: efficacious therapies for myositis; understanding of the molecular features important for disease pathogenesis; and potential molecular biomarkers for characterizing inflammatory myopathies to aid in clinical development. Methods In this study, we developed a simple model and predicted that 1) leukocyte-specific transcripts (including both protein-coding transcripts and microRNAs) should be coherently overexpressed in myositis muscle and 2) the level of over-expression of these transcripts should be correlated with leukocyte infiltration. We applied this model to assess immune cell infiltration in myositis by examining mRNA and microRNA (miRNA) expression profiles in muscle biopsies from 31 myositis patients and 5 normal controls. Results Several gene signatures, including a leukocyte index, type 1 interferon (IFN), MHC class I, and immunoglobulin signature, were developed to characterize myositis patients at the molecular level. The leukocyte index, consisting of genes predominantly associated with immune function, displayed strong concordance with pathological assessment of immune cell infiltration. This leukocyte index was subsequently utilized to differentiate transcriptional changes due to leukocyte infiltration from other alterations in myositis muscle. Results from this differentiation revealed biologically relevant differences in the relationship between the type 1 IFN pathway, miR-146a, and leukocyte infiltration within various myositis subtypes. Conclusions Results indicate that a likely interaction between miR-146a expression and the type 1 IFN pathway is confounded by the level of leukocyte infiltration into muscle tissue. Although the role of miR-146a in myositis remains uncertain, our results highlight the potential benefit of deconvoluting the source of

Clinical significance of determination of changes of serum SOD and T-cell subsets distribution type after leukocyte-deduced red blood cell transfusion in patients with lung cancer

International Nuclear Information System (INIS)

Yu Zhengqin; Li Keqin; Xiang Hengquan

2006-01-01

Objective: To investigate the changes of serum SOD contents and T-cell subsets distribution type after leukocyte-deduced red blood cell transfusion in patients with lung cancer. Methods: Serum SOD levels was measured with RIA and T-cell subsets distribution type was detected with monoclonal antibody technic both before and after leukocyte-deduced red blood cell transfusion in 32 patients with lung cancer and 35 normal controls. Results: Before treatment, the serum levels of SOD and T-cell CIM/ CD8 value were significantly lower in the patients than those in controls (P 0.05). Conclusion: Determination of serum SOD level and T-cell subsets distribution type is clinically useful in the management of patients with lung cancer. (authors)

Lymphatic pump treatment mobilizes leukocytes from the gut associated lymphoid tissue into lymph.

Science.gov (United States)

Hodge, Lisa M; Bearden, Melissa K; Schander, Artur; Huff, Jamie B; Williams, Arthur; King, Hollis H; Downey, H Fred

2010-06-01

Lymphatic pump techniques (LPT) are used clinically by osteopathic practitioners for the treatment of edema and infection; however, the mechanisms by which LPT enhances lymphatic circulation and provides protection during infection are not understood. Rhythmic compressions on the abdomen during LPT compress the abdominal area, including the gut-associated lymphoid tissues (GALT), which may facilitate the release of leukocytes from these tissues into the lymphatic circulation. This study is the first to document LPT-induced mobilization of leukocytes from the GALT into the lymphatic circulation. Catheters were inserted into either the thoracic or mesenteric lymph ducts of dogs. To determine if LPT enhanced the release of leukocytes from the mesenteric lymph nodes (MLN) into lymph, the MLN were fluorescently labeled in situ. Lymph was collected during 4 min pre-LPT, 4 min LPT, and 10 min following cessation of LPT. LPT significantly increased lymph flow and leukocytes in both mesenteric and thoracic duct lymph. LPT had no preferential effect on any specific leukocyte population, since neutrophil, monocyte, CD4+ T cell, CD8+ T cell, IgG+B cell, and IgA+B cell numbers were similarly increased. In addition, LPT significantly increased the mobilization of leukocytes from the MLN into lymph. Lymph flow and leukocyte counts fell following LPT treatment, indicating that the effects of LPT are transient. LPT mobilizes leukocytes from GALT, and these leukocytes are transported by the lymphatic circulation. This enhanced release of leukocytes from GALT may provide scientific rationale for the clinical use of LPT to improve immune function.

Insulin radioreceptor assay on murine splenic leukocytes and peripheral erythrocytes

International Nuclear Information System (INIS)

Shimizu, F.; Kahn, R.

1982-01-01

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH 4 Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free [ 125 I]iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 x 10 7 /ml) consisted of incubation with [ 125 I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific [ 125 I]iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 x 10 9 /ml) were incubated with [ 125 ]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific [ 125 I]iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner

[Effects of HiLo for two weeks on erythrocyte immune adhesion and leukocyte count of swimmers].

Science.gov (United States)

Zhao, Yong-Cai; Gao, Bing-Hong; Wu, Ge-Lin; Zhang, Jiu-Li

2012-07-01

To investigate the effects of living high-training low (HiLo) on innate immunity in blood of elite swimmers. Six female swimmers undertook HiLo for two weeks, erythrocyte adhesion function and counts of leukocyte were tested in different time of training period. Red blood cell C3b receptor ring rate (RBC-C3bRR) decreased and red blood cell immune complex matter ring rate (RBC-ICR) increased significantly (P < 0.05), the two markers returned to base line 1 week after training. Counts of leukocyte and granulocyte decreased significantly (P < 0.05), and they recovered 1 week after training; Counts of lymphocyte and monocyte decreased without significance during training and did not recovered after training. Immunity of erythrocyte and granulocyte decreased quickly, but lymphocyte and monocyte recovered slowly, swimmers were adaptive to the training.

Hug tightly and say goodbye: role of endothelial ICAM-1 in leukocyte transmigration.

Science.gov (United States)

Rahman, Arshad; Fazal, Fabeha

2009-04-01

Stable adhesion of leukocytes to endothelium is crucial for transendothelial migration (TEM) of leukocytes evoked during inflammatory responses, immune surveillance, and homing and mobilization of hematopoietic progenitor cells. The basis of stable adhesion involves expression of intercellular adhesion molecule-1 (ICAM-1), an inducible endothelial adhesive protein that serves as a counter-receptor for beta(2)-integrins on leukocytes. Interaction of ICAM-1 with beta(2)-integrins enables leukocytes to adhere firmly to the vascular endothelium and subsequently, to migrate across the endothelial barrier. The emerging paradigm is that ICAM-1, in addition to firmly capturing leukocytes, triggers intracellular signaling events that may contribute to active participation of the endothelium in facilitating the TEM of adherent leukocytes. The nature, duration, and intensity of ICAM-1-dependent signaling events may contribute to the determination of the route (paracellular vs. transcellular) of leukocyte passage; these aspects of ICAM-1 signaling may in turn be influenced by density and distribution of ICAM-1 on the endothelial cell surface, the source of endothelial cells it is present on, and the type of leukocytes with which it is engaged. This review summarizes our current understanding of the "ICAM-1 paradigm" of TEM with an emphasis on the signaling events mediating ICAM-1 expression and activated by ICAM-1 engagement in endothelial cells.

Inflammation Scan Using {sup 99m}Tc-HMPAO Labelled Leukocytes

Energy Technology Data Exchange (ETDEWEB)

Yang, Woo Jin; Chung, Soo Kyo; Shinn, Kyung Sub; Bahk, Yong Whee; Kim, Hoon Kyo [Catholic University College of Medicine, Seoul (Korea, Republic of)

1989-07-15

Inflammation scan using radiolabelled leukocytes has high sensitivity and specificity. Several methods for labelling leukocytes have been evaluated using P-32 diisopropyl fluorophosphate (DFP -32), H-3 thymidine, Cr-51 chromate, Ga-67 citrate and {sup 99m}Tc-sulfur colloid. In-111-oxine has proved so far to be the most reliable agent for labelling leukocytes. In-111-oxine is, however, expensive, not easily available when needed, and its radiation dose to leukocytes is relatively high. Moreover, resolution of the resultant image is relatively poor. {sup 99m}Tc is still the agent of choice because of, as compared with the indium, its favorable physical characteristics, lower cost and availability. Now the technique for labelling the leukocytes with technetium is successfully obtained using the lipophilic HMPAO with higher efficiency for granulocytes than for other cells. With this technique it is possible to label leukocytes in plans to improve the viability of the leukocytes. Inflammation scan using {sup 99m}Tc-HMPAO has been evaluated in several laboratories, and difference in methods for separation and labelling accounts for difference in efficiency, viability and biodistribution of the labelled leukocytes. We performed inflammation scan using leukocytes labelled with {sup 99m}Tc-HMPAO in three dogs 24 hours after inoculation of live E. Coli and S. Aureus in their right abdominal wall. We separated mixed leukocytes by simple sedimentation using 6% hetastarch (HES) and labelled the leukocytes with {sup 99m}Tc-HMPAO in 20% cell free plasma diluted with phosphate buffer solution. Uptake was high in the liver and spleen but is was minimal in the lungs on whole body scan. Kidneys and intestine showed minimal activity although it was high in the urinary bladder. Uptake of labelled leukocytes in the inflammation site was definite on 2 hour-postinjection scan and abscess was clearly delineated on 24 hour-delayed scan with high target-to-nontarget ratio. 4). Inflammation

Leukocyte depletion results in improved lung function and reduced inflammatory response after cardiac surgery

NARCIS (Netherlands)

Gu, YJ; Boonstra, PW; vanOeveren, W

Leukocyte depletion during cardiopulmonary bypass has been demonstrated in animal experiments to improve pulmonary function, Conflicting results have been reported, however, with clinical depletion by arterial line filter of leukocytes at the beginning of cardiopulmonary bypass. In this study, we

Mechanisms of transcriptional regulation and prognostic significance of activated leukocyte cell adhesion molecule in cancer

Directory of Open Access Journals (Sweden)

Chen Hairu

2010-10-01

Full Text Available Abstract Background Activated leukocyte cell adhesion molecule (ALCAM is implicated in the prognosis of multiple cancers with low level expression associated with metastasis and early death in breast cancer. Despite this significance, mechanisms that regulate ALCAM gene expression and ALCAM's role in adhesion of pre-metastatic circulating tumor cells have not been defined. We studied ALCAM expression in 20 tumor cell lines by real-time PCR, western blot and immunochemistry. Epigenetic alterations of the ALCAM promoter were assessed using methylation-specific PCR and bisulfite sequencing. ALCAM's role in adhesion of tumor cells to the vascular wall was studied in isolated perfused lungs. Results A common site for transcription initiation of the ALCAM gene was identified and the ALCAM promoter sequenced. The promoter contains multiple cis-active elements including a functional p65 NF-κB motif, and it harbors an extensive array of CpG residues highly methylated exclusively in ALCAM-negative tumor cells. These CpG residues were modestly demethylated after 5-aza-2-deoxycytidine treatment. Restoration of high-level ALCAM expression using an ALCAM cDNA increased clustering of MDA-MB-435 tumor cells perfused through the pulmonary vasculature of ventilated rat lungs. Anti-ALCAM antibodies reduced the number of intravascular tumor cell clusters. Conclusion Our data suggests that loss of ALCAM expression, due in part to DNA methylation of extensive segments of the promoter, significantly impairs the ability of circulating tumor cells to adhere to each other, and may therefore promote metastasis. These findings offer insight into the mechanisms for down-regulation of ALCAM gene expression in tumor cells, and for the positive prognostic value of high-level ALCAM in breast cancer.

A human thymic epithelial cell culture system for the promotion of lymphopoiesis from hematopoietic stem cells.

Science.gov (United States)

Beaudette-Zlatanova, Britte C; Knight, Katherine L; Zhang, Shubin; Stiff, Patrick J; Zúñiga-Pflücker, Juan Carlos; Le, Phong T

2011-05-01

A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs). Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis. In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage. We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy. Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.

Transformation of ATLA-negative leukocytes by blood components from anti-ATLA-positive donors in vitro.

Science.gov (United States)

Miyamoto, K; Tomita, N; Ishii, A; Nishizaki, T; Kitajima, K; Tanaka, T; Nakamura, T; Watanabe, S; Oda, T

1984-06-15

Anti-ATLA-positive blood components transformed healthy human leukocytes in vitro. Blood components examined were packed red cells, whole blood, platelet concentrate and fresh frozen plasma. Leukocytes present in anti-ATLA-positive blood components such as packed red cells, whole blood and platelet concentrate easily transformed anti-ATLA-negative leukocytes. Co-culture in fresh frozen plasma, however, did not transform recipient leukocytes, and leukocytes of anti-ATLA-positive recipients proved refractory to transformation. The transformed cells were morphologically lymphoid, grew in suspension, and possessed normal recipient karyotypes except in the case of three platelet concentrates. A high proportion of all the transformed populations formed E-rosettes with neuraminidase-treated sheep erythrocytes. The cytoplasm of over 90% of each recipient was stained brilliantly with antibodies against ATLV-determined antigens. Electron microscopy of these transformed cells revealed many C-type virus particles in the extracellular space. Blood components, such as packed red cells, whole blood and platelet concentrate, containing leukocytes from anti-ATLA-positive donors, should be used cautiously to prevent the transmission on ATLV to anti-ATLA-negative recipients.

Comparison of Plasmagel with LeucoPREP-Macrodex methods for separation of leukocytes for virus isolation.

Science.gov (United States)

Woods, G L; Proffitt, M R

1987-10-01

Plasmagel (Cellular Products, Inc., Buffalo, NY), which can separate both polymorphonuclear leukocytes (PMN) and mononuclear cells from other blood components, and LeucoPREP (Becton Dickinson Immunocytometry Systems, Mountain View, CA), which can separate mononuclear cells from other blood components, were used to harvest leukocytes from whole blood for the purpose of virus isolation. Macrodex was combined with the later, in a second step, for recovery of PMN. Of 90 peripheral blood specimens examined, cytomegalovirus was recovered from 10: in six by both methods, in three from Plasmagel prepared cells only, and in one from cells from the LeucoPREP-Macrodex preparation only. Total leukocyte counts, differential counts, and leukocyte viability did not differ significantly for the two methods. Plasmagel provided an efficient, inexpensive means of harvesting leukocytes from whole blood for virus isolation.

TNFα promotes CAR-dependent migration of leukocytes across epithelial monolayers

Science.gov (United States)

Morton, Penny E.; Hicks, Alexander; Ortiz-Zapater, Elena; Raghavan, Swetavalli; Pike, Rosemary; Noble, Alistair; Woodfin, Abigail; Jenkins, Gisli; Rayner, Emma; Santis, George; Parsons, Maddy

2016-01-01

Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKCδ pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings. PMID:27193388

Difference in membrane repair capacity between cancer cell lines and a normal cell line

DEFF Research Database (Denmark)

Frandsen, Stine Krog; McNeil, Anna K.; Novak, Ivana

2016-01-01

repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique...... cancer cell lines (p immortalized cell line (p

A Novel Centrifugation Method Using a Cell Salvage Device Offers an Alternative to the Use of Leukocyte-Depleting Filters for Autologous Blood Transfusions.

Science.gov (United States)

Barchilon, Michael; Gaspar, Cristina; Mexas, Angela; Nieter, Don

2016-12-01

Autotransfusion protocols often use the use of costly filters, such as leukocyte-depleting filters (LDFs), to minimize reinfusion of activated leukocytes and inflammatory mediators associated with reperfusion injury (RI). LDFs are used extensively in hospital settings; however, they represent an additional capital expenditure for hospitals, as well as a constraint on the reinfusion rate of blood products for health-care providers. We compared a commonly used LDF to a novel centrifugation method employing a widely used cell salvage device. Complete blood counts and enzyme-linked immunosorbent assays (ELISAs) measuring tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) were performed to compare the efficacy of these methodologies. The LDF removed, on average, 94% of all leukocytes, including 96% of neutrophils. The centrifugation method removed, on average, 89% of all leukocytes, including 91% of neutrophils and resulted in a highly concentrated red blood cell product. Our results suggest both methods offer equivalent leukocyte reduction. TNF-α was also comparably reduced following our novel centrifugation method and the LDF method and IL-2 levels were undetectable in all samples. These results indicate our novel centrifugation method may preclude the need for a LDF during select autotransfusion applications.

The Effect of Physical Activity agains the Telomere Length in the Leukocytes Cells of KONI Athletes

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Endang Purwaningsih

2017-07-01

Full Text Available Telomeres are strands of non coding DNA at the ends of chromosomes that have the primary function to protect DNA from damage and maintain chromosomal stability. Physical exercise will increase the antioxidant activity can increase telomere proteins, lengthen telomeres and or protein networks associated with telomere so that the telomere remains long, or stopping telomere shortening. Telomere length was also associated with age. The purpose of the research was to determine telomere length of leukocyte cells in the KONI (Indonesian National Sports Committee athletes in Jakarta. The research method is descriptive, by measuring telomere length using quantitative PCR on leukocyte cells. Samples are KONI athletes from several sports, including men and women athletes, with ages between 15-20 years. Used a control group (not athletes is students of the Faculty of Medicine, University of YARSI. The results showed that there was no significant difference (p> 0.05 between telomere length group of athletes with the control group in both sexes. Similarly, telomere length between athlete male with female athletes also showed no significant difference (p> 0.05. It was concluded that physical exercise in athletes KONI at the age of 15- 20 years had no effect on telomere length in leukocytes. The results of this study provide information about the telomere length in Indonesian athletes at an early age.

Fine-grained leukocyte classification with deep residual learning for microscopic images.

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Qin, Feiwei; Gao, Nannan; Peng, Yong; Wu, Zizhao; Shen, Shuying; Grudtsin, Artur

2018-08-01

Leukocyte classification and cytometry have wide applications in medical domain, previous researches usually exploit machine learning techniques to classify leukocytes automatically. However, constrained by the past development of machine learning techniques, for example, extracting distinctive features from raw microscopic images are difficult, the widely used SVM classifier only has relative few parameters to tune, these methods cannot efficiently handle fine-grained classification cases when the white blood cells have up to 40 categories. Based on deep learning theory, a systematic study is conducted on finer leukocyte classification in this paper. A deep residual neural network based leukocyte classifier is constructed at first, which can imitate the domain expert's cell recognition process, and extract salient features robustly and automatically. Then the deep neural network classifier's topology is adjusted according to the prior knowledge of white blood cell test. After that the microscopic image dataset with almost one hundred thousand labeled leukocytes belonging to 40 categories is built, and combined training strategies are adopted to make the designed classifier has good generalization ability. The proposed deep residual neural network based classifier was tested on microscopic image dataset with 40 leukocyte categories. It achieves top-1 accuracy of 77.80%, top-5 accuracy of 98.75% during the training procedure. The average accuracy on the test set is nearly 76.84%. This paper presents a fine-grained leukocyte classification method for microscopic images, based on deep residual learning theory and medical domain knowledge. Experimental results validate the feasibility and effectiveness of our approach. Extended experiments support that the fine-grained leukocyte classifier could be used in real medical applications, assist doctors in diagnosing diseases, reduce human power significantly. Copyright © 2018 Elsevier B.V. All rights reserved.

Unsupervised explorative data analysis of normal human leukocytes and BCR/ABL positive leukemic cells mid-infrared spectra

NARCIS (Netherlands)

Bellisola, G.; Bolomini-Vittori, M.; Cinque, G.; Dumas, P.; Fiorini, Z.; Laudanna, C.; Mirenda, M.; Sandt, C.; Silvestri, G.; Tomasello, L.; Vezzalini, M.; Wehbe, K.; Sorio, C.

2015-01-01

We proved the ability of Fourier Transform Infrared microspectroscopy (microFTIR) complemented by Principal Component Analysis (PCA) to detect protein phosphorylation/de-phosphorylation in mammalian cells. We analyzed by microFTIR human polymorphonuclear neutrophil (PMNs) leukocytes, mouse-derived

Effect of vitamin C on innate immune responses of rainbow trout (Oncorhynchus mykiss) leukocytes.

Science.gov (United States)

Leal, Esther; Zarza, Carlos; Tafalla, Carolina

2017-08-01

Vitamin C, also known as ascorbic acid, is an essential micronutrient that influences a wide variety of physiological processes, including immunological functions. Although the positive effects of vitamin C supplementation on the immunological status of fish has been established in different species, the bases for these positive effects are still unknown. Hence, the aim of our study was to evaluate the in vitro effect of vitamin C on several innate immune functions of rainbow trout (Oncorhynchus mykiss) leukocyte populations. For this, we assessed the effects exerted on the established rainbow trout monocyte-macrophage cell line RTS11, and compared them to those observed in trout head kidney leukocytes. Our results demonstrate that vitamin C increases the production of reactive oxygen species and the percentage of phagocytic cells in both cell populations. On the other hand, vitamin C had no effect on the surface MHC II levels and only in the case of RTS11 cells increased the capacity of these cells to migrate towards the CK9 chemokine. Finally, vitamin C also increased the transcription of several pro-inflammatory and antimicrobial genes elicited by Escherichia coli, with some differences depending on the cell population studied. Our results contribute to further understand how vitamin C supplementation regulates the fish immune system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Leukocyte adhesion deficiencies

NARCIS (Netherlands)

van de Vijver, Edith; van den Berg, Timo K.; Kuijpers, Taco W.

2013-01-01

During inflammation, leukocytes play a key role in maintaining tissue homeostasis through elimination of pathogens and removal of damaged tissue. Leukocytes migrate to the site of inflammation by crawling over and through the blood vessel wall, into the tissue. Leukocyte adhesion deficiencies (ie,

Leukocyte Reference Intervals for Free-Ranging Hummingbirds in Northern California.

Science.gov (United States)

Safra, Noa; Christopher, Mary M; Ernest, Holly B; Bandivadekar, Ruta; Tell, Lisa A

2018-04-04

  Hummingbirds are specialized nectarivores and important ecological pollinators that are the focus of conservation efforts as well as scientific investigations of metabolism and flight dynamics. Despite their importance, basic information is lacking about hummingbird blood cells. We aimed to establish reference intervals for total and differential leukocyte counts from healthy free-ranging hummingbirds in northern California. Hummingbirds were captured in five counties in spring and summer of 2012. A drop of blood was used to prepare smears for total white blood cell estimate and 200-cell differential leukocyte counts. Reference Value Advisor was used for descriptive statistics and calculation of reference intervals. Blood smears from 42 Anna's Hummingbirds ( Calypte anna) and 33 Black-chinned Hummingbirds ( Archilochus alexandri) were included. The only significant differences in leukocyte counts were due to age, and juvenile hummingbirds had significantly higher lymphocyte counts than adult hummingbirds ( Phummingbirds.

Hyperbaric environment up-regulates CD15s expression on leukocytes, down-regulates CD77 expression on renal cells and up-regulates CD34 expression on pulmonary and cardiac cells in rat

Directory of Open Access Journals (Sweden)

Danka Đevenica

2016-08-01

Full Text Available Objective(s: The aim of this study was to estimate effects of hyperbaric (HB treatment by determination of CD15s and CD11b leukocyte proinflammatory markers expression.  In addition, this study describes changes in CD77 and CD34 expression on rat endothelial cells in renal, pulmonary and cardiac tissue following exposure to hyperbaric pressure. Materials and Methods:Expression of CD11b and CD15s on leukocytes, as well as CD77 and CD34 expression on endothelial cells in cell suspensions of renal, pulmonary and cardiac tissue in rats after hyperbaric treatment and in control rats were determined by flow cytometry. Results: Hyperbaric treatment significantly increased percentage of leukocytes expressing CD15s+CD11b- (from 1.71±1.11 to 23.42±2.85, P

Tracking flow of leukocytes in blood for drug analysis

Science.gov (United States)

Basharat, Arslan; Turner, Wesley; Stephens, Gillian; Badillo, Benjamin; Lumpkin, Rick; Andre, Patrick; Perera, Amitha

2011-03-01

Modern microscopy techniques allow imaging of circulating blood components under vascular flow conditions. The resulting video sequences provide unique insights into the behavior of blood cells within the vasculature and can be used as a method to monitor and quantitate the recruitment of inflammatory cells at sites of vascular injury/ inflammation and potentially serve as a pharmacodynamic biomarker, helping screen new therapies and individualize dose and combinations of drugs. However, manual analysis of these video sequences is intractable, requiring hours per 400 second video clip. In this paper, we present an automated technique to analyze the behavior and recruitment of human leukocytes in whole blood under physiological conditions of shear through a simple multi-channel fluorescence microscope in real-time. This technique detects and tracks the recruitment of leukocytes to a bioactive surface coated on a flow chamber. Rolling cells (cells which partially bind to the bioactive matrix) are detected counted, and have their velocity measured and graphed. The challenges here include: high cell density, appearance similarity, and low (1Hz) frame rate. Our approach performs frame differencing based motion segmentation, track initialization and online tracking of individual leukocytes.

Stress-induced enhancement of leukocyte trafficking into sites of surgery or immune activation

Science.gov (United States)

Viswanathan, Kavitha; Dhabhar, Firdaus S.

2005-04-01

Effective immunoprotection requires rapid recruitment of leukocytes into sites of surgery, wounding, infection, or vaccination. In contrast to immunosuppressive chronic stressors, short-term acute stressors have immunoenhancing effects. Here, we quantify leukocyte infiltration within a surgical sponge to elucidate the kinetics, magnitude, subpopulation, and chemoattractant specificity of an acute stress-induced increase in leukocyte trafficking to a site of immune activation. Mice acutely stressed before sponge implantation showed 200-300% higher neutrophil, macrophage, natural killer cell, and T cell infiltration than did nonstressed animals. We also quantified the effects of acute stress on lymphotactin- (LTN; a predominantly lymphocyte-specific chemokine), and TNF-- (a proinflammatory cytokine) stimulated leukocyte infiltration. An additional stress-induced increase in infiltration was observed for neutrophils, in response to TNF-, macrophages, in response to TNF- and LTN, and natural killer cells and T cells in response to LTN. These results show that acute stress initially increases trafficking of all major leukocyte subpopulations to a site of immune activation. Tissue damage-, antigen-, or pathogen-driven chemoattractants subsequently determine which subpopulations are recruited more vigorously. Such stress-induced increases in leukocyte trafficking may enhance immunoprotection during surgery, vaccination, or infection, but may also exacerbate immunopathology during inflammatory (cardiovascular disease or gingivitis) or autoimmune (psoriasis, arthritis, or multiple sclerosis) diseases. chemokine | psychophysiological stress | surgical sponge | wound healing | lymphotactin

Advances in RNAi therapeutic delivery to leukocytes using lipid nanoparticles.

Science.gov (United States)

Ramishetti, Srinivas; Landesman-Milo, Dalit; Peer, Dan

2016-11-01

Small interfering RNAs (siRNAs) therapeutics has advanced into clinical trials for liver diseases and solid tumors, but remain a challenge for manipulating leukocytes fate due to lack of specificity and safety issues. Leukocytes ingest pathogens and defend the body through a complex network. They are also involved in the pathogeneses of inflammation, viral infection, autoimmunity and cancers. Modulating gene expression in leukocytes using siRNAs holds great promise to treat leukocyte-mediated diseases. Leukocytes are notoriously hard to transduce with siRNAs and are spread throughout the body often located deep in tissues, therefore developing an efficient systemic delivery strategy is still a challenge. Here, we discuss recent advances in siRNA delivery to leukocyte subsets such as macrophages, monocytes, dendritic cells and lymphocytes. We focus mainly on lipid-based nanoparticles (LNPs) comprised of new generation of ionizable lipids and their ability to deliver siRNA to primary or malignant leukocytes in a targeted manner. Special emphasis is made on LNPs targeted to subsets of leukocytes and we detail a novel microfluidic mixing technology that could aid in changing the landscape of process development of LNPs from a lab tool to a potential novel therapeutic modality.

Radiosensitivity of mesothelioma cell lines

International Nuclear Information System (INIS)

Haekkinen, A.M.; Laasonen, A.; Linnainmaa, K.; Mattson, K.; Pyrhoenen, S.

1996-01-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters α and β of the linear quadratic model (LQ-model) and mean inactivation dose (D MID ) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean α value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The α/β ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.)

Radiosensitivity of mesothelioma cell lines

Energy Technology Data Exchange (ETDEWEB)

Haekkinen, A.M. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland); Laasonen, A. [Dept. of Pathology, Central Hospital of Etelae-Pohjanmaa, Seinaejoki (Finland); Linnainmaa, K. [Dept. of Industrial Hygiene and Toxicology, Inst. of Occupational Health, Helsinki (Finland); Mattson, K. [Dept. Pulmonary Medicine, Univ. Central Hospital, Helsinki (Finland); Pyrhoenen, S. [Dept. of Oncology, Univ. Central Hospital, Helsinki (Finland)

1996-10-01

The present study was carried out in order to examine the radiosensitivity of malignant pleural mesothelioma cell lines. Cell kinetics, radiation-induced delay of the cell cycle and DNA ploidy of the cell lines were also determined. For comparison an HeLa and a human foetal fibroblast cell line were simultaneously explored. Six previously cytogenetically and histologically characterized mesothelioma tumor cell lines were applied. A rapid tiazolyl blue microtiter (MTT) assay was used to analyze radiosensitivity and cell kinetics and DNA ploidy of the cultured cells were determined by flow cytometry. The survival fraction after a dose of 2 Gy (SF2), parameters {alpha} and {beta} of the linear quadratic model (LQ-model) and mean inactivation dose (D{sub MID}) were also estimated. The DNA index of four cell lines equaled 1.0 and two cell lines equaled 1.5 and 1.6. Different mesothelioma cell lines showed a great variation in radiosensitivity. Mean survival fraction after a radiation dose of 2 Gy (SF2) was 0.60 and ranged from 0.36 to 0.81 and mean {alpha} value was 0.26 (range 0.48-0.083). The SF2 of the most sensitive diploid mesothelioma cell line was 0.36: Less than that of the foetal fibroblast cell line (0.49). The survival fractions (0.81 and 0.74) of the two most resistant cell lines, which also were aneuploid, were equal to that of the HeLa cell line (0.78). The {alpha}/{beta} ratios of the most sensitive cell lines were almost an order of magnitude greater than those of the two most resistant cell lines. Radiation-induced delay of the most resistant aneuploid cell line was similar to that of HeLa cells but in the most sensitive (diploid cells) there was practically no entry into the G1 phase following the 2 Gy radiation dose during 36 h. (orig.).

Role of early growth response 1 in arteriogenesis: impact on vascular cell proliferation and leukocyte recruitment in vivo.

Science.gov (United States)

Pagel, Judith-Irina; Ziegelhoeffer, Tibor; Heil, Matthias; Fischer, Silvia; Fernández, Borja; Schaper, Wolfgang; Preissner, Klaus T; Deindl, Elisabeth

2012-03-01

Based on previous findings that early growth response 1 (Egr-1) participates in leukocyte recruitment and cell proliferation in vitro, this study was designed to investigate its mode of action during arteriogenesis in vivo. In a model of peripheral arteriogenesis, Egr-1 was significantly upregulated in growing collaterals of wild-type (WT) mice, both on mRNA and protein level. Egr-1(-/-) mice demonstrated delayed arteriogenesis after femoral artery ligation. They further showed increased levels of monocytes and granulocytes in the circulation, but reduced levels in adductor muscles under baseline conditions. After femoral artery ligation, elevated numbers of macrophages were detected in the perivascular zone of collaterals in Egr-1(-/-) mice and mRNA of leukocyte recruitment mediators was upregulated. Other Egr family members (Egr-2 to -4) were significantly upregulated only in Egr-1(-/-) mice, suggesting a mechanism of counterbalancing Egr-1 deficiency. Moreover, splicing factor-1, downregulated in WT mice after femoral artery ligation in the process of increased vascular cell proliferation, was upregulated in Egr-1(-/-) mice. αSM-actin on the other hand, significantly downregulated in WT mice, showed no differential expression in Egr-1(-/-) mice. While cell cycle regulator cyclin E and cdc20 were upregulated in Egr-1(-/-) mice, cyclin D1 expression decreased below the detection limit in collaterals, and the proliferation marker ki67 was not differentially expressed. In conclusion, compensation for deficiency in Egr-1 function in leukocyte recruitment can presumably be mediated by other transcription factors; however, Egr-1 is indispensable for effective vascular cell cycle progression in arteriogenesis.

Flow cytofluorometric assay of human whole blood leukocyte DNA degradation in response to Yersinia pestis and Staphylococcus aureus

Science.gov (United States)

Kravtsov, Alexander L.; Grebenyukova, Tatyana P.; Bobyleva, Elena V.; Golovko, Elena M.; Malyukova, Tatyana A.; Lyapin, Mikhail N.; Kostyukova, Tatyana A.; Yezhov, Igor N.; Kuznetsov, Oleg S.

2001-05-01

Human leukocytes containing less than 2C DNA per cell (damaged or dead cells) were detected and quantified by flow cytometry and DNA-specific staining with ethidium bromide and mithramycin in whole blood infected with Staphylococcus aureus or Yersinia pestis. Addition of live S. aureus to the blood (100 microbe cells per one leukocyte) resulted in rapid degradation of leukocyte DNA within 3 to 6 hours of incubation at 37 degree(s)C. However, only about 50 percent cells were damaged and the leukocytes with the intact genetic apparatus could be found in the blood for a period up to 24 hours. The leukocyte injury was preceded by an increase of DNA per cell content (as compared to the normal one) that was likely to be connected with the active phagocytosis of S. aureus by granulocytes (2C DNA of diploid phagocytes plus the all bacterial DNA absorbed). In response to the same dose of actively growing (at 37 degree(s)C) virulent Y. pestis cells, no increase in DNA content per cell could be observed in the human blood leukocytes. The process of the leukocyte DNA degradation started after a 6-hour incubation, and between 18 to 24 hours of incubation about 90 percent leukocytes (phagocytes and lymphocytes) lost their specific DNA fluorescence. These results demonstrated a high potential of flow cytometry in comparative analysis in vitro of the leukocyte DNA degradation process in human blood in response to bacteria with various pathogenic properties. They agree with the modern idea of an apoptotic mechanism of immunosuppression in plague.

Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

Science.gov (United States)

Henschler, R; Rüster, B; Steimle, A; Hansmann, H L; Walker, W; Montag, T; Seifried, E

2005-08-01

Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

Shorter leukocyte telomere length is associated with higher risk of infections

DEFF Research Database (Denmark)

Helby, Jens; Nordestgaard, Børge G; Benfield, Thomas

2017-01-01

In the general population, older age is associated with short leukocyte telomere length and with high risk of infections. In a recent study of allogeneic hematopoietic cell transplantation for severe aplastic anemia, long donor leukocyte telomere length was associated with improved survival...

Electrophoretic detection of protein p53 in human leukocytes

International Nuclear Information System (INIS)

Paponov, V.D.; Kupsik, E.G.; Shcheglova, E.G.; Yarullin, N.N.

1986-01-01

The authors have found an acid-soluble protein with mol. wt. of about 53 kD in peripheral blood leukocytes of persons with Down's syndrome. It was present in different quantities in all 20 patients tested, but was virtually not discovered in 12 healthy blood donors. This paper determines the possible identity of this protein with protein p53 from mouse ascites carcinoma by comparing their electrophoretic mobilities, because the accuracy of electrophoretic determination of the molecular weight of proteins is not sufficient to identify them. The paper also describes experiments to detect a protein with electrophoretic mobility identical with that of a protein in the leukocytes of patients with Down's syndrome in leukocytes of patients with leukemia. To discover if protein p53 is involved in cell proliferation, the protein composition of leukocytes from healthy blood donors, cultured in the presence and absence of phytohemagglutinin (PHA), was compared. Increased incorporation of H 3-thymidine by leukocytes of patients with Down's syndrome is explained by the presence of a population of immature leukocytes actively synthesizing DNA in the peripheral blood of these patients, and this can also explain the presence of protein p53 in the leukocytes of these patients

Exposure to ultrafine particles, intracellular production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells

International Nuclear Information System (INIS)

Jantzen, Kim; Møller, Peter; Karottki, Dorina Gabriela; Olsen, Yulia; Bekö, Gabriel; Clausen, Geo; Hersoug, Lars-Georg; Loft, Steffen

2016-01-01

Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related to leukocyte-mediated oxidative stress. The study utilized a cross sectional design performed in 58 study participants from a larger cohort. Levels of circulating endothelial progenitor cells, defined as either late (CD34 + KDR + cells) or early (CD34 + CD133 + KDR + cells) subsets were measured using polychromatic flow cytometry. We additionally measured production of reactive oxygen species in leukocyte subsets (lymphocytes, monocytes and granulocytes) by flow cytometry using intracellular 2′,7′-dichlorofluoroscein. The measurements encompassed both basal levels of reactive oxygen species production and capacity for reactive oxygen species production for each leukocyte subset. We found that the late endothelial progenitor subset was negatively associated with levels of ultrafine particles measured within the participant residences and with reactive oxygen species production capacity in lymphocytes. Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate that exposure to fine and ultrafine particles derived from indoor sources may have adverse effects on human vascular health.

Modulation of interferon-gamma-induced HLA-DR expression on the human keratinocyte cell line SCC-13 by ultraviolet radiation

International Nuclear Information System (INIS)

Khan, I.U.; Boehm, K.D.; Elmets, C.A.

1993-01-01

Cell surface expression of major histocompatibility determinants on epidermal keratinocytes is a characteristic feature of a number of inflammatory dermatoses and in all likelihood is caused by diffusion of human leukocyte antigen (HLA)-DR-inducing cytokines from cells present in the dermal mononuclear cell infiltrate. Many of these same disorders respond to ultraviolet (UV) radiation phototherapy. Using the human SCC-13 keratinocyte cell line as a model, UV radiation was found to inhibit interferon-gamma-induced HLA-DR expression. Inhibition correlated closely with decreased steady-state levels of HLA-DR mRNA. These findings provide evidence that the therapeutic effect of UV radiation phototherapy may be mediated by its capacity to down-regulate cytokine-induced keratinocyte HLA-DR expression. (Author)

The molecule HLA-G: radiosensitivity indicator of a human melanoma cell line

International Nuclear Information System (INIS)

Michelin, S.C.; Gallegos, C.E.; Dubner, D.L.; Baffa Trasci, S.; Favier, B.; Carosella, E.D.

2010-01-01

The physiological and pathological relevance of the HLA-G molecule (non-classical Human Leukocyte Antigen) has been motif of important research studies. Its distribution is restricted to only few tissues. HLA-G takes part in the implantation after in vitro fecundation, in graft tolerance, in auto-immune diseases, and in tumoral immune escape. Its expression has been demonstrated in more than 30% of tumors of 15 different histological types. Gamma radiation modulates HLA-G expression at the cell surface. However, its involvement in tumoral radiosensitivity has not been demonstrated yet. The objective of this work was to demonstrate if the HLA-G molecule intervenes in the radiosensibility of human melanoma cells cultured in vitro. For this purpose we used the human melanoma cell line M8, which was transfected with the plasmid containing the HLA-G gene (M8 HLA-G+) or with the plasmid alone, without the HLA-G gene (M8 pc DNA). Both cell lines were irradiated with 0, 2, 5 y 10 Gy and in all cases survival frequency was determined with the clonogenic assay. We observed a significant reduction in M8 HLA-G+ survival with respect to M8 pc DNA for all irradiation doses and was independent of doses. These results, if confirmed in other histological types, could postulate the HLA-G molecule as a tumoral radiosensitivity marker. The specific mechanism involved in the radiosensibility modification exerted by HLA-G has not been elucidated yet. (authors) [es

Arachidonic acid triggers an oxidative burst in leukocytes

Directory of Open Access Journals (Sweden)

Pompeia C.

2003-01-01

Full Text Available The change in cellular reducing potential, most likely reflecting an oxidative burst, was investigated in arachidonic acid- (AA stimulated leukocytes. The cells studied included the human leukemia cell lines HL-60 (undifferentiated and differentiated into macrophage-like and polymorphonuclear-like cells, Jurkat and Raji, and thymocytes and macrophages from rat primary cultures. The oxidative burst was assessed by nitroblue tetrazolium reduction. AA increased the oxidative burst until an optimum AA concentration was reached and the burst decreased thereafter. In the leukemia cell lines, optimum concentration ranged from 200 to 400 µM (up to 16-fold, whereas in rat cells it varied from 10 to 20 µM. Initial rates of superoxide generation were high, decreasing steadily and ceasing about 2 h post-treatment. The continuous presence of AA was not needed to stimulate superoxide generation. It seems that the NADPH oxidase system participates in AA-stimulated superoxide production in these cells since the oxidative burst was stimulated by NADPH and inhibited by N-ethylmaleimide, diphenyleneiodonium and superoxide dismutase. Some of the effects of AA on the oxidative burst may be due to its detergent action. There apparently was no contribution of other superoxide-generating systems such as xanthine-xanthine oxidase, cytochromes P-450 and mitochondrial electron transport chain, as assessed by the use of inhibitors. Eicosanoids and nitric oxide also do not seem to interfere with the AA-stimulated oxidative burst since there was no systematic effect of cyclooxygenase, lipoxygenase or nitric oxide synthase inhibitors, but lipid peroxides may play a role, as indicated by the inhibition of nitroblue tetrazolium reduction promoted by tocopherol.

Lignans From Forsythia x Intermedia Leaves and Flowers Attenuate the Pro-inflammatory Function of Leukocytes and Their Interaction With Endothelial Cells

OpenAIRE

Barbara Michalak; Agnieszka Filipek; Piotr Chomicki; Małgorzata Pyza; Marta Woźniak; Barbara Żyżyńska-Granica; Jakub P. Piwowarski; Agnieszka Kicel; Monika A. Olszewska; Anna K. Kiss

2018-01-01

Aim of the study: Taking into account that overactivated leukocytes are an important factor in the development of many chronic diseases, we investigated the activity of phytochemically characterized (HPLC-DAD-MSn) extracts from forsythia leaves and flowers on the pro- and anti-inflammatory functions of leukocytes (effects on IL-1β, IL-8, TNF-α, and TGFβ release) and their adherence to endothelial cells. Using bio-guided fractionation, we isolated the active compounds and determined their biol...

Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

Science.gov (United States)

Pearson, Helen; Britt, Rodney D; Pabelick, Christine M; Prakash, Y S; Amrani, Yassine; Pandya, Hitesh C

2015-12-01

Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

Single-cell Raman and fluorescence microscopy reveal the association of lipid bodies with phagosomes in leukocytes

Science.gov (United States)

van Manen, Henk-Jan; Kraan, Yvonne M.; Roos, Dirk; Otto, Cees

2005-01-01

Cellular imaging techniques based on vibrational spectroscopy have become powerful tools in cell biology because the molecular composition of subcellular compartments can be visualized without the need for labeling. Using high-resolution, nonresonant confocal Raman microscopy on individual cells, we demonstrate here that lipid bodies (LBs) rich in arachidonate as revealed by their Raman spectra associate with latex bead-containing phagosomes in neutrophilic granulocytes. This finding was corroborated in macrophages and in PLB-985 cells, which can be induced to differentiate into neutrophil-like cells, by selective staining of LBs and visualization by confocal fluorescence microscopy. We further show that the accumulation of LBs near phagosomes is mediated at least in part by the flavohemoprotein gp91phox (in which “phox” is phagocyte oxidase), because different LB distributions around phagocytosed latex beads were observed in WT and gp91phox-deficient PLB-985 cells. gp91phox, which accumulates in the phagosomal membrane, is the catalytic subunit of the leukocyte NADPH oxidase, a critical enzyme in the innate immune response. Finally, time-lapse fluorescence microscopy experiments on neutrophils revealed that the LB-phagosome association is transient, similar to the “kiss-and-run” behavior displayed by endosomes involved in phagosome maturation. Because arachidonic acid (AA) has been shown to be involved in NADPH oxidase activation and phagosome maturation in neutrophils and macrophages, respectively, the findings reported here suggest that LBs may provide a reservoir of AA for local activation of these essential leukocyte functions. PMID:16002471

Discriminating between Interstitial and Circulating Leukocytes in Tissues of the Murine Oral Mucosa Avoiding Nasal-Associated Lymphoid Tissue Contamination.

Science.gov (United States)

Bittner-Eddy, Peter D; Fischer, Lori A; Tu, Andy A; Allman, Daniel A; Costalonga, Massimo

2017-01-01

Periodontitis is a chronic inflammatory response to a microbial biofilm that destroys bone and soft tissues supporting the teeth. Murine models of periodontitis based on Porphyromonas gingivalis ( Pg ) colonization have shown that extravasation of leukocytes into oral tissue is critical to driving alveolar bone destruction. Identifying interstitial leukocytes is key to understanding the immunopathogenesis of periodontitis. Here, we describe a robust flow cytometry assay based on intravenous FITC-conjugated anti-mouse CD45 mAb that distinguishes interstitial leukocytes in the oral mucosa of mice from those circulating within the vasculature or in post-dissection contaminating blood. Unaccounted circulating leukocytes skewed the relative frequency of B cells and granulocytes and inflated the numbers of all leukocyte cell types. We also describe a dissection technique that avoids contamination of oral mucosal tissues with nasal-associated lymphoid tissues (NALT), a B cell rich organ that can inflate leukocyte numbers at least 10-fold and skew the assessment of interstitial CD4 T cell phenotypes. Unlike circulating CD4 T cells, interstitial CD4 T cells were almost exclusively antigen-experienced cells (CD44 hi ). We report for the first time the presence of antigen-experienced Pg -specific CD4 T cells in NALT following oral feeding of mice with Pg . This new combined flow cytometry and dissection approach allows identification of leukocytes infiltrating the connective tissues of the murine oral mucosa and avoids confounding analyses of leukocytes not recruited to inflamed oral mucosal tissues in disease conditions like periodontitis, candidiasis, or sialadenitis.

Cell lines authentication and mycoplasma detection as minimun quality control of cell lines in biobanking.

Science.gov (United States)

Corral-Vázquez, C; Aguilar-Quesada, R; Catalina, P; Lucena-Aguilar, G; Ligero, G; Miranda, B; Carrillo-Ávila, J A

2017-06-01

Establishment of continuous cell lines from human normal and tumor tissues is an extended and useful methodology for molecular characterization of cancer pathophysiology and drug development in research laboratories. The exchange of these cell lines between different labs is a common practice that can compromise assays reliability due to contamination with microorganism such as mycoplasma or cells from different flasks that compromise experiment reproducibility and reliability. Great proportions of cell lines are contaminated with mycoplasma and/or are replaced by cells derived for a different origin during processing or distribution process. The scientific community has underestimated this problem and thousand of research experiment has been done with cell lines that are incorrectly identified and wrong scientific conclusions have been published. Regular contamination and authentication tests are necessary in order to avoid negative consequences of widespread misidentified and contaminated cell lines. Cell banks generate, store and distribute cell lines for research, being mandatory a consistent and continuous quality program. Methods implementation for guaranteeing both, the absence of mycoplasma and authentication in the supplied cell lines, has been performed in the Andalusian Health System Biobank. Specifically, precise results were obtained using real time PCR detection for mycoplasma and 10 STRs identification by capillary electrophoresis for cell line authentication. Advantages and disadvantages of these protocols are discussed.

Suppression of leukocyte inhibitory factor (LIF) production and [3H]thymidine incorporation by concanavalin A-activated mononuclear cells

International Nuclear Information System (INIS)

Lomnitzer, R.; Rabson, A.R.

1979-01-01

The capacity of human mononuclear (MN) cells pretreated with concanavalin A (Con A) to suppress the activity of fresh phytohemagglutinin (PHA)-pulsed mononuclear cells was assessed. Con A-pretreated MN cells suppressed leukocyte inhibitory factor (LIF) activity in supernatants of PHA-pulsed cell cultures and [ 3 H]thymidine incorporation by these cells. Suppression was obtained in both allogeneic and autologous systems with mitomycin-treated, irradiated, or untreated Con A-induced cells. Lymphocytes from two patients that, following treatment with Con A, did not suppress mitogen-induced proliferative response of normal cells also did not suppress LIF production

Radiation sensitivity of Merkel cell carcinoma cell lines

Energy Technology Data Exchange (ETDEWEB)

Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

1995-07-30

Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

Radiation sensitivity of Merkell cell carcinoma cell lines

International Nuclear Information System (INIS)

Leonard, J. Helen; Ramsay, Jonathan R.; Kearsley, John H.; Birrell, Geoff W.

1995-01-01

Purpose: Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Methods and Materials: Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after γ irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. Results: We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to γ irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Conclusion: Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution

Inhibition of nitric oxide synthesis enhances leukocyte rolling and adhesion in human microvasculature

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Hossain Mokarram

2012-07-01

Full Text Available Abstract Background Nitric oxide (NO is a multifunctional signaling molecule that regulates important cellular events in inflammation including leukocyte recruitment. Previous studies have shown that pharmacological inhibition of NO synthesis induces leukocyte recruitment in various in vitro and animal models. However, it is not known whether NO modulation has similar effects on leukocyte-endothelial cell interactions within the human microvasculature. The present study explored the effect of systemic L-NAME treatment on leukocyte recruitment in the SCID-hu mouse model. Methods Human skin xenografts were transplanted in SCID mice to study human leukocyte dynamics in human vasculature. Early events of human leukocyte recruitment in human vasculature were studied using intravital microscopy. NO synthesis was pharmacologically inhibited using NG-nitro-L-arginine methyl ester (L-NAME. Immunohistochemical analysis was performed to elucidate E-selectin expression in human xenograft skin. Human neutrophil-endothelial cell interactions were also studied in an in vitro flow chamber assay system. P- and E-selectin expression on cultured human umbilical vein endothelial cells (HUVECs was measured using ELISA. Platelet-activating factor (PAF synthesis was detected using a TLC-based assay. Results L-NAME treatment significantly enhanced the rolling and adhesion of human leukocytes to the human vasculature. Functional blocking of P- and E-selectins significantly inhibited rolling but not adhesion induced by inhibition of NO synthesis. Systemic L-NAME treatment enhanced E-selectin expression in human xenograft skin. L-NAME treatment significantly enhanced P- and E-selectin expression on HUVECs. L-NAME treatment did not significantly modify neutrophil rolling or adhesion to HUVECs indicating that L-NAME−induced subtle P- and E-selectin expression was insufficient to elicit dynamic neutrophil-HUVEC interactions in vitro. Moreover, synthesis of endothelial

In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

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Erika Evangelina Coronado-Cerda

2016-01-01

Full Text Available Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE or IMMUNEPOTENT CRP® (ICRP is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM, cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients.

In Vivo Chemoprotective Activity of Bovine Dialyzable Leukocyte Extract in Mouse Bone Marrow Cells against Damage Induced by 5-Fluorouracil

Science.gov (United States)

Coronado-Cerda, Erika Evangelina; Franco-Molina, Moisés Armides; Mendoza-Gamboa, Edgar; Prado-García, Heriberto; Rivera-Morales, Lydia Guadalupe; Zapata-Benavides, Pablo; Rodríguez-Salazar, María del Carmen; Caballero-Hernandez, Diana; Tamez-Guerra, Reyes Silvestre; Rodríguez-Padilla, Cristina

2016-01-01

Chemotherapy treatments induce a number of side effects, such as leukopenia neutropenia, peripheral erythropenia, and thrombocytopenia, affecting the quality of life for cancer patients. 5-Fluorouracil (5-FU) is wieldy used as myeloablative model in mice. The bovine dialyzable leukocyte extract (bDLE) or IMMUNEPOTENT CRP® (ICRP) is an immunomodulatory compound that has antioxidants and anti-inflammatory effects. In order to investigate the chemoprotection effect of ICRP on bone marrow cells in 5-FU treated mice, total bone marrow (BM) cell count, bone marrow colony forming units-granulocyte/macrophage (CFU-GM), cell cycle, immunophenotypification, ROS/superoxide and Nrf2 by flow cytometry, and histological and hematological analyses were performed. Our results demonstrated that ICRP increased BM cell count and CFU-GM number, arrested BM cells in G0/G1 phase, increased the percentage of leukocyte, granulocytic, and erythroid populations, reduced ROS/superoxide formation and Nrf2 activation, and also improved hematological levels and weight gain in 5-FU treated mice. These results suggest that ICRP has a chemoprotective effect against 5-FU in BM cells that can be used in cancer patients. PMID:27191003

Leukocyte scintigraphy with 99mTc-exametazime-labeled leukocytes is not useful for follow-up of systemic vasculitis

International Nuclear Information System (INIS)

Staudenherz, A.; Kletter, K.; Deicher, R.; Haas, M.; Hoerl, W.H.; Jilma, B.; Becherer, A.; Dudczak, R.

2002-01-01

Background: The prognosis of systemic vasculitis, for instance Wegener's granulomatosis (WG), was greatly improved by the introduction of immunosuppressive treatment. However, relapses are frequent and predictors are scarce. 111 In-leukocytes have been found to indicate unknown manifestations of WG and to predict later relapse. We prospectively investigated the value of 99m Tc-Exametazime ( 99m Tc-HMPAO)-labeled leukocytes with regard to specific patterns and for their usefulness in the follow-up of patients with WG. Methods: The vasculitis group consisted of 8 patients with WG and 2 with idiopathic necrotizing glomerulonephritis (ING). Seven patients with different inflammatory diseases served as controls. Leukocyte labeling with 99m Tc-HMPAO was done using a slightly modified Hammersmith protocol. Cell viability after labeling was verified in vivo by the exclusion of early lung and splenic uptake and in vitro by means of propidium iodide and FACS analysis. Static gamma camera images from the head, chest, abdomen, and pelvis were obtained up to 18 hours after injection of approximately 300 MBq 99m Tc-HMPAO-labeled leukocytes. Scintigrams were analyzed visually; for semiquantitative analysis ROIs were drawn over the nasal region, the right lung, kidneys, and liver. Results: Increased nasal leukocyte accumulation was found in 7/8 patients with WG and in 2/2 patients with ING. Of 2 patients who had a relapse 6 months later, one presented with, and one without nasal uptake. The kidney/liver ratio was higher in controls (0.24 ± 0.07 vs. 0.37 ± 0.11, p 99m Tc-HMPAO leukocyte scintigraphy failed to indicate or exclude a later relapse and is therefore not suitable as a diagnostic tool in the management of patients with systemic vasculitis. (author)

Leukocyte scintigraphy with 99mTc-exametazime-labeled leukocytes is not useful for follow-up of systemic vasculitis

International Nuclear Information System (INIS)

Becherer, A.; Dudczak, R.; Deicher, R.; Haas, M.; Hoerl, W.H.; Jilma, B.; Staudenherz, A.; Kletter, K.

2002-01-01

The prognosis of systemic vasculitis, for instance Wegener's granulomatosis (WG), was greatly improved by the introduction of immunosuppressive treatment. However, relapses are frequent and predictors are scarce. 111 In-leukocytes have been found to indicate unknown manifestations of WG and to predict later relapse. We prospectively investigated the value of 99m Tc-Exametazime ( 99m Tc-HMPAO)-labeled leukocytes with regard to specific patterns and for their usefulness in the follow-up of patients with WG. The vasculitis group consisted of 8 patients with WG and 2 with idiopathic necrotizing glomerulonephritis (ING). Seven patients with different inflammatory diseases served as controls. Leukocyte labeling with 99m Tc-HMPAO was done using a slightly modified Hammersmith protocol. Cell viability after labeling was verified in vivo by the exclusion of early lung and splenic uptake and in vitro by means of propidium iodide and FACS analysis. Static gamma camera images from the head, chest, abdomen, and pelvis were obtained up to 18 hours after injection of approximately 300 MBq 99m Tc-HMPAO-labeled leukocytes. Scintigrams were analyzed visually; for semiquantitative analysis ROls were drawn over the nasal region, the right lung, kidneys, and liver. Increased nasal leukocyte accumulation was found in 7/8 patients with WG and in 2/2 patients with ING. Of 2 patients who had a relapse 6 months later, one presented with, and one without nasal uptake. The kidney/liver ratio was higher in controls (0.24 ± 0.07 vs. 0.37 ± 0.11, p 99m Tc-HMPAO leukocyte scintigraphy failed to indicate or exclude a later relapse and is therefore not suitable as a diagnostic tool in the management of patients with systemic vasculitis. (author)

Cell Line Data Base: structure and recent improvements towards molecular authentication of human cell lines.

Science.gov (United States)

Romano, Paolo; Manniello, Assunta; Aresu, Ottavia; Armento, Massimiliano; Cesaro, Michela; Parodi, Barbara

2009-01-01

The Cell Line Data Base (CLDB) is a well-known reference information source on human and animal cell lines including information on more than 6000 cell lines. Main biological features are coded according to controlled vocabularies derived from international lists and taxonomies. HyperCLDB (http://bioinformatics.istge.it/hypercldb/) is a hypertext version of CLDB that improves data accessibility by also allowing information retrieval through web spiders. Access to HyperCLDB is provided through indexes of biological characteristics and navigation in the hypertext is granted by many internal links. HyperCLDB also includes links to external resources. Recently, an interest was raised for a reference nomenclature for cell lines and CLDB was seen as an authoritative system. Furthermore, to overcome the cell line misidentification problem, molecular authentication methods, such as fingerprinting, single-locus short tandem repeat (STR) profile and single nucleotide polymorphisms validation, were proposed. Since this data is distributed, a reference portal on authentication of human cell lines is needed. We present here the architecture and contents of CLDB, its recent enhancements and perspectives. We also present a new related database, the Cell Line Integrated Molecular Authentication (CLIMA) database (http://bioinformatics.istge.it/clima/), that allows to link authentication data to actual cell lines.

Long-term leukocyte reconstitution in NSG mice transplanted with human cord blood hematopoietic stem and progenitor cells.

Science.gov (United States)

Audigé, Annette; Rochat, Mary-Aude; Li, Duo; Ivic, Sandra; Fahrny, Audrey; Muller, Christina K S; Gers-Huber, Gustavo; Myburgh, Renier; Bredl, Simon; Schlaepfer, Erika; Scherrer, Alexandra U; Kuster, Stefan P; Speck, Roberto F

2017-05-30

Humanized mice (hu mice) are based on the transplantation of hematopoietic stem and progenitor cells into immunodeficient mice and have become important pre-clinical models for biomedical research. However, data about their hematopoiesis over time are scarce. We therefore characterized leukocyte reconstitution in NSG mice, which were sublethally irradiated and transplanted with human cord blood-derived CD34+ cells at newborn age, longitudinally in peripheral blood and, for more detailed analyses, cross-sectionally in peripheral blood, spleen and bone marrow at different time points. Human cell chimerism and absolute human cell count decreased between week 16 and 24 in the peripheral blood of hu mice, but were stable thereafter as assessed up to 32 weeks. Human cell chimerism in spleen and bone marrow was maintained over time. Notably, human cell chimerism in peripheral blood and spleen as well as bone marrow positively correlated with each other. Percentage of B cells decreased between week 16 and 24, whereas percentage of T cells increased; subsequently, they levelled off with T cells clearly predominating at week 32. Natural killer cells, monocytes and plasmacytoid dendritic cells (DCs) as well as CD1c + and CD141+ myeloid DCs were all present in hu mice. Proliferative responses of splenic T cells to stimulation were preserved over time. Importantly, the percentage of more primitive hematopoietic stem cells (HSCs) in bone marrow was maintained over time. Overall, leukocyte reconstitution was maintained up to 32 weeks post-transplantation in our hu NSG model, possibly explained by the maintenance of HSCs in the bone marrow. Notably, we observed great variation in multi-lineage hematopoietic reconstitution in hu mice that needs to be taken into account for the experimental design with hu mice.

Signaling through intercellular adhesion molecule 1 (ICAM-1) in a B cell lymphoma line

DEFF Research Database (Denmark)

Holland, J; Owens, T

1997-01-01

Intercellular adhesion molecule 1 (ICAM-1) (CD54) is an adhesion molecule of the immunoglobulin superfamily. The interaction between ICAM-1 on B lymphocytes and leukocyte function-associated antigen 1 on T cells plays a major role in several aspects of the immune response, including T-dependent B...... cell activation. While it was originally believed that ICAM-1 played a purely adhesive role, recent evidence suggests that it can itself transduce biochemical signals. We demonstrate that cross-linking of ICAM-1 results in the up-regulation of class II major histocompatibility complex, and we...... investigate the biochemical mechanism for the signaling role of ICAM-1. We show that cross-linking of ICAM-1 on the B lymphoma line A20 induces an increase in tyrosine phosphorylation of several cellular proteins, including the Src family kinase p53/p56(lyn). In vitro kinase assays showed that Lyn kinase...

Observing a fictitious stressful event: haematological changes, including circulating leukocyte activation.

Science.gov (United States)

Mian, Rubina; Shelton-Rayner, Graham; Harkin, Brendan; Williams, Paul

2003-03-01

The aim of this study was to assess the effect of watching a psychological stressful event on the activation of leukocytes in healthy human volunteers. Blood samples were obtained from 32 healthy male and female subjects aged between 20 and 26 years before, during and after either watching an 83-minute horror film that none of the subjects had previously seen (The Texas Chainsaw Massacre, 1974) or by sitting quietly in a room (control group). Total differential cell counts, leukocyte activation as measured by the nitroblue tetrazolium (NBT) test, heart rate and blood pressure (BP) measurements were taken at defined time points. There were significant increases in peripheral circulating leukocytes, the number of activated circulating leukocytes, haemoglobin (Hb) concentration and haematocrit (Hct) in response to the stressor. These were accompanied by significant increases in heart rate, systolic and diastolic BP (P<0.05 from baseline). This is the first reported study on the effects of observing a psychologically stressful, albeit fictitious event on circulating leukocyte numbers and the state of leukocyte activation as determined by the nitrotetrazolium test.

Oligonol Supplementation Affects Leukocyte and Immune Cell Counts after Heat Loading in Humans

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Jeong Beom Lee

2014-06-01

Full Text Available Oligonol is a low-molecular-weight form of polyphenol and has antioxidant and anti-inflammatory activity, making it a potential promoter of immunity. This study investigates the effects of oligonol supplementation on leukocyte and immune cell counts after heat loading in 19 healthy male volunteers. The participants took a daily dose of 200 mg oligonol or a placebo for 1 week. After a 2-week washout period, the subjects were switched to the other study arm. After each supplement, half-body immersion into hot water was made, and blood was collected. Then, complete and differential blood counts were performed. Flow cytometry was used to enumerate and phenotype lymphocyte subsets. Serum concentrations of interleukin (IL-1β and IL-6 in blood samples were analyzed. Lymphocyte subpopulation variables included counts of total T cells, B cells, and natural killer (NK cells. Oligonol intake attenuated elevations in IL-1β (an 11.1-fold change vs. a 13.9-fold change immediately after heating; a 12.0-fold change vs. a 12.6-fold change 1h after heating and IL-6 (an 8.6-fold change vs. a 9.9-fold change immediately after heating; a 9.1-fold change vs. a 10.5-fold change 1h after heating immediately and 1 h after heating in comparison to those in the placebo group. Oligonol supplementation led to significantly higher numbers of leukocytes (a 30.0% change vs. a 21.5% change immediately after heating; a 13.5% change vs. a 3.5% change 1h after heating and lymphocytes (a 47.3% change vs. a 39.3% change immediately after heating; a 19.08% change vs. a 2.1% change 1h after heating relative to those in the placebo group. Oligonol intake led to larger increases in T cells, B cells, and NK cells at rest (p < 0.05, p < 0.05, and p < 0.001, respectively and immediately after heating (p < 0.001 in comparison to those in the placebo group. In addition, levels of T cells (p < 0.001 and B cells (p < 0.001 were significantly higher 1 h after heating in comparison to those in

Effect of irradiation and leukocyte filtration on red cell transfusion for premature infants in an incubator

Energy Technology Data Exchange (ETDEWEB)

Sawa, Fumihiro; Iwasita, Yoichi; Motegi, Saori; Nakajima, Tomoko; Sawai, Kiyoshi; Sawada, Ken [Toho Univ., Chiba (Japan). Sakura Hospital

1999-06-01

We investigated the effect of irradiation and leukocyte filtration on red cells in MAP solution (RC-MAP) for premature infants. RC-MAPs were stored for 3 or 7 days and pretreated with 15-Gy irradiation and a leukocyte depletion filter, with either the irradiation or filtration performed first. Infusion was performed using an infusion pump for 8 hr at a speed of 2 ml/hr through a 4 ml, 100 cm tube and a 24 G needle passing into an infant incubator warmed to 34degC. Free hemoglobin concentration in the supernatant of tested RC-MAP stored 7 days and irradiated after filtration was increased to the maximum level of 42.6 mg/dl. Potassium ion level in the supernatants and ATP and 2, 3-DPG concentration in red cells from tested RC-MAP were similar to pretreated values. Maximum potassium ion level was increased to 23.5 mEq/l. Our results showed that both irradiation and filtration against RC-MAP solutions stored for 3 or 7 days is safe for use with premature infants in warmed incubators. However, further investigation is necessary to clarify the risk of bacterial contamination in such transfusion situations. (author)

Measurement of leukocyte rheology in vascular disease: clinical rationale and methodology. International Society of Clinical Hemorheology.

Science.gov (United States)

Wautier, J L; Schmid-Schönbein, G W; Nash, G B

1999-01-01

The measurement of leukocyte rheology in vascular disease is a recent development with a wide range of new opportunities. The International Society of Clinical Hemorheology has asked an expert panel to propose guidelines for the investigation of leukocyte rheology in clinical situations. This article first discusses the mechanical, adhesive and related functional properties of leukocytes (especially neutrophils) which influence their circulation, and establishes the rationale for clinically-related measurements of parameters which describe them. It is concluded that quantitation of leukocyte adhesion molecules, and of their endothelial receptors may assist understanding of leukocyte behaviour in vascular disease, along with measurements of flow resistance of leukocytes, free radical production, degranulation and gene expression. For instance, vascular cell adhesion molecule (VCAM-1) is abnormally present on endothelial cells in atherosclerosis, diabetes mellitus and inflammatory conditions. Soluble forms of intercellular adhesion molecule (ICAM-1) or VCAM can be found elevated in the blood of patients with rheumatoid arthritis or infections disease. In the second part of the article, possible technical approaches are presented and possible avenues for leukocyte rheological investigations are discussed.

Dark chocolate consumption improves leukocyte adhesion factors and vascular function in overweight men.

Science.gov (United States)

Esser, Diederik; Mars, Monica; Oosterink, Els; Stalmach, Angelique; Müller, Michael; Afman, Lydia A

2014-03-01

Flavanol-enriched chocolate consumption increases endothelium-dependent vasodilation. Most research so far has focused on flow-mediated dilation (FMD) only; the effects on other factors relevant to endothelial health, such as inflammation and leukocyte adhesion, have hardly been addressed. We investigated whether consumption of regular dark chocolate also affects other markers of endothelial health, and whether chocolate enrichment with flavanols has additional benefits. In a randomized double-blind crossover study, the effects of acute and of 4 wk daily consumption of high flavanol chocolate (HFC) and normal flavanol chocolate (NFC) on FMD, augmentation index (AIX), leukocyte count, plasma cytokines, and leukocyte cell surface molecules in overweight men (age 45-70 yr) were investigated. Sensory profiles and motivation scores to eat chocolate were also collected. Findings showed that a 4 wk chocolate intake increased FMD by 1%, which was paralleled by a decreased AIX of 1%, decreased leukocyte cell count, decreased plasma sICAM1 and sICAM3, and decreased leukocyte adhesion marker expression (Peffect), with no difference between HFC and NFC consumption. Flavanol enrichment did affect taste and negatively affected motivation to consume chocolate. This study provides new insights on how chocolate affects endothelial health by demonstrating that chocolate consumption, besides improving vascular function, also lowers the adherence capacity of leukocytes in the circulation.

99mTc-HMPAO Labelled WBC Scan in Experimental Abscess by Labelling Autologous Leukocytes with In-House-Synthesized HMPAO

International Nuclear Information System (INIS)

Lee, Dong Soo; Shin, Hyung Sik; Ahn, Curie; Chung, June Key; Lee, Myung Chul; Choi, Kang Won; Koh, Chang Soon; Jung, Jae Min; Chung, Eun Ju

1991-01-01

With HMPAO we have synthesized in our laboratory, we labelled 99m Tc to canine leukocytes. Experimental abscess made by subcutaneous injection with Staphylococcus aureus was imaged with these 99m Tc labelled leukocytes. Labelling efficiency of HMPAO with 99m Tc was 66.2% ± 14.6% (N=9). Labelling efficiency of leukocytes with 99m Tc-HMPAO was 54% ± 7.79 (N=7). Cell bound radio activity in 99m Tc-HMPAO labelled leukocytes was around 80%. when these cells were incubated in plasma in vitro at 37 .deg. C for 5 hours. In vivo cell bound activity was over 80% at 24 hours after injection. One day and four days after inoculation, uptake at the inflammatory focus was found with 99m Tc labelled leukocytes. Uptake showed up in 4 hour image, and the uptake at the lesion was most prominent in 24 hour image. These findings show that in-house-synthesized HMPAO could be used for labelling leukocytes with 99m Tc, and that 99m Tc-HMPAO-labelled leukocytes were so stable and viable that inflammatory focus could be visualized with these 99m Tc-labelled leukocytes.

CLO : The cell line ontology

NARCIS (Netherlands)

Sarntivijai, Sirarat; Lin, Yu; Xiang, Zuoshuang; Meehan, Terrence F.; Diehl, Alexander D.; Vempati, Uma D.; Schuerer, Stephan C.; Pang, Chao; Malone, James; Parkinson, Helen; Liu, Yue; Takatsuki, Terue; Saijo, Kaoru; Masuya, Hiroshi; Nakamura, Yukio; Brush, Matthew H.; Haendel, Melissa A.; Zheng, Jie; Stoeckert, Christian J.; Peters, Bjoern; Mungall, Christopher J.; Carey, Thomas E.; States, David J.; Athey, Brian D.; He, Yongqun

2014-01-01

Background: Cell lines have been widely used in biomedical research. The community-based Cell Line Ontology (CLO) is a member of the OBO Foundry library that covers the domain of cell lines. Since its publication two years ago, significant updates have been made, including new groups joining the CLO

An agent-based model of leukocyte transendothelial migration during atherogenesis.

Directory of Open Access Journals (Sweden)

Rita Bhui

2017-05-01

Full Text Available A vast amount of work has been dedicated to the effects of hemodynamics and cytokines on leukocyte adhesion and trans-endothelial migration (TEM and subsequent accumulation of leukocyte-derived foam cells in the artery wall. However, a comprehensive mechanobiological model to capture these spatiotemporal events and predict the growth and remodeling of an atherosclerotic artery is still lacking. Here, we present a multiscale model of leukocyte TEM and plaque evolution in the left anterior descending (LAD coronary artery. The approach integrates cellular behaviors via agent-based modeling (ABM and hemodynamic effects via computational fluid dynamics (CFD. In this computational framework, the ABM implements the diffusion kinetics of key biological proteins, namely Low Density Lipoprotein (LDL, Tissue Necrosis Factor alpha (TNF-α, Interlukin-10 (IL-10 and Interlukin-1 beta (IL-1β, to predict chemotactic driven leukocyte migration into and within the artery wall. The ABM also considers wall shear stress (WSS dependent leukocyte TEM and compensatory arterial remodeling obeying Glagov's phenomenon. Interestingly, using fully developed steady blood flow does not result in a representative number of leukocyte TEM as compared to pulsatile flow, whereas passing WSS at peak systole of the pulsatile flow waveform does. Moreover, using the model, we have found leukocyte TEM increases monotonically with decreases in luminal volume. At critical plaque shapes the WSS changes rapidly resulting in sudden increases in leukocyte TEM suggesting lumen volumes that will give rise to rapid plaque growth rates if left untreated. Overall this multi-scale and multi-physics approach appropriately captures and integrates the spatiotemporal events occurring at the cellular level in order to predict leukocyte transmigration and plaque evolution.

Signaling through MyD88 regulates leukocyte recruitment after brain injury

DEFF Research Database (Denmark)

Babcock, Alicia A; Toft-Hansen, Henrik; Owens, Trevor

2008-01-01

hippocampus. We now show that significant leukocyte entry into the EC occurs within 3-12 h of stab injury. Whereas T cells showed small, gradual increases over 8 days, macrophage infiltration was pronounced and peaked within 12-24 h. MyD88 deficiency significantly reduced macrophage and T cell recruitment...... for TNF-alpha, IL-1beta, and CCL2, which increased >50-fold after stab injury in C57BL/6 mice at the time of peak expression, were severely reduced in injured MyD88 knockout mice. Leukocyte recruitment and gene expression were unaffected in TLR2-deficient or TLR4 mutant mice. No significant differences...... in gene expression were observed in mice lacking IL-1R or IL-18R. These data show that MyD88-dependent signaling mediates proinflammatory gene expression and leukocyte recruitment after CNS injury....

Comparative genome analysis of three eukaryotic parasites with differing abilities to transform leukocytes reveals key mediators of theileria-induced leukocyte transformation

KAUST Repository

Hayashida, Kyoko; Hara, Yuichiro; Abe, Takashi; Yamasaki, Chisato; Toyoda, Atsushi; Kosuge, Takehide; Suzuki, Yutaka; Sato, Yoshiharu; Kawashima, Shuichi; Katayama, Toshiaki; Wakaguri, Hiroyuki; Inoue, Noboru; Homma, Keiichi; Tada-Umezaki, Masahito; Yagi, Yukio; Fujii, Yasuyuki; Habara, Takuya; Kanehisa, Minoru; Watanabe, Hidemi; Ito, Kimihito; Gojobori, Takashi; Sugawara, Hideaki; Imanishi, Tadashi; Weir, William; Gardner, Malcolm; Pain, Arnab; Shiels, Brian; Hattori, Masahira; Nene, Vishvanath; Sugimoto, Chihiro

2012-01-01

. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within

Identifying the rules of engagement enabling leukocyte rolling, activation, and adhesion.

Directory of Open Access Journals (Sweden)

Jonathan Tang

2010-02-01

Full Text Available The LFA-1 integrin plays a pivotal role in sustained leukocyte adhesion to the endothelial surface, which is a precondition for leukocyte recruitment into inflammation sites. Strong correlative evidence implicates LFA-1 clustering as being essential for sustained adhesion, and it may also facilitate rebinding events with its ligand ICAM-1. We cannot challenge those hypotheses directly because it is infeasible to measure either process during leukocyte adhesion following rolling. The alternative approach undertaken was to challenge the hypothesized mechanisms by experimenting on validated, working counterparts: simulations in which diffusible, LFA1 objects on the surfaces of quasi-autonomous leukocytes interact with simulated, diffusible, ICAM1 objects on endothelial surfaces during simulated adhesion following rolling. We used object-oriented, agent-based methods to build and execute multi-level, multi-attribute analogues of leukocytes and endothelial surfaces. Validation was achieved across different experimental conditions, in vitro, ex vivo, and in vivo, at both the individual cell and population levels. Because those mechanisms exhibit all of the characteristics of biological mechanisms, they can stand as a concrete, working theory about detailed events occurring at the leukocyte-surface interface during leukocyte rolling and adhesion experiments. We challenged mechanistic hypotheses by conducting experiments in which the consequences of multiple mechanistic events were tracked. We quantified rebinding events between individual components under different conditions, and the role of LFA1 clustering in sustaining leukocyte-surface adhesion and in improving adhesion efficiency. Early during simulations ICAM1 rebinding (to LFA1 but not LFA1 rebinding (to ICAM1 was enhanced by clustering. Later, clustering caused both types of rebinding events to increase. We discovered that clustering was not necessary to achieve adhesion as long as LFA1 and

Modulation of gap junctional intercellular communication between human smooth muscle cells by leukocyte-derived growth factors and cytokines in relation to atherogenesis

NARCIS (Netherlands)

Mensink, A.

1997-01-01

In this thesis, the effect of leukocyte-derived growth factors and cytokines on GJIC between SMC was investigated. GJIC is regarded as an important mechanism in the control of cell growth, cell differentiation and tissue homeostasis. Disturbance of SMC growth control is regarded to be a

Authentication of M14 melanoma cell line proves misidentification of MDA‐MB‐435 breast cancer cell line

Science.gov (United States)

Korch, Christopher; Hall, Erin M.; Dirks, Wilhelm G.; Ewing, Margaret; Faries, Mark; Varella‐Garcia, Marileila; Robinson, Steven; Storts, Douglas; Turner, Jacqueline A.; Wang, Ying; Burnett, Edward C.; Healy, Lyn; Kniss, Douglas; Neve, Richard M.; Nims, Raymond W.; Reid, Yvonne A.; Robinson, William A.

2017-01-01

A variety of analytical approaches have indicated that melanoma cell line UCLA‐SO‐M14 (M14) and breast carcinoma cell line MDA‐MB‐435 originate from a common donor. This indicates that at some point in the past, one of these cell lines became misidentified, meaning that it ceased to correspond to the reported donor and instead became falsely identified (through cross‐contamination or other means) as a cell line from a different donor. Initial studies concluded that MDA‐MB‐435 was the misidentified cell line and M14 was the authentic cell line, although contradictory evidence has been published, resulting in further confusion. To address this question, we obtained early samples of the melanoma cell line (M14), a lymphoblastoid cell line from the same donor (ML14), and donor serum preserved at the originator's institution. M14 samples were cryopreserved in December 1975, before MDA‐MB‐435 cells were established in culture. Through a series of molecular characterizations, including short tandem repeat (STR) profiling and cytogenetic analysis, we demonstrated that later samples of M14 and MDA‐MB‐435 correspond to samples of M14 frozen in 1975, to the lymphoblastoid cell line ML14, and to the melanoma donor's STR profile, sex and blood type. This work demonstrates conclusively that M14 is the authentic cell line and MDA‐MB‐435 is misidentified. With clear provenance information and authentication testing of early samples, it is possible to resolve debates regarding the origins of problematic cell lines that are widely used in cancer research. PMID:28940260

Quantitative and qualitative changes in leukocytes of psoriatic patients

International Nuclear Information System (INIS)

Mahesar, S.M.; Khand, A.A.

2011-01-01

Background: Psoriasis is a disease concerned with inflammation and scaling of skin. In psoriasis, cells of the skin come on surface quickly before their complete maturation. In psoriatic patients, T-cells produce an abnormally large amount of toxic chemicals and cause inflammation. This study was undertaken to find out values of prognostic significance for worsening of the disease at early stage and to evaluate the changes (quantitative and qualitative) occurring in white blood cells of psoriatic patients. Methods: A total of 158 subjects, 79 psoriatic patients (44 males and 35 females) and same numbers of normal control volunteers were recruited. Total and Differential Leukocyte Counts (TLC and DLC) were determined. Morphological examination was also undertaken. All results of patients were compared with normal control volunteers. Results : In 47.7% male and 54.2% female patients TLC was higher than controls while variation in differential count was observed in 61.3% male and 62.8% female patients. Overall, neutrophils in 45% patients, basophils in 30.3%, eosinophils in 65.8%, and monocytes in 15% of patients were elevated. In 77.2% psoriatic patients, lymphocytes were decreased. In volunteers total and differential leukocyte counts were within normal range. Total leukocyte count in normal males was 5,136 +- 31, and in psoriatic male subjects it was 10,498 +- 43, and it was 5,023 +- 35 against 11,390 +- 31 in normal versus psoriatic females ( p<0.001). Conclusion: Total leukocyte count was elevated in psoriatics while on DLC neutrophils, eosinophils and neutrophils were significantly raised where as lymphocytes were significantly decreased in psoriatic patients. Morphological changes were also noted. (author)

Decline of blood leukocyte counts 1947-59, Hiroshima and Nagasaki

Energy Technology Data Exchange (ETDEWEB)

Ichimaru, Michito; Ueda, Shoichi; Blaisdell, R K

1963-03-03

Earlier reports of progressive decline in leukocyte counts in Hiroshima from about 1948 to 1954 have been confirmed. A similar phenomenon has been observed in Nagasaki. Analysis indicates that this decline in white cell count with time is not related to exposure to the 1945 atomic bombs, to sex, to age, to commonly diagnosed diseases, or to the disproportionate influence of a subgroup. The principal white cells affected were neutrophils, lymphocytes and eosinophils. The precise etiologic factors accounting for the decline, and the biological significance of the present lower range of leukocyte values in Hiroshima and Nagasaki remain to be determined. 16 references, 5 figures, 5 tables.

The random co-polymer glatiramer acetate rapidly kills primary human leukocytes through sialic-acid-dependent cell membrane damage

DEFF Research Database (Denmark)

Christiansen, Stig Hill; Zhang, Xianwei; Juul-Madsen, Kristian

2017-01-01

in innate immunity. It shares the positive charge and amphipathic character of GA, and, as shown here, also the ability to kill human leukocyte. The cytotoxicity of both compounds depends on sialic acid in the cell membrane. The killing was associated with the generation of CD45 + debris, derived from cell...... membrane deformation. Nanoparticle tracking analysis confirmed the formation of such debris, even at low GA concentrations. Electric cell-substrate impedance sensing measurements also recorded stable alterations in T lymphocytes following such treatment. LL-37 forms oligomers through weak hydrophobic...

Studies for labelling of leukocytes with sup 99m Tc-HM-PAO in vitro and animal experiments

Energy Technology Data Exchange (ETDEWEB)

Zhaoxiang, Gu; Xiangtong, Lin [Shanghai Medical Univ. (China). Huashan Hospital

1989-05-01

A technigue for in vitro labelling of human leukocytes with {sup 99m}Tc-HM-PAO is described. The percentage of labelled leukocytes is 43.0 +- 5.0 (mean +- SD, n = 6). Cell function was not impaired by the labelling procedure. Sterility and exclusion of bacterial endotoxins in the final cell suspensions were demonstrated. In experiments on dogs with abscess, scintigraphic imaging showed accumulation of radioactivity in inflammation lesions, indicating the viability of the labelled leukocytes.

Leukocyte infiltration and tumor cell plasticity are parameters of aggressiveness in primary cutaneous melanoma.

NARCIS (Netherlands)

Hillen, F.; Baeten, C.I.M.; Winkel, van de A.; Creytens, D.; Schaft, van der D.W.J.; Winnepenninckx, V.; Griffioen, A.W.

2008-01-01

Various clinical and experimental observations detected an immunological host defense in cutaneous melanoma. In order to investigate the prognostic value of leukocyte effector mechanisms, we examined the presence of different subsets of leukocytes in tumor samples of 58 patients diagnosed with

Leukocyte removal efficiency of cell-washed and unwashed whole blood: an in vitro study.

NARCIS (Netherlands)

Brinke, M. ten; Weerwind, P.W.; Teerenstra, S.; Feron, JC; Meer, W. van der; Brouwer, René

2005-01-01

Leukocyte filtration of the cardiopulmonary bypass (CPB) perfusate after cardiac surgery has evolved as an important technique to prevent effector functions mediated by activated leukocytes. However, little is known about the filtration efficiency. Therefore, an in vitro study was conducted to

Photoperiod affects the expression of sex and species differences in leukocyte number and leukocyte trafficking in congeneric hamsters.

Science.gov (United States)

Bilbo, S D; Dhabhar, F S; Viswanathan, K; Saul, A; Nelson, R J

2003-11-01

Sex differences in immune function are well documented. These sex differences may be modulated by social and environmental factors. Individuals of polygynous species generally exhibit more pronounced sex differences in immune parameters than individuals of monogamous species, often displaying an energetic trade-off between enhanced immunity and high mating success. During winter, animals contend with environmental conditions (e.g. low temperatures and decreased food availability) that evoke energetic-stress responses; many mammals restrict reproduction in response to photoperiod as part of an annual winter coping strategy. To test the hypothesis that extant sex and species differences in immune surveillance may be modulated by photoperiod, we examined leukocyte numbers in males and females of two closely related hamster species (Phodopus). As predicted, uniparental P. sungorus exhibited a robust sex difference, with total white blood cells, total lymphocytes, T cells, and B cells higher in females than males, during long days when reproduction occurs, but not during short days when reproduction usually stops. In contrast, biparental male and female P. campbelli exhibited comparable leukocyte numbers during both long and short days. To study sex differences in stress responses, we also examined immune cell trafficking in response to an acute (2 h) restraint stressor. During stressful challenges, it appears beneficial for immune cells to exit the blood and move to primary immune defense areas such as the skin, in preparation for potential injury or infection. Acute stress moved lymphocytes and monocytes out of the blood in all animals. Blood cortisol concentrations were increased in P. sungorus females compared to males at baseline (52%) and in response to restraint stress (38%), but only in long days. P. campbelli males and females exhibited comparable blood cortisol and stress responses during both long and short days. Our results suggest that interactions among

Meisoindigo, but not its core chemical structure indirubin, inhibits zebrafish interstitial leukocyte chemotactic migration.

Science.gov (United States)

Ye, Baixin; Xiong, Xiaoxing; Deng, Xu; Gu, Lijuan; Wang, Qiongyu; Zeng, Zhi; Gao, Xiang; Gao, Qingping; Wang, Yueying

2017-12-01

Inflammatory disease is a big threat to human health. Leukocyte chemotactic migration is required for efficient inflammatory response. Inhibition of leukocyte chemotactic migration to the inflammatory site has been shown to provide therapeutic targets for treating inflammatory diseases. Our study was designed to discover effective and safe compounds that can inhibit leukocyte chemotactic migration, thus providing possible novel therapeutic strategy for treating inflammatory diseases. In this study, we used transgenic zebrafish model (Tg:zlyz-EGFP line) to visualize the process of leukocyte chemotactic migration. Then, we used this model to screen the hit compound and evaluate its biological activity on leukocyte chemotactic migration. Furthermore, western blot analysis was performed to evaluate the effect of the hit compound on the AKT or ERK-mediated pathway, which plays an important role in leukocyte chemotactic migration. In this study, using zebrafish-based chemical screening, we identified that the hit compound meisoindigo (25 μM, 50 μM, 75 μM) can significantly inhibit zebrafish leukocyte chemotactic migration in a dose-dependent manner (p = 0.01, p = 0.0006, p migration (p = 0.43). Furthermore, our results unexpectedly showed that indirubin, the core structure of meisoindigo, had no significant effect on zebrafish leukocyte chemotactic migration (p = 0.6001). Additionally, our results revealed that meisoindigo exerts no effect on the Akt or Erk-mediated signalling pathway. Our results suggest that meisoindigo, but not indirubin, is effective for inhibiting leukocyte chemotactic migration, thus providing a potential therapeutic agent for treating inflammatory diseases.

Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection.

Science.gov (United States)

Deuschle, Eva; Keller, Birgit; Siegfried, Alexandra; Manncke, Birgit; Spaeth, Tanja; Köberle, Martin; Drechsler-Hake, Doreen; Reber, Julia; Böttcher, Ralph T; Autenrieth, Stella E; Autenrieth, Ingo B; Bohn, Erwin; Schütz, Monika

2016-02-01

Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors. Copyright © 2015. Published by Elsevier GmbH.

HDAC inhibitors: modulating leukocyte differentiation, survival, proliferation and inflammation.

Science.gov (United States)

Sweet, Matthew J; Shakespear, Melanie R; Kamal, Nabilah A; Fairlie, David P

2012-01-01

Therapeutic effects of histone deacetylase (HDAC) inhibitors in cancer models were first linked to their ability to cause growth arrest and apoptosis of tumor cells. It is now clear that these agents also have pleiotropic effects on angiogenesis and the immune system, and some of these properties are likely to contribute to their anti-cancer activities. It is also emerging that inhibitors of specific HDACs affect the differentiation, survival and/or proliferation of distinct immune cell populations. This is true for innate immune cells such as macrophages, as well as cells of the acquired immune system, for example, T-regulatory cells. These effects may contribute to therapeutic profiles in some autoimmune and chronic inflammatory disease models. Here, we review our current understanding of how classical HDACs (HDACs 1-11) and their inhibitors impact on differentiation, survival and proliferation of distinct leukocyte populations, as well as the likely relevance of these effects to autoimmune and inflammatory disease processes. The ability of HDAC inhibitors to modulate leukocyte survival may have implications for the rationale of developing selective inhibitors as anti-inflammatory drugs.

Inhibition of the mixed leukocyte reaction by alloantisera in man

International Nuclear Information System (INIS)

Jonker, M.; Leeuwen, A. van; Rood, J.J. van

1977-01-01

The incidence of MLC(mixed leukocyte culture)-inhibiting antibodies was determined in 42 pregnancy sera. MLC's were carried out between the cells from the serum donor and her husband in the presence of nonimmune AB serum and the test serum. Fifty per cent of the sera reduced the MLC response to less than 40% of the control values. Only four sera had lymphocytotoxic activity. The inhibition was strong against the specific immunizor, less strong against random unrelated cells and weak against cells which were SD(serologically defined)-identical with the serum donor. Absorptions with lymphocytes and platelets were carried out. Lymphocytes removed activity in three of the four sera tested. Platelets removed activity from one serum. It was concluded that both anti-LD(lymphocyte defined) and anti-SD antibodies were able to inhibit the MLR (mixed leukocyte reaction) at the stimulator cell level. (author)

The hemostatic agent ethamsylate promotes platelet/leukocyte aggregate formation in a model of vascular injury.

Science.gov (United States)

Hernandez, Maria Rosa; Alvarez-Guerra, Miriam; Escolar, Ginés; Chiavaroli, Carlo; Hannaert, Patrick; Garay, Ricardo P

2004-08-01

The hemostatic agent ethamsylate enhances membrane expression of P-selectin in human platelets, but whether this promotes platelet-leukocyte aggregate formation is unknown. Here we investigated this point by flow cytometry determination of human platelet-leukocyte aggregates under basal conditions and after whole-blood perfusion through a damaged rabbit aorta segment. Actions of ethamsylate on adhesive molecules of platelets and leukocytes were investigated in parallel. Under basal conditions, ethamsylate was unable to modify whole-blood platelet-leukocyte aggregation, but following whole-blood perfusion through a damaged vessel, ethamsylate produced a modest, but significant increase in platelet-leukocyte aggregates (48+/-21 and 45+/-26% above control levels at ethamsylate 20 and 40 microm respectively). In isolated leukocyte plasma membranes, 14C-ethamsylate specifically bound up to an amount of 660 pmol/mg protein. Moreover, at concentrations > or =1 microm, ethamsylate induced an important (100-200%) and significant increase in the P-selectin glycoprotein ligand 1 (PSGL-1) fluorescence signal in isolated leukocytes and was unable to significantly modify the percentage of CD11b-positive cells. However, no significant changes in aggregate formation were found when ethamsylate was incubated with isolated leukocytes and blood was reconstituted and perfused. In isolated platelet cell membranes, anti-P-selectin antibody and the anti-integrin RGD-containing pentapeptide (GRDGS) were unable to displace 14C-ethamsylate binding. In conclusion, ethamsylate specifically binds to plasma membranes of leukocytes, enhances membrane PSGL-1 expression and promotes leukocyte-platelet aggregation in whole-blood perfused through a damaged vascular segment. These results together with the previously observed enhancement of platelet P-selectin membrane expression [Thromb. Res. (2002)107:329-335] confirms and extends the view that ethamsylate acts on the first step of hemostasis, by

Hypoxia, leukocytes, and the pulmonary circulation.

Science.gov (United States)

Stenmark, Kurt R; Davie, Neil J; Reeves, John T; Frid, Maria G

2005-02-01

Data are rapidly accumulating in support of the idea that circulating monocytes and/or mononuclear fibrocytes are recruited to the pulmonary circulation of chronically hypoxic animals and that these cells play an important role in the pulmonary hypertensive process. Hypoxic induction of monocyte chemoattractant protein-1, stromal cell-derived factor-1, vascular endothelial growth factor-A, endothelin-1, and tumor growth factor-beta(1) in pulmonary vessel wall cells, either directly or indirectly via signals from hypoxic lung epithelial cells, may be a critical first step in the recruitment of circulating leukocytes to the pulmonary circulation. In addition, hypoxic stress appears to induce release of increased numbers of monocytic progenitor cells from the bone marrow, and these cells may have upregulated expression of receptors for the chemokines produced by the lung circulation, which thus facilitates their specific recruitment to the pulmonary site. Once present, macrophages/fibrocytes may exert paracrine effects on resident pulmonary vessel wall cells stimulating proliferation, phenotypic modulation, and migration of resident fibroblasts and smooth muscle cells. They may also contribute directly to the remodeling process through increased production of collagen and/or differentiation into myofibroblasts. In addition, they could play a critical role in initiating and/or supporting neovascularization of the pulmonary artery vasa vasorum. The expanded vasa network may then act as a conduit for further delivery of circulating mononuclear cells to the pulmonary arterial wall, creating a feedforward loop of pathological remodeling. Future studies will need to determine the mechanisms that selectively induce leukocyte/fibrocyte recruitment to the lung circulation under hypoxic conditions, their direct role in the remodeling process via production of extracellular matrix and/or differentiation into myofibroblasts, their impact on the phenotype of resident smooth muscle

The adaptor protein SAP directly associates with PECAM-1 and regulates PECAM-1-mediated-cell adhesion in T-like cell lines.

Science.gov (United States)

Proust, Richard; Crouin, Catherine; Gandji, Leslie Yewakon; Bertoglio, Jacques; Gesbert, Franck

2014-04-01

SAP is a small cytosolic adaptor protein expressed in hematopoietic lineages whose main function is to regulate intracellular signaling pathways induced by the triggering of members of the SLAM receptor family. In this paper, we have identified the adhesion molecule PECAM-1 as a new partner for SAP in a conditional yeast two-hybrid screen. PECAM-1 is an immunoglobulin-like molecule expressed by endothelial cells and leukocytes, which possesses both pro- and anti-inflammatory properties. However, little is known about PECAM-1 functions in T cells. We show that SAP directly and specifically interacts with the cytosolic tyrosine 686 of PECAM-1. We generated different T-like cell lines in which SAP or PECAM-1 are expressed or down modulated and we demonstrate that a diminished SAP expression correlates with a diminished PECAM-1-mediated adhesion. Although SAP has mainly been shown to associate with SLAM receptors, we evidence here that SAP is a new actor downstream of PECAM-1. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rescue from acute neuroinflammation by pharmacological chemokine-mediated deviation of leukocytes

Directory of Open Access Journals (Sweden)

Berghmans Nele

2012-10-01

Full Text Available Abstract Background Neutrophil influx is an important sign of hyperacute neuroinflammation, whereas the entry of activated lymphocytes into the brain parenchyma is a hallmark of chronic inflammatory processes, as observed in multiple sclerosis (MS and its animal models of experimental autoimmune encephalomyelitis (EAE. Clinically approved or experimental therapies for neuroinflammation act by blocking leukocyte penetration of the blood brain barrier. However, in view of unsatisfactory results and severe side effects, complementary therapies are needed. We have examined the effect of chlorite-oxidized oxyamylose (COAM, a potent antiviral polycarboxylic acid on EAE. Methods EAE was induced in SJL/J mice by immunization with spinal cord homogenate (SCH or in IFN-γ-deficient BALB/c (KO mice with myelin oligodendrocyte glycoprotein peptide (MOG35-55. Mice were treated intraperitoneally (i.p. with COAM or saline at different time points after immunization. Clinical disease and histopathology were compared between both groups. IFN expression was analyzed in COAM-treated MEF cell cultures and in sera and peritoneal fluids of COAM-treated animals by quantitative PCR, ELISA and a bioassay on L929 cells. Populations of immune cell subsets in the periphery and the central nervous system (CNS were quantified at different stages of disease development by flow cytometry and differential cell count analysis. Expression levels of selected chemokine genes in the CNS were determined by quantitative PCR. Results We discovered that COAM (2 mg i.p. per mouse on days 0 and 7 protects significantly against hyperacute SCH-induced EAE in SJL/J mice and MOG35-55-induced EAE in IFN-γ KO mice. COAM deviated leukocyte trafficking from the CNS into the periphery. In the CNS, COAM reduced four-fold the expression levels of the neutrophil CXC chemokines KC/CXCL1 and MIP-2/CXCL2. Whereas the effects of COAM on circulating blood and splenic leukocytes were limited, significant

HLA engineering of human pluripotent stem cells.

Science.gov (United States)

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-06-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I-negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8(+) T cell responses were reduced in class I-negative cells that had undergone differentiation in embryoid bodies. These B2M(-/-) ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines.

HLA Engineering of Human Pluripotent Stem Cells

Science.gov (United States)

Riolobos, Laura; Hirata, Roli K; Turtle, Cameron J; Wang, Pei-Rong; Gornalusse, German G; Zavajlevski, Maja; Riddell, Stanley R; Russell, David W

2013-01-01

The clinical use of human pluripotent stem cells and their derivatives is limited by the rejection of transplanted cells due to differences in their human leukocyte antigen (HLA) genes. This has led to the proposed use of histocompatible, patient-specific stem cells; however, the preparation of many different stem cell lines for clinical use is a daunting task. Here, we develop two distinct genetic engineering approaches that address this problem. First, we use a combination of gene targeting and mitotic recombination to derive HLA-homozygous embryonic stem cell (ESC) subclones from an HLA-heterozygous parental line. A small bank of HLA-homozygous stem cells with common haplotypes would match a significant proportion of the population. Second, we derive HLA class I–negative cells by targeted disruption of both alleles of the Beta-2 Microglobulin (B2M) gene in ESCs. Mixed leukocyte reactions and peptide-specific HLA-restricted CD8+ T cell responses were reduced in class I–negative cells that had undergone differentiation in embryoid bodies. These B2M−/− ESCs could act as universal donor cells in applications where the transplanted cells do not express HLA class II genes. Both approaches used adeno-associated virus (AAV) vectors for efficient gene targeting in the absence of potentially genotoxic nucleases, and produced pluripotent, transgene-free cell lines. PMID:23629003

Cloning of a glutathione S-transferase decreasing during differentiation of HL60 cell line

Energy Technology Data Exchange (ETDEWEB)

Kim, Jae Chul; Park, In Kyu; Lee, Kyu Bo; Sohn, Sang Kyun; Kim, Moo Kyu; Kim, Jung Chul [College of Medicine, Kyungpook National Univ., Taegu (Korea, Republic of)

1999-06-01

By sequencing the Expressed Sequence Tags of human dermal papilla cDNA library, we identified a clone named K872 of which the expression decreased during differentiation of HL60 cell line. K872 plasmid DNA was isolated according to QIA plasmid extraction kit (Qiagen GmbH, Germany). The nucleotide sequencing was performed by Sanger's method with K872 plasmid DNA. The most updated GenBank EMBL necleic acid banks were searched through the internet by using BLAST (Basic Local Alignment Search Tools) program. Northern bots were performed using RNA isolated from various human tissues and cancer cell lines. The gene expression of the fusion protein was achieved by His-Patch Thiofusion expression system and the protein product was identified on SDS-PAGE. K872 clone is 1006 nucleotides long, and has a coding region of 675 nucleotides and a 3' non-coding region of 280 nucleotides. The presumed open reading frame starting at the 5' terminus of K872 encodes 226 amino acids, including the initiation methionine residue. The amino acid sequence deduced from the open reading frame of K872 shares 70% identity with that of rat glutathione S-transferase kappa 1 (rGSTK1). The transcripts were expressed inh a variety of human tissues and cancer cells. The levels of transcript were relatively high in those tissues such as heart, skeletal muscle, and peripheral blood leukocyte. It is noteworthy that K872 was found to be abundantly expressed in colorectal cancer and melanoma cell lines. Homology search result suggests that K872 clone is the human homolog of the rGSTK1 which is known to be involved in the resistance of cytotoxic therapy. We propose that meticulous functional analysis should be followed to confirm that.

Effect of leukocyte hydrolases on bacteria

International Nuclear Information System (INIS)

Cohen, D.; Michel, J.; Ferne, M.; Bergner-Rabinowitz, S.; Ginsburg, I.

1979-01-01

Leukocyte extracts, trypsin, and lysozyme are all capable of releasing the bulk of the LPS from S. typhi, S. typhimurium, and E. coli. Bacteria which have been killed by heat, ultraviolet irradiation, or by a variety of metabolic inhibitors and antibiotics which affect protein, DNA, RNA, and cell wall synthesis no longer yield soluble LPS following treatment with the releasing agents. On the other hand, bacteria which are resistant to certain of the antibiotics yield nearly the full amount of soluble LPS following treatment, suggesting that certain heatabile endogenous metabolic pathways collaborate with the releasing agents in the release of LPS from the bacteria. It is suggested that some of the beneficial effects of antibiotics on infections with gram-negative bacteria may be the prevention of massive release of endotoxin by leukocyte enzymes in inflammatory sites

Expression of Tac antigen component of bovine interleukin-2 receptor in different leukocyte populations infected with Theileria parva or Theileria annulata.

Science.gov (United States)

Dobbelaere, D A; Prospero, T D; Roditi, I J; Kelke, C; Baumann, I; Eichhorn, M; Williams, R O; Ahmed, J S; Baldwin, C L; Clevers, H

1990-01-01

The Tac antigen component of the bovine interleukin-2 receptor was expressed as a Cro-beta-galactosidase fusion protein in Escherichia coli and used to raise antibodies in rabbits. These antibodies were used for flow cytofluorimetric analysis to investigate the expression of Tac antigen in a variety of Theileria parva-infected cell lines and also in three Theileria annulata-infected cell lines. Cells expressing Tac antigen on their surface were found in all T. parva-infected cell lines tested whether these were of T- or B-cell origin. T cells expressing Tac antigen could be CD4- CD8-, CD4+ CD8-, CD4- CD8+, or CD4+ CD8+. Tac antigen expression was observed both in cultures which had been maintained in the laboratory for several years and in transformed cell lines which had recently been established by infection of lymphocytes in vitro with T. parva. Northern (RNA) blot analysis demonstrated Tac antigen transcripts in RNA isolated from all T. parva-infected cell lines. Three T. annulata-infected cell lines which were not of T-cell origin were also tested. Two of them expressed Tac antigen on their surface. Abundant Tac antigen mRNA was detected in these T. annulata-infected cell lines, but only trace amounts were demonstrated in the third cell line, which contained very few Tac antigen-expressing cells. In all cell lines tested, whether cloned or uncloned, a proportion of the cells did not express detectable levels of Tac antigen on their surface. This was also the case for a number of other leukocyte surface markers. In addition, we showed that the interleukin-2 receptors were biologically functional, because addition of recombinant interleukin-2 to cultures stimulated cell proliferation. Recombinant interleukin-2 treatment also resulted in increased amounts of steady-state Tac antigen mRNA. The relevance of interleukin-2 receptor expression on Theileria-infected cells is discussed. Images PMID:1979317

Prevention of alloimmunization by ultraviolet-B irradiation. Inactivation of leukocytes and the generation of active oxygen and radicals

International Nuclear Information System (INIS)

Takahashi, Tsuneo; Mogi, Yuko; Sekiguchi, Sadayoshi; Akasaka, Junichi; Kamo, Naoki; Kuwabara, Mikinori.

1994-01-01

UV-B irradiation of platelet concentrates (PC) has been tried in several institutes to inactivate leukocytes in PC and prevent alloimmunization on platelet transfusion. However, the mechanism of inactivation of leukocytes contaminating PC has not been fully understood. It is known that UV-B light is absorbed by photosensitizers in cells and produces active oxygen and radicals, such as singlet oxygen, superioxide anions and hydroxyl radicals. These active oxygen or radicals should injure cellular components and this could cause the suppression of cellular functions. In this study, we investigated the relationships among UV-B irradiation, free radical generation and leukocyte inactivation. We found the evidence that active oxygen and radicals were produced in peripheral blood mononuclear cells by UV-B irradiation. UV-B irradiation suppressed the stimulatory function of leukocytes in a mixed lymphocyte reaction (MLR), and the suppression depended on the dosage of UV-B. Even a low dosage of UV-B, 10 J/m 2 , could inhibit the MLR if the irradiated cells were incubated at 37degC for 24 hours before co-culture with responder cells. Treatments of cells with the exogenous singlet oxygen or superoxide anions also caused suppression of the stimulatory function in the MLR, inhibition of capping formation of HLA-DR antigens, and an increase of intracellular free Ca 2+ levels as did the UV-B treatment. These results indicate that the active oxygen or radicals generated in UV-B-irradiated leukocytes could be one of the causes of leukocyte inactivation. (author0

Studies on N5-methyltetrahydrofolate-homocystein methyltransferase in normal and leukemia leukocytes.

Science.gov (United States)

Peytremann, R; Thorndike, J; Beck, W S

1975-11-01

A cobalamin-dependent N5-methyltetra-hydrofolate-homocysteine methyltransferase (methyl-transferase) was demonstrated in unfractioned extracts of human normal and leukemia leukocytes. Activity was substantially reduced in the absence of an added cobalamin derivative. Presumably, this residual activity reflects the endogeneous level of holoenzyme. Enzyme activity was notably higher in lymphoid cells than in myeloid cells. Thus, mean specific activities (+/-SD) were: chronic lymphocytic leukemia lymphocytes, 2.15+/-1.16; normal lymphocytes, 0.91+/-0.59; normal mature granulocytes, 0.15+/-0.10; chronic myelocytic leukemia granulocytes, barely detectable activity. Properties of leukocytes enzymes resembled those of methyltransferases previously studied in bacteria and other animal cells. Granulocytes and chronic myelocytic leukemia cells contain a factor or factors that inhibits Escherichia coli enzyme. The data suggest that the prominence of this cobalamin-dependent enzyme in lymphocytes and other mononuclear cell types may be related to their potential for cell division.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

Energy Technology Data Exchange (ETDEWEB)

Giovannelli, Lisa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisag@pharm.unifi.it; Bellandi, Serena [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Pitozzi, Vanessa [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Fabbri, Paolo [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Dolara, Piero [Department of Preclinical and Clinical Pharmacology, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Viale Pieraccini 6, 50139 Florence (Italy)

2004-11-22

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo.

Increased oxidative DNA damage in mononuclear leukocytes in vitiligo

International Nuclear Information System (INIS)

Giovannelli, Lisa; Bellandi, Serena; Pitozzi, Vanessa; Fabbri, Paolo; Dolara, Piero; Moretti, Silvia

2004-01-01

Vitiligo is an acquired pigmentary disorder of the skin of unknown aetiology. The autocytotoxic hypothesis suggests that melanocyte impairment could be related to increased oxidative stress. Evidences have been reported that in vitiligo oxidative stress might also be present systemically. We used the comet assay (single cell alkaline gel electrophoresis) to evaluate DNA strand breaks and DNA base oxidation, measured as formamidopyrimidine DNA glycosylase (FPG)-sensitive sites, in peripheral blood cells from patients with active vitiligo and healthy controls. The basal level of oxidative DNA damage in mononuclear leukocytes was increased in vitiligo compared to normal subjects, whereas DNA strand breaks (SBs) were not changed. This alteration was not accompanied by a different capability to respond to in vitro oxidative challenge. No differences in the basal levels of DNA damage in polymorphonuclear leukocytes were found between patients and healthy subjects. Thus, this study supports the hypothesis that in vitiligo a systemic oxidative stress exists, and demonstrates for the first time the presence of oxidative alterations at the nuclear level. The increase in oxidative DNA damage shown in the mononuclear component of peripheral blood leukocytes from vitiligo patients was not particularly severe. However, these findings support an adjuvant role of antioxidant treatment in vitiligo

Tuft (caveolated) cells in two human colon carcinoma cell lines.

OpenAIRE

Barkla, D. H.; Whitehead, R. H.; Foster, H.; Tutton, P. J.

1988-01-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting f...

Lignans From Forsythia x Intermedia Leaves and Flowers Attenuate the Pro-inflammatory Function of Leukocytes and Their Interaction With Endothelial Cells

Science.gov (United States)

Michalak, Barbara; Filipek, Agnieszka; Chomicki, Piotr; Pyza, Małgorzata; Woźniak, Marta; Żyżyńska-Granica, Barbara; Piwowarski, Jakub P.; Kicel, Agnieszka; Olszewska, Monika A.; Kiss, Anna K.

2018-01-01

Aim of the study: Taking into account that overactivated leukocytes are an important factor in the development of many chronic diseases, we investigated the activity of phytochemically characterized (HPLC-DAD-MSn) extracts from forsythia leaves and flowers on the pro- and anti-inflammatory functions of leukocytes (effects on IL-1β, IL-8, TNF-α, and TGFβ release) and their adherence to endothelial cells. Using bio-guided fractionation, we isolated the active compounds and determined their biological activity, and we included the positive control quercetin. Methods: The effect on IL-1β, TNF-α, IL-8, and TGF-α production by leukocytes was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesion molecules was analyzed with flow cytometry, and the neutrophil attachment to the endothelial cells was assessed fluorimetrically. The effects on p38MAPK, ERK1/2 and JNK phosphorylation were determined using western blots. Results: Leaf extracts had the effect of decreasing TNF-α production in neutrophils and monocyte/macrophage cells. The bio-guided fractionation led to the isolation of the following lignan aglycones: (+)-pinoresinol, (+)-epipinoresinol, (−)-matairesinol, (+)-phillygenin, and (−)-arctigenin. Only phillygenin was able to stimulate the anti-inflammatory function of macrophages by inducing TGF-β release and IL-10 receptor surface expression. Arctigenin, phillygenin, and a metabolite produced by the gut microbiota, enterolactone, decreased TNF-α and IL-1β production and neutrophil adhesion to endothelial cells, probably by attenuating the p38 and ERK kinase pathways. Conclusion: Forsythia x intermedia is a valuable source of active lignans, which may be potential candidates for treating inflammatory diseases that are associated with the excessive production of cytokines such as TNF-α and IL-1β. PMID:29740324

Lignans From Forsythia x Intermedia Leaves and Flowers Attenuate the Pro-inflammatory Function of Leukocytes and Their Interaction With Endothelial Cells

Directory of Open Access Journals (Sweden)

Barbara Michalak

2018-04-01

Full Text Available Aim of the study: Taking into account that overactivated leukocytes are an important factor in the development of many chronic diseases, we investigated the activity of phytochemically characterized (HPLC-DAD-MSn extracts from forsythia leaves and flowers on the pro- and anti-inflammatory functions of leukocytes (effects on IL-1β, IL-8, TNF-α, and TGFβ release and their adherence to endothelial cells. Using bio-guided fractionation, we isolated the active compounds and determined their biological activity, and we included the positive control quercetin.Methods: The effect on IL-1β, TNF-α, IL-8, and TGF-α production by leukocytes was measured by enzyme-linked immunosorbent assay (ELISA. The surface expression of adhesion molecules was analyzed with flow cytometry, and the neutrophil attachment to the endothelial cells was assessed fluorimetrically. The effects on p38MAPK, ERK1/2 and JNK phosphorylation were determined using western blots.Results: Leaf extracts had the effect of decreasing TNF-α production in neutrophils and monocyte/macrophage cells. The bio-guided fractionation led to the isolation of the following lignan aglycones: (+-pinoresinol, (+-epipinoresinol, (−-matairesinol, (+-phillygenin, and (−-arctigenin. Only phillygenin was able to stimulate the anti-inflammatory function of macrophages by inducing TGF-β release and IL-10 receptor surface expression. Arctigenin, phillygenin, and a metabolite produced by the gut microbiota, enterolactone, decreased TNF-α and IL-1β production and neutrophil adhesion to endothelial cells, probably by attenuating the p38 and ERK kinase pathways.Conclusion:Forsythia x intermedia is a valuable source of active lignans, which may be potential candidates for treating inflammatory diseases that are associated with the excessive production of cytokines such as TNF-α and IL-1β.

Lignans From Forsythia x Intermedia Leaves and Flowers Attenuate the Pro-inflammatory Function of Leukocytes and Their Interaction With Endothelial Cells.

Science.gov (United States)

Michalak, Barbara; Filipek, Agnieszka; Chomicki, Piotr; Pyza, Małgorzata; Woźniak, Marta; Żyżyńska-Granica, Barbara; Piwowarski, Jakub P; Kicel, Agnieszka; Olszewska, Monika A; Kiss, Anna K

2018-01-01

Aim of the study: Taking into account that overactivated leukocytes are an important factor in the development of many chronic diseases, we investigated the activity of phytochemically characterized (HPLC-DAD-MS n ) extracts from forsythia leaves and flowers on the pro- and anti-inflammatory functions of leukocytes (effects on IL-1β, IL-8, TNF-α, and TGFβ release) and their adherence to endothelial cells. Using bio-guided fractionation, we isolated the active compounds and determined their biological activity, and we included the positive control quercetin. Methods: The effect on IL-1β, TNF-α, IL-8, and TGF-α production by leukocytes was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesion molecules was analyzed with flow cytometry, and the neutrophil attachment to the endothelial cells was assessed fluorimetrically. The effects on p38MAPK, ERK1/2 and JNK phosphorylation were determined using western blots. Results: Leaf extracts had the effect of decreasing TNF-α production in neutrophils and monocyte/macrophage cells. The bio-guided fractionation led to the isolation of the following lignan aglycones: (+)-pinoresinol, (+)-epipinoresinol, (-)-matairesinol, (+)-phillygenin, and (-)-arctigenin. Only phillygenin was able to stimulate the anti-inflammatory function of macrophages by inducing TGF-β release and IL-10 receptor surface expression. Arctigenin, phillygenin, and a metabolite produced by the gut microbiota, enterolactone, decreased TNF-α and IL-1β production and neutrophil adhesion to endothelial cells, probably by attenuating the p38 and ERK kinase pathways. Conclusion: Forsythia x intermedia is a valuable source of active lignans, which may be potential candidates for treating inflammatory diseases that are associated with the excessive production of cytokines such as TNF-α and IL-1β.

IgE production by normal human B cells induced by alloreactive T cell clones is mediated by IL-4 and suppressed by IFN-gamma

NARCIS (Netherlands)

Pène, J.; Rousset, F.; Brière, F.; Chrétien, I.; Paliard, X.; Banchereau, J.; Spits, H.; de Vries, J. E.

1988-01-01

Seven T cell clones were established from mixed leukocyte cultures in which PBMC from two healthy donors and from one patient suffering from the hyper-IgE syndrome were stimulated by the irradiated EBV-transformed B cell lines JY or UD53. Five of seven T cell clones, after activation by

Role of flexural stiffness of leukocyte microvilli in adhesion dynamics

Science.gov (United States)

Wu, Tai-Hsien; Qi, Dewei

2018-03-01

Previous work reported that microvillus deformation has an important influence on dynamics of cell adhesion. However, the existing studies were limited to the extensional deformation of microvilli and did not consider the effects of their bending deformation on cell adhesion. This Rapid Communication investigates the effects of flexural stiffness of microvilli on the rolling process related to adhesion of leukocytes by using a lattice-Boltzmann lattice-spring method (LLM) combined with adhesive dynamics (AD) simulations. The simulation results reveal that the flexural stiffness of microvilli and their bending deformation have a profound effect on rolling velocity and adhesive forces. As the flexural stiffness of the microvilli decreases, their bending angles increase, resulting in an increase in the number of receptor-ligand bonds and adhesive bonding force and a decrease in the rolling velocity of leukocytes. The effects of flexural stiffness on deformation and adhesion represent crucial factors involved in cell adhesion.

HK2 Recruitment to Phospho-BAD Prevents Its Degradation, Promoting Warburg Glycolysis by Theileria-Transformed Leukocytes.

Science.gov (United States)

Haidar, Malak; Lombès, Anne; Bouillaud, Frédéric; Kennedy, Eileen J; Langsley, Gordon

2017-03-10

Theileria annulata infects bovine leukocytes, transforming them into invasive, cancer-like cells that cause the widespread disease called tropical theileriosis. We report that in Theileria-transformed leukocytes hexokinase-2 (HK2) binds to B cell lymphoma-2-associated death promoter (BAD) only when serine (S) 155 in BAD is phosphorylated. We show that HK2 recruitment to BAD is abolished by a cell-penetrating peptide that acts as a nonphosphorylatable BAD substrate that inhibits endogenous S155 phosphorylation, leading to complex dissociation and ubiquitination and degradation of HK2 by the proteasome. As HK2 is a critical enzyme involved in Warburg glycolysis, its loss forces Theileria-transformed macrophages to switch back to HK1-dependent oxidative glycolysis that down-regulates macrophage proliferation only when they are growing on glucose. When growing on galactose, degradation of HK2 has no effect on Theileria-infected leukocyte proliferation, because metabolism of this sugar is independent of hexokinases. Thus, targeted disruption of the phosphorylation-dependent HK2/BAD complex may represent a novel approach to control Theileria-transformed leukocyte proliferation.

Analysis of Swine Leukocyte Antigen Peptide Binding Profiles and the Identification of T cell Epitopes by Tetramer Staining

DEFF Research Database (Denmark)

Pedersen, Lasse Eggers

class I peptide binding characteristics in relation to immune responses to vaccination or infection. Applying proven technologies to newly produced, recombinant swine leukocyte antigen (SLA) class I proteins yielded a body of data for peptide:SLA:β2m (pSLA) complex affinity and stability. Mapping...... system to specifically identify and react upon non-self peptide fragments unique only to the foreign intruder. The polymorphism of the MHC molecule effectively individualizes the immune response of each member of any given species. Moreover, responding T cells recognize antigen ligands, only...... in the context of peptide:MHC:β2m (pMHC) complex. The gene encoding the MHC is one of the most polymorphic regions of the genome known. Despite thousands of different human leukocyte antigen (HLA) variants identified, each member of a species only inherits and expresses a few of these MHC alleles. The “MHC...

Activation of human leukocytes on tantalum trabecular metal in comparison to commonly used orthopedic metal implant materials.

Science.gov (United States)

Schildhauer, T A; Peter, E; Muhr, G; Köller, M

2009-02-01

We analyzed leukocyte functions and cytokine response of human leukocytes toward porous tantalum foam biomaterial (Trabecular Metaltrade mark, TM) in comparison to equally sized solid orthopedic metal implant materials (pure titanium, titanium alloy, stainless steel, pure tantalum, and tantalum coated stainless steel). Isolated peripheral blood mononuclear cells (PBMC) and polymorphonuclear neutrophil leukocytes (PMN) were cocultured with equally sized metallic test discs for 24 h. Supernatants were analyzed for cytokine content by enzyme-linked immunosorbent assay. Compared to the other used test materials there was a significant increase in the release of IL (interleukin)-1ra and IL-8 from PMN, and of IL-1ra, IL-6, and TNF-alpha from PBMC in response to the TM material. The cytokine release correlated with surface roughness of the materials. In contrast, the release of IL-2 was not induced showing that mainly myeloid leukocytes were activated. In addition, supernatants of these leukocyte/material interaction (conditioned media, CM) were subjected to whole blood cell function assays (phagocytosis, chemotaxis, bacterial killing). There was a significant increase in the phagocytotic capacity of leukocytes in the presence of TM-conditioned media. The chemotactic response of leukocytes toward TM-conditioned media was significantly higher compared to CM obtained from other test materials. Furthermore, the bactericidal capacity of whole blood was enhanced in the presence of TM-conditioned media. These results indicate that leukocyte activation at the surface of TM material induces a microenvironment, which may enhance local host defense mechanisms.

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)

Characterization of a Merkel Cell Polyomavirus-Positive Merkel Cell Carcinoma Cell Line CVG-1.

Science.gov (United States)

Velásquez, Celestino; Amako, Yutaka; Harold, Alexis; Toptan, Tuna; Chang, Yuan; Shuda, Masahiro

2018-01-01

Merkel cell polyomavirus (MCV) plays a causal role in ∼80% of Merkel cell carcinomas (MCC). MCV is clonally integrated into the MCC tumor genome, which results in persistent expression of large T (LT) and small T (sT) antigen oncoproteins encoded by the early locus. In MCV-positive MCC tumors, LT is truncated by premature stop codons or deletions that lead to loss of the C-terminal origin binding (OBD) and helicase domains important for replication. The N-terminal Rb binding domain remains intact. MCV-positive cell lines derived from MCC explants have been valuable tools to study the molecular mechanism of MCV-induced Merkel cell carcinogenesis. Although all cell lines have integrated MCV and express truncated LT antigens, the molecular sizes of the LT proteins differ between cell lines. The copy number of integrated viral genome also varies across cell lines, leading to significantly different levels of viral protein expression. Nevertheless, these cell lines share phenotypic similarities in cell morphology, growth characteristics, and neuroendocrine marker expression. Several low-passage MCV-positive MCC cell lines have been established since the identification of MCV. We describe a new MCV-positive MCV cell line, CVG-1, with features distinct from previously reported cell lines. CVG-1 tumor cells grow in more discohesive clusters in loose round cell suspension, and individual cells show dramatic size heterogeneity. It is the first cell line to encode an MCV sT polymorphism resulting in a unique leucine (L) to proline (P) substitution mutation at amino acid 144. CVG-1 possesses a LT truncation pattern near identical to that of MKL-1 cells differing by the last two C-terminal amino acids and also shows an LT protein expression level similar to MKL-1. Viral T antigen knockdown reveals that, like other MCV-positive MCC cell lines, CVG-1 requires T antigen expression for cell proliferation.

Variation in sister chromatid exchange frequencies between human and pig whole blood, plasma leukocyte, and mononuclear leukocyte cultures

International Nuclear Information System (INIS)

Larramendy, M.L.; Reigosa, M.A.

1986-01-01

Sister chromatid exchange (SCE) induction by ultraviolet (UV) light was studied in both human and pig whole blood cultures (WBC) and plasma leukocyte cultures (PLC). No variation in SCE frequency was observed between pig WBC and PLC in control as well as in treated cells. Conversely, SCE frequencies of human PLC were consistently higher than those of WBC in control and UV-exposed cells. Thus, red blood cells (RBCs) do not influence the sensitivity of lymphocytes to UV LIGHT exposure, and there must be some different culture condition(s) in the inducation of SCEs between human WBC and PLC but not in swine lymphocyte cultures. Since the BrdUrd/lymphocyte ratio of WBC was halved in PLC, the effect of BrdUrd concentration in inducing the SCE baseline frequency of PLC may be ruled out. Neither the cell separation technique nor polymorphonuclear leukocytes had a significant role in the elevated SCE frequency of human PLC or MLC. Experiments where human RBCs were titrated into human PLC showed that the induction of an elevated SCE frequency of PLC was suppressed in a dose-dependent manner by the presence of RBCs in the culture medium. Since the incorporation of pig or human RBCs into human PLC as well as into MLC reduced the SCE frequency to that of WBC, a common component and/or function existing in these cells is suggested. Analysis of different RBC components showed that RBCs, specifically RBC ghosts, release a diffusible but not dialyzable corrective factor into culture medium that is able to reduce the SCE frequencies of PLC

The Cellosaurus, a Cell-Line Knowledge Resource

Science.gov (United States)

Bairoch, Amos

2018-01-01

The Cellosaurus is a knowledge resource on cell lines. It aims to describe all cell lines used in biomedical research. Its scope encompasses both vertebrates and invertebrates. Currently, information for >100,000 cell lines is provided. For each cell line, it provides a wealth of information, cross-references, and literature citations. The Cellosaurus is available on the ExPASy server (https://web.expasy.org/cellosaurus/) and can be downloaded in a variety of formats. Among its many uses, the Cellosaurus is a key resource to help researchers identify potentially contaminated/misidentified cell lines, thus contributing to improving the quality of research in the life sciences. PMID:29805321

Reduced antibody-dependent cellular cytotoxicity to herpes simplex virus-infected cells of salivary polymorphonuclear leukocytes and inhibition of peripheral blood polymorphonuclear leukocyte cytotoxicity by saliva.

Science.gov (United States)

Ashkenazi, M; Kohl, S

1990-06-15

Blood polymorphonuclear leukocytes (BPMN) have been shown to mediate antibody-dependent cellular cytotoxicity (ADCC) against HSV-infected cells. Although HSV infections are frequently found in the oral cavity, the ADCC capacity of salivary PMN (SPMN) has not been studied, mainly because methods to isolate SPMN were not available. We have recently developed a method to isolate SPMN, and in this study have evaluated their ADCC activity against HSV-infected cells. SPMN were obtained by repeated washings of the oral cavity, and separated from epithelial cells by nylon mesh filtration. ADCC was quantitatively determined by 51Cr release from HSV-infected Chang liver cells. SPMN in the presence of antibody were able to destroy HSV-infected cells, but SPMN were much less effective in mediating ADCC than BPMN (3.4% vs 40.7%, p less than 0.0001). In the presence of antiviral antibody, SPMN were able to adhere to HSV-infected cells, but less so than BPMN (34% vs 67%), and specific antibody-induced adherence was significantly lower in SPMN (p less than 0.04). The spontaneous adherence to HSV-infected cells was higher for SPMN than BPMN. SPMN demonstrated up-regulation of the adhesion glycoprotein CD18, but down-regulation of the FcRIII receptor. Incubation with saliva decreased ADCC capacity of BPMN, up-regulated CD18 expression, and down-regulated FcRIII expression.

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/....

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...

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

Science.gov (United States)

Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

2009-04-01

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

Monitoring of benzene-induced hematotoxicity in mice by serial leukocyte counting using a microcavity array.

Science.gov (United States)

Hosokawa, Masahito; Asami, Marie; Yoshino, Tomoko; Tsujimura, Noriyuki; Takahashi, Masayuki; Nakasono, Satoshi; Tanaka, Tsuyoshi; Matsunaga, Tadashi

2013-02-15

Monitoring of hematotoxicity, which requires serial blood collection, is difficult to carry out in small animals due to a lack of non-invasive, individual animal-appropriate techniques that enable enumeration of leukocyte subsets from limited amounts of whole blood. In this study, a microfluidic device equipped with a microcavity array that enables highly efficient separation of leukocytes from submicroliters of whole blood was applied for hematotoxicity monitoring in mice. The microcavity array can specifically separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Mouse leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered almost 100% of mouse leukocytes in 0.1 μL of whole blood without the effect of serial blood collection such as changes in body weight and total leukocyte count. We assessed benzene-associated hematotoxicity in mice using this system. Mice were administered with benzene once daily and the depression of leukocyte numbers induced in individual mice was successfully monitored from tail vein blood collected every other day for 2 weeks. Serial monitoring of the leukocyte number in individual mice will contribute to the understanding of hematotoxicity and reduction of the number of animal experiment trials. Copyright © 2012 Elsevier B.V. All rights reserved.

Rapid, high-efficiency labeling of leukocytes with In-111 after hemolytic removal of erythrocytes

International Nuclear Information System (INIS)

Karesh, S.M.; Henkin, R.E.

1985-01-01

During the labeling of leukocytes with Indium-111, conventional methodology involves separation and washing to remove red cells. This technique results in the loss of a significant number of leukocytes. Citrated whole blood of ten normal volunteers was studied for an alternate labeling method following sedimentation for 30 to 45 minutes and low speed centrifugation of the leukocyte-rich plasma. The average labeling for these ten volunteers by Indium-111 was 90% versus 60% by the older technique. Viability as measured by the trypan blue exclusion test was greater than 95%, WBC losses were essentially zero, and no WBC clumping was observed. Eighteen patients referred for leukocyte imaging were studied by this method. In this patient population, there was 91% labeling with viability greater than 95% and no evidence of clumping. Less than 5% RBC's were noted in any lot. Indium-111 WBC activity 20 minutes post injection averaged 79% of whole blood activity. This modification results in decreased losses of white cells, reduces preparation time to less than 2 hours, and significantly improves the labeling efficiency of the final product. Liver/spleen ratios and image quality were unchanged from the original method

Effect of radiographic contrast agents on leukocyte metabolic response

International Nuclear Information System (INIS)

Hernanz-Schulman, M.; Vanholder, R.; Waterloos, M.A.; Hakim, R.; Schulman, G.

2000-01-01

Barium, at clinical dilutions, causes a significant increase of baseline ''resting state'' phagocytic activity, which in turn leads to significant blunting of subsequent response to phagocytic challenge and adversely affects the response to all bacteria tested. There is no baseline activation of leukocytes by the water-soluble media, although there was some inhibition (rather than activation) of leukocyte metabolic activity. The effect of the water-soluble media in bacteria was more complex (although inhibition is minor compared to barium). Our data demonstrate that barium is a significant activator of phagocytic cells, which results in deactivation of phagocytic response when challenged; these data serve to explain the enhanced adverse effect of barium in cased of fecal peritonitis. (orig.)

Infiltrating leukocytes confound the detection of E-cadherin promoter methylation in tumors

International Nuclear Information System (INIS)

Lombaerts, Marcel; Middeldorp, Janneke W.; Weide, Esther van der; Philippo, Katja; Wezel, Tom van; Smit, Vincent T.H.B.M.; Cornelisse, Cees J.; Cleton-Jansen, Anne-Marie

2004-01-01

Promoter hypermethylation is known to result in transcriptional downregulation of many genes including the CDH1 gene. In this study we set out to determine CDH1 promoter methylation in breast tumors with decreased or absent E-cadherin protein expression and without CDH1 gene mutations by methylation-specific PCR (MSP). Interestingly, some tumor samples with normal E-cadherin expression yielded a methylation-specific PCR product. We hypothesized that other cells than tumor cells contribute to these products. Since in normal breast tissue no CDH1 promoter methylation is detected, infiltrating leukocytes, often present in tumors, might account for these methylation-specific fragments. Indeed, a methylation-specific fragment is found in all twelve leukocyte samples tested. Furthermore, activated T-cells also yielded a methylation-specific fragment. Sequencing of these fragments reveals two distinct methylation profiles. Leukocytes have only partial methylation of some CpGs, while the tumor-associated methylation profile shows complete methylation of most CpGs. Therefore, to assess whether CDH1 methylation is tumor associated, sequencing of MSP products is a prerequisite. Here we show that out of six lobular tumors lacking E-cadherin protein expression, three have tumor-associated CDH1 promoter methylation while in three other tumors no methylation is detected

Scintigraphy with In-111 labeled leukocytes

International Nuclear Information System (INIS)

Itoh, Kazuo; Tsukamoto, Eriko; Furudate, Masayori; Saito, Chihoko.

1987-01-01

With increasing necessity for In-111 labeled leukocyte scintigraphy (ILLS) as a routine examination, a problem of complicated labeling of leukocytes has arisen. In this study, simplified labeling of leukocytes was examined with respect to its ability to detect abscesses. Simplified labeling method yielded significantly satisfactory results for recovery and labeling rates of leukocytes, as compared with conventional recommended method. Therefore, ILLS by simplified technique was clinically applied in 58 patients with suppurative or non-suppurative diseases who gave informed consent. In an analysis of ILLS for detecting suppurative region, the sensitivity, specificity, and corrected specificity were found to be 81 %, 75 %, and 82 %, respectively. (Namekawa, K.)

Monitoring cell line identity in collections of human induced pluripotent stem cells

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Raquel Sarafian

2018-04-01

Full Text Available The ability to reprogram somatic cells into induced pluripotent stem cells (hiPSCs has led to the generation of large collections of cell lines from thousands of individuals with specific phenotypes, many of which will be shared among different research groups as invaluable tools for biomedical research. As hiPSC-based research involves extensive culture of many cell lines, the issue periodic cell line identification is particularly important to ensure that cell line identity remains accurate. Here we analyzed the different commercially available genotyping methods considering ease of in-house genotyping, cost and informativeness, and applied one of them in our workflow for hiPSC generation. We show that the chosen STR method was able to establish a unique DNA profile for each of the 35 individuals/hiPSC lines at the examined sites, as well as identify two discrepancies resulting from inadvertently exchanged samples. Our results highlight the importance of hiPSC line genotyping by an in-house method that allows periodic cell line identification and demonstrate that STR is a useful approach to supplement less frequent karyotyping and epigenetic evaluations. Keywords: Induced pluripotent stem cells, Genotyping, Cell line identification, Short tandem repeats, Quality control

Cell surface expression level variation between two common Human Leukocyte Antigen alleles, HLA-A2 and HLA-B8, is dependent on the structure of the C terminal part of the alpha 2 and the alpha 3 domains

DEFF Research Database (Denmark)

Dellgren, Christoffer; Nehlin, Jan O; Barington, Torben

2015-01-01

Constitutive cell surface expression of Human Leukocyte Antigen (HLA) class I antigens vary extremely from tissue to tissue and individual antigens may differ widely in expression levels. Down-regulation of class I expression is a known immune evasive mechanism used by cancer cells and viruses....... Moreover, recent observations suggest that even minor differences in expression levels may influence the course of viral infections and the frequency of complications to stem cell transplantation. We have shown that some human multipotent stem cells have high expression of HLA-A while HLA-B is only weakly...... expressed, and demonstrate here that this is also the case for the human embryonic kidney cell line HEK293T. Using quantitative flow cytometry and quantitative polymerase chain reaction we found expression levels of endogenous HLA-A3 (median 71,204 molecules per cell) 9.2-fold higher than the expression of...

A microautoradiographic method permitting the study of 99m-technetium labelled leukocytes

International Nuclear Information System (INIS)

Colas-Linhart, N.; Perianin, A.; Petiet, A.; Bretillon, A.; Bok, B.

1985-01-01

Cell migration studies are very important in inflammatory phenomena. Methods currently used are not quantitative and have been subject to much controversy. Homogeneity of sup(99m)Tc leukocyte labelling was verified by a microautoradiographic method (MAR), which was performed in our laboratory. This method was used in cell migration studies to verify if the migrating cells were indeed the labelled cells [fr

Human Leukocyte Antigen-G and Regulatory T Cells during Specific Immunotherapy for Pollen Allergy

DEFF Research Database (Denmark)

Sørensen, Anja Elaine; Johnsen, Claus R; Dalgaard, Louise Torp

2013-01-01

of the cytokine profile towards a TH1-polarized immune response. We investigated the effects of SIT on T cells, on immunomodulation of human leukocyte antigen (HLA)-G, which has been associated with allergy, on regulatory cytokine expression, and on serum allergen-specific antibody subclasses (IgE and IgG4......). Methods: Eleven birch and/or grass pollen-allergic patients and 10 healthy nonatopic controls were studied before and during SIT. Tregs, chemokine receptors, soluble HLA-G (sHLA-G), Ig-like transcript (ILT) 2, specific IgE, and IgG4 were studied. Peripheral blood mononuclear cells (PBMCs) were stimulated...... with pollen extract in vitro and immune factors were evaluated. Results: During SIT, the main changes in the peripheral blood were an increase in CXCR3+CD4+CD25+CD127low/- Tregs and a decrease in CCR4+CD4+CD25+CD127low/- Tregs, an increase in allergen-specific IgG4, and a decrease in sHLA-G during the first...

Pulmonary leukocytic responses are linked to the acquired immunity of mice vaccinated with irradiated cercariae of Schistosoma mansoni

International Nuclear Information System (INIS)

Aitken, R.; Coulson, P.S.; Wilson, R.A.

1988-01-01

Pulmonary cellular responses in C57BL/6 mice exposed to Schistosoma mansoni have been investigated by sampling cells from the respiratory airways with bronchoalveolar lavage. Mice exposed to cercariae attenuated with 20 krad gamma-radiation developed stronger and more persistent pulmonary leukocytic responses than animals exposed to equal numbers of normal parasites. Although vaccination with irradiated cercariae also stimulated T cell responses of greater magnitude and duration than normal infection, the lymphocytic infiltrate elicited by each regimen did not differ substantially in its composition, 5 wk after exposure. Studies with cercariae attenuated by different treatments established that a link exists between the recruitment of leukocytes to the lungs of vaccinated mice and resistance to reinfection. There was a strong association between pulmonary leukocytic responses and the elimination of challenge infections by vaccinated mice. Animals exposed to irradiated cercariae of S. mansoni were resistant to homologous challenge infection but were not protected against Schistosoma margrebowiei. Homologous challenge of vaccinated mice stimulated anamnestic leukocytic and T lymphocytic responses in the lungs, 2 wk postinfection, but exposure of immunized animals to the heterologous species failed to trigger an expansion in these populations of cells. Our studies indicate that pulmonary leukocytes and T lymphocytes are intimately involved in the mechanism of vaccine-induced resistance to S. mansoni. It remains unclear whether these populations of cells initiate protective inflammatory reactions against challenge parasites in the lungs, or accumulate in response to the activation of the protective mechanism by other means

Establishment of cell lines with rat spermatogonial stem cell characteristics

NARCIS (Netherlands)

van Pelt, Ans M. M.; Roepers-Gajadien, Hermien L.; Gademan, Iris S.; Creemers, Laura B.; de Rooij, Dirk G.; van Dissel-Emiliani, Federica M. F.

2002-01-01

Spermatogonial cell lines were established by transfecting a mixed population of purified rat A(s) (stem cells), A(pr) and A(al) spermatogonia with SV40 large T antigen. Two cell lines were characterized and found to express Hsp90alpha and oct-4, specific markers for germ cells and A spermatogonia,

Identification of a second putative receptor of platelet activating factor on human polymorphonuclear leukocytes

International Nuclear Information System (INIS)

Hwang, S.B.

1987-01-01

Due to multiple molecular species of platelet activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors on human leukocytes and platelets. Human PMN leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (Kd) of 4.7 (+/- 1.4) x 10 -10 M. The maximal number (B/sub max/) of receptor sites was estimated to be 3.13 (+/- 1.4) x 10 -13 mol/mg protein. They compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One antagonist (Ono-6240) was found to be 8 times less potent at inhibiting the [ 3 H]PAF specific receptor binding to human leukocytes than to human platelets. Mg 2+ , Ca 2+ and K + ions potentiated the [ 3 H]PAF specific binding in both systems. Na + ions inhibited the [ 3 H]PAF specific binding to human platelets but showed no effects in human leukocytes. K + ions decreased the Mg 2+ -potentiated [ 3 H]PAF binding in human leukocytes but showed no effects in human platelets. These results suggest that the PAF specific receptors in human leukocytes are different structurally and possibly functionally from the receptors identified in human platelets

The recognition of adsorbed and denatured proteins of different topographies by β2 integrins and effects on leukocyte adhesion and activation

DEFF Research Database (Denmark)

Brevig, T.; Holst, B.; Ademovic, Z.

2005-01-01

Leukocyte beta(2) integrins Mac-1 and p150,95 are promiscuous cell-surface receptors that recognise and mediate cell adhesion to a variety of adsorbed and denatured proteins. We used albumin as a model protein to study whether leukocyte adhesion and activation depended on the nm-scale topography...

Comparison of steroid receptors from the androgen responsive DDT1 cell line and the nonresponsive HVP cell line.

Science.gov (United States)

Norris, J S; Kohler, P O

1978-01-01

Two hamster cell lines have been isolated from androgen target tissue. The DDT1 cells derived from ductus deferens tissue exhibit a growth response to androgens, while the HVP cells derived from ventral prostate are androgen unresponsive. Both cell lines contain androgen receptors, that are similar when compared by kinetic methods, sedimentation velocity, chromatographic procedures or nuclear translocation ability. The forms of the high salt extracted nuclear receptors are indistinguishable chromatographically. Therefore, we postulate that the lesion preventing androgen induced growth in the HVP cell line is subseqent to nuclear translocation of the steroid receptor complex.

Total and Differential Leukocyte Counts in Relation to Incidence of Diabetes Mellitus: A Prospective Population-Based Cohort Study.

Science.gov (United States)

Borné, Yan; Smith, J Gustav; Nilsson, Peter M; Melander, Olle; Hedblad, Bo; Engström, Gunnar

2016-01-01

High concentrations of leukocytes in blood have been associated with diabetes mellitus. This prospective study aimed to explore whether total and differential leukocyte counts are associated with incidence of diabetes. A missense variant R262W in the SH2B3 (SH2B adaptor protein 3) gene, coding for a protein that negatively regulates hematopoietic cell proliferation, was also studied in relation to incidence of diabetes. Leukocyte count and its subtypes (neutrophils, lymphocytes and mixed cells) were analyzed in 26,667 men and women, 45-73 years old, from the population-based Malmö Diet and Cancer study. Information about the R262W polymorphism (rs3184504) in SH2B3 was genotyped in 24,489 subjects. Incidence of diabetes was studied during a mean follow-up of 14 years. Cox proportional hazards regression was used to examine incidence of diabetes by total and differential leukocyte counts. Mendelian randomization analysis using R262W as an instrumental variable was performed with two-stage least squares regression. A total of 2,946 subjects developed diabetes during the follow-up period. After taking several possible confounders into account, concentrations of total leukocyte count, neutrophils and lymphocytes were all significantly associated with incidence of diabetes. The adjusted hazard ratios (95% confidence interval; quartile 4 vs quartile 1) were 1.37 (1.22-1.53) for total leukocytes, 1.33 (1.19-1.49) for neutrophils and 1.29 (1.15-1.44) for lymphocytes. The R262W polymorphism was strongly associated with leukocytes (0.11x109 cells/l per T allele, p = 1.14 x10-12), lymphocytes (p = 4.3 x10-16), neutrophils (p = 8.0 x10-6) and mixed cells (p = 3.0 x10-6). However, there was no significant association between R262W and fasting glucose, HbA1c or incidence of diabetes. Concentrations of total leukocytes, neutrophils and lymphocytes are associated with incidence of diabetes. However, the lack of association with the R262W polymorphism suggests that the associations

Therapeutic relevance of penicillin-induced hypersensitivity of Staphylococcus aureus to killing by polymorphonuclear leukocytes.

OpenAIRE

Lam, C; Georgopoulos, A; Laber, G; Schütze, E

1984-01-01

There is an overwhelming body of evidence that certain Staphylococcus aureus strains become more sensitive to killing by polymorphonuclear leukocytes after their growth in media containing subinhibitory concentrations of penicillin. However, it is not clear to what extent this phenomenon contributes to the curative effect of penicillin in vivo. To explore its therapeutic relevance, we evaluated the interaction of staphylococci pretreated with penicillin in vitro with leukocytes in cell-proof ...

Expression Levels of Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166 in Primary Breast Carcinoma and Distant Breast Cancer Metastases

Directory of Open Access Journals (Sweden)

M. Ihnen

2010-01-01

Full Text Available Introduction: Activated Leukocyte Cell Adhesion Molecule (ALCAM/CD166 gained increasing attention regarding tumorprogression and metastatic spread in breast cancer. The aim of this study was to examine ALCAM expression levels in primary breast cancer and distant metastases of the same patient within 29 autopsy cases to better understand the underlying mechanisms of metastases and the role of adhesion molecules in this process.

Pathway-specific differences between tumor cell lines and normal and tumor tissue cells

Directory of Open Access Journals (Sweden)

Tozeren Aydin

2006-11-01

Full Text Available Abstract Background Cell lines are used in experimental investigation of cancer but their capacity to represent tumor cells has yet to be quantified. The aim of the study was to identify significant alterations in pathway usage in cell lines in comparison with normal and tumor tissue. Methods This study utilized a pathway-specific enrichment analysis of publicly accessible microarray data and quantified the gene expression differences between cell lines, tumor, and normal tissue cells for six different tissue types. KEGG pathways that are significantly different between cell lines and tumors, cell lines and normal tissues and tumor and normal tissue were identified through enrichment tests on gene lists obtained using Significance Analysis of Microarrays (SAM. Results Cellular pathways that were significantly upregulated in cell lines compared to tumor cells and normal cells of the same tissue type included ATP synthesis, cell communication, cell cycle, oxidative phosphorylation, purine, pyrimidine and pyruvate metabolism, and proteasome. Results on metabolic pathways suggested an increase in the velocity nucleotide metabolism and RNA production. Pathways that were downregulated in cell lines compared to tumor and normal tissue included cell communication, cell adhesion molecules (CAMs, and ECM-receptor interaction. Only a fraction of the significantly altered genes in tumor-to-normal comparison had similar expressions in cancer cell lines and tumor cells. These genes were tissue-specific and were distributed sparsely among multiple pathways. Conclusion Significantly altered genes in tumors compared to normal tissue were largely tissue specific. Among these genes downregulation was a major trend. In contrast, cell lines contained large sets of significantly upregulated genes that were common to multiple tissue types. Pathway upregulation in cell lines was most pronounced over metabolic pathways including cell nucleotide metabolism and oxidative

Characterization of stem-like cells in a new astroblastoma cell line

Energy Technology Data Exchange (ETDEWEB)

Coban, Esra Aydemir; Kasikci, Ezgi [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Karatas, Omer Faruk [Molecular Biology and Genetics Department, Erzurum Technical University, Erzurum (Turkey); Suakar, Oznur; Kuskucu, Aysegul [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey); Altunbek, Mine [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Türe, Uğur [Department of Neurosurgery, Yeditepe University School of Medicine, Istanbul (Turkey); Sahin, Fikrettin [Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University, Istanbul (Turkey); Bayrak, Omer Faruk, E-mail: ofbayrak@yeditepe.edu.tr [Department of Medical Genetics, Yeditepe University Medical School and Yeditepe University Hospital, Istanbul (Turkey)

2017-03-15

Cell lines established from tumors are the most commonly used models in cancer research, and their use in recent years has enabled a greater understanding of the biology of cancer and the means to develop effective treatment strategies. Astroblastomas are uncommon neuroepithelial tumors of glial origin, predominantly affecting young people, mainly teenagers and children, predominantly females. To date, only a single study has reported that astroblastomas contain a large number of neural stem-like cells, which had only a partial proliferation capacity and differentiation. Our objective was to establish an astroblastoma cell line to investigate the presence of astroblastic cells and cancer stem-like cells. The migratory and invasion abilities of the cells were quantified with invasion and migration assays and compared to a glioblastoma cell line. The presence of stem cells was detected with surface-marker analysis by using flow cytometry, and measuring the differentiation ability with a differentiation assay and the self-renewal capacity with a sphere-forming assay. These characteristics may determine whether this novel cell line is a model for astroblastomas that may have stem-cell characteristics. With this novel cell line, scientists can investigate the molecular pathways underlying astroblastomas and develop new therapeutic strategies for patients with these tumors. - Highlights: • An establishment of a novel astroblastoma cell line was proposed. • The presence of astroblastic cells and cancer stem-like cells was investigated. • The molecular pathways underlying astroblastomas may be investigated. • New therapeutic strategies for patients with astroblastoma may be developed.

CD44 antibody stimulates adhesion of peripheral blood T cells to keratinocytes through the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 pathway

NARCIS (Netherlands)

Bruynzeel, I.; Koopman, G.; van der Raaij, L. M.; Pals, S. T.; Willemze, R.

1993-01-01

Close contact between T lymphocytes and keratinocytes is an important feature of many inflammatory skin diseases. In vitro studies showed that stimulation of keratinocytes with interferon-gamma or tumor necrosis factor-alpha and of T cells with phorbol esters results in a leukocyte

Effect of radiographic contrast agents on leukocyte metabolic response

Energy Technology Data Exchange (ETDEWEB)

Hernanz-Schulman, M. [Dept. of Pediatric Radiology, Vanderbilt Children' s Hospital, Nashville, TN (United States); Vanholder, R.; Waterloos, M.A. [Dept. of Internal Medicine, Nephrology Section, University Hospital, Gent (Belgium); Hakim, R.; Schulman, G. [Department of Nephrology, Vanderbilt University Medical Center, Nashville, TN (United States)

2000-06-01

Barium, at clinical dilutions, causes a significant increase of baseline ''resting state'' phagocytic activity, which in turn leads to significant blunting of subsequent response to phagocytic challenge and adversely affects the response to all bacteria tested. There is no baseline activation of leukocytes by the water-soluble media, although there was some inhibition (rather than activation) of leukocyte metabolic activity. The effect of the water-soluble media in bacteria was more complex (although inhibition is minor compared to barium). Our data demonstrate that barium is a significat activator of phagocytic cells, which results in deactivation of phagocytic response when challenged; these dsata serve to explain the enhanced adverse effect of barium in cased of fecal peritonitis. (orig.)

Phosphatidylcholine reverses ethanol-induced increase in transepithelial endotoxin permeability and abolishes transepithelial leukocyte activation

DEFF Research Database (Denmark)

Mitscherling, K.; Volynets, V.; Parlesak, Alexandr

2009-01-01

BACKGROUND: Chronic alcohol abuse increases both intestinal bacterial overgrowth and intestinal permeability to macromolecules. Intestinal permeability of endotoxin, a component of the outer cell membrane of Gram-negative bacteria, plays a crucial role in the development of alcohol-induced liver...... disease (ALD). As impaired bile flow leads to endotoxemia and the bile component phosphatidylcholine (PC) is therapeutically active in ALD, we tested the hypothesis that conjugated primary bile salts (CPBS) and PC inhibit ethanol-enhanced transepithelial permeability of endotoxin and the subsequent...... transepithelial activation of human leukocytes. METHODS: For this purpose, we used a model in which intestinal epithelial cells (Caco-2) were basolaterally cocultivated with mononuclear leukocytes. Cells were challenged apically with endotoxin from Escherichia coli K12 and were incubated with or without...

Adhesion of leukocytes under oscillating stagnation point conditions: a numerical study.

Science.gov (United States)

Walker, P G; Alshorman, A A; Westwood, S; David, T

2002-01-01

Leukocyte recruitment from blood to the endothelium plays an important role in atherosclerotic plaque formation. Cells show a primary and secondary adhesive process with primary bonds responsible for capture and rolling and secondary bonds for arrest. Our objective was to investigate the role played by this process on the adhesion of leukocytes in complex flow. Cells were modelled as rigid spheres with spring like adhesion molecules which formed bonds with endothelial receptors. Models of bond kinetics and Newton's laws of motion were solved numerically to determine cell motion. Fluid force was obtained from the local shear rate obtained from a CFD simulation of the flow over a backward facing step.In stagnation point flow the shear rate near the stagnation point has a large gradient such that adherent cells in this region roll to a high shear region preventing permanent adhesion. This is enhanced if a small time dependent perturbation is imposed upon the stagnation point. For lower shear rates the cell rolling velocity may be such that secondary bonds have time to form. These bonds resist the lower fluid forces and consequently there is a relatively large permanent adhesion region.

Effect of lithium carbonate on leukocyte number after influence of ionizing radiation. 2. Influence of lithium carbonate on peripheral leukocytes

Energy Technology Data Exchange (ETDEWEB)

Rose, H.; Kehrberg, G.; Saul, G.; Pradel, I. (Humboldt-Universitaet, Berlin (German Democratic Republic). Bereich Medizin (Charite))

1985-01-01

The increase of leukocyte number in peripheral blood, found after application of lithium carbonate, is attributed to a rise in granulocytes first of all. The reduced period of acute leukopenia after whole-body irradiation, caused by lithium, is the result of the stimulating the myeloid progenitor cells. Increased syntheses of colony stimulating factor or influencing factors on the microecology of bone marrow are discussed.

Human leukocyte mobilization and morphology in nickel contact allergy using a skin chamber technique

DEFF Research Database (Denmark)

Lerche, A; Bisgaard, H; Christensen, J D

1981-01-01

An improved skin chamber technique has been devised and used for quantitative evaluation of the leukocyte mobilization rate (LMR). The method was applied in 10 nickel-hypersensitive patients exposed to nickel sulphate. Each patient served as his own control and for additional control purpose, 5...... healthy individuals without nickel hypersensitivity were studied. The kinetics of the mobilized leukocytes were followed over a 48-hour period. After an initial lag phase of 2-4 hours, maximum migration was observed from the 24th to the 48th hour, with a wide interindividual variability in the number...... of mobilized cells at the time of maximum LMR response. The median cumulative leukocyte count was 1.412 x 10(6) leukocytes/cm2/48 h. In the same period a statistically significant increase in the basophils for all the nickel allergic patients was observed. In 8 out of 10 patients a statistically significant...

Activation of lysosomal cathepsins in pregnant bovine leukocytes.

Science.gov (United States)

Talukder, Md Abdus Shabur; Balboula, Ahmed Zaky; Shirozu, Takahiro; Kim, Sung Woo; Kunii, Hiroki; Suzuki, Toshiyuki; Ito, Tsukino; Kimura, Koji; Takahashi, Masashi

2018-06-01

In ruminants, interferon-tau (IFNT) - mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy. © 2018 Society for Reproduction and Fertility.

Cell fusion induced by ionizing radiation in various cell lines

International Nuclear Information System (INIS)

Khair, M.B.

1994-07-01

Cell fusion induced by ionizing radiation has been studied in rat's hepatocytes in vivo and in different cell lines in vitro. These cell lines were: Hela cells, V-79 fibroblasts, human and rat lymphocytes. For irradiation, 0.85 MeV fission neutrons and 14 MeV fast neutrons were used. Cell analyses were performed by fluorescent dyes using immunofluorescent microscope and flow cytometre. Our results in vivo showed that, regardless the dose-rate, a dose of 1 Gy approximately was enough to induce a significant level of cell fusion depending on neutron energy and the age of rats. The level of cell fusion was also significant in Hela cells at a dose of 0.5 Gy. Similar effect, but to a lesser extent, was observed in V-79 cells. Whereas, in lymphocytes insignificant cell fusion was noticed. The varying levels of cell-fusion in different cell lines could be attributed to the type of cells and mutual contact between cells. Furthermore irradiation did not show any influence on cell division ability in both hepatocytes and Hela cells and that fused cells were also able to divide forming a new generation of cells. (author). 36 refs., 8 figs., 10 tabs

Failure to demonstrate a major role for Kupffer cells and radiosensitive leukocytes in immunoglobulin-mediated elimination of Trypanosoma musculi

International Nuclear Information System (INIS)

Kongshavn, P.A.; Shaw, K.; Ghadirian, E.; Ulczak, O.

1990-01-01

Previous studies have indicated that elimination of parasitemia in Trypanosoma musculi infection is brought about by immunoglobulin G2a antibodies, C3, and an effector cell. Experiments were designed to identify the putative effector cell by using several approaches. Infected C5-deficient or C5-sufficient mice treated with silica particles or given 900 rads of radiation 3 days earlier effectively eliminated trypanosomes following administration of immune plasma (IP). Silica-treated, noninfected mice given T. musculi preincubated with IP also cleared the parasites. Radiolabeling studies revealed that uptake of the cleared trypanosomes by the liver in normal mice was relatively low and fell only slightly (19%) in silica-treated mice. In contrast, uptake of radiolabeled sheep erythrocytes by the liver was normally much higher and fell drastically (7%) in silica-treated mice. Mice were then immunocompromised by 900 rads of radiation, silica particles, and anti-platelet serum combined before IP-sensitized trypanosomes were given. Leukocyte and platelet counts were both reduced by 95% and sheep erythrocyte uptake by the liver fell from 77 to 5%; however, greater than 99% of the injected trypanosomes were cleared in these mice and uptake of radiolabeled trypanosomes by the liver was similar to that of normal mice. Lastly, in anesthetized mice in which Kupffer cells were excluded surgically from the circulation, greater than 99% of the IP-sensitized trypanosomes disappeared rapidly from the blood. Only 7% of the radiolabel was found in the liver versus 60% in sham-operated mice. The results are interpreted as showing that hepatic Kupffer cells play a minor role in the immune elimination of T. musculi. Likewise, radiosensitive leukocytes and platelets are unlikely to be sole candidates for the putative effector cell that mediates a cure of murine trypanosomiasis

[The use of programmed microcalculators for automation of leukocyte count].

Science.gov (United States)

Plykin, D L

1989-01-01

Soviet programmed microcalculators are recommended to be used for the calculation of the leukocytic formulae when making serial blood analyses at clinical laboratories. The suggested program helps completely automate the process of estimating the leukocyte types, detectable in microscopic examination of the blood smears; the results may be obtained as a per cent ratio of the cells (a form most prevalent nowadays) and as their quantity per microliter of blood. The presence of service elements in the program essentially simplifies the work, making it convenient for an untrained user of the microcalculator. Since commercial Soviet programmed microcalculators somewhat differ in the systems of program steps, two variants of the program are suggested, adapted to the two most prevalent designs.

Human T-Lymphotropic Virus Type 1-Induced Overexpression of Activated Leukocyte Cell Adhesion Molecule (ALCAM) Facilitates Trafficking of Infected Lymphocytes through the Blood-Brain Barrier.

Science.gov (United States)

Curis, Céline; Percher, Florent; Jeannin, Patricia; Montange, Thomas; Chevalier, Sébastien A; Seilhean, Danielle; Cartier, Luis; Couraud, Pierre-Olivier; Gout, Olivier; Gessain, Antoine; Ceccaldi, Pierre-Emmanuel; Afonso, Philippe V

2016-08-15

Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease develops upon infiltration of HTLV-1-infected lymphocytes into the central nervous system, mostly the thoracic spinal cord. The central nervous system is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. In this study, we investigated the role of activated leukocyte cell adhesion molecule (ALCAM/CD166), a member of the immunoglobulin superfamily, in the crossing of the BBB by HTLV-1-infected lymphocytes. We demonstrated that ALCAM is overexpressed on the surface of HTLV-1-infected lymphocytes, both in chronically infected cell lines and in primary infected CD4(+) T lymphocytes. ALCAM overexpression results from the activation of the canonical NF-κB pathway by the viral transactivator Tax. In contrast, staining of spinal cord sections of HAM/TSP patients showed that ALCAM expression is not altered on the BBB endothelium in the context of HTLV-1 infection. ALCAM blockade or downregulation of ALCAM levels significantly reduced the migration of HTLV-1-infected lymphocytes across a monolayer of human BBB endothelial cells. This study suggests a potential role for ALCAM in HAM/TSP pathogenesis. Human T-lymphotropic virus type 1 (HTLV-1) is the etiological agent of a slowly progressive neurodegenerative disease, HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). This disease is the consequence of the infiltration of HTLV-1-infected lymphocytes into the central nervous system (CNS), mostly the thoracic spinal cord. The CNS is normally protected by a physiological structure called the blood-brain barrier (BBB), which consists primarily of a continuous endothelium with tight junctions. The mechanism of migration of lymphocytes into the CNS is unclear

A Metabolic Biofuel Cell: Conversion of Human Leukocyte Metabolic Activity to Electrical Currents

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Cui X Tracy

2011-05-01

Full Text Available Abstract An investigation of the electrochemical activity of human white blood cells (WBC for biofuel cell (BFC applications is described. WBCs isolated from whole human blood were suspended in PBS and introduced into the anode compartment of a proton exchange membrane (PEM fuel cell. The cathode compartment contained a 50 mM potassium ferricyanide solution. Average current densities between 0.9 and 1.6 μA cm-2 and open circuit potentials (Voc between 83 and 102 mV were obtained, which were both higher than control values. Cyclic voltammetry was used to investigate the electrochemical activity of the activated WBCs in an attempt to elucidate the mechanism of electron transfer between the cells and electrode. Voltammograms were obtained for the WBCs, including peripheral blood mononuclear cells (PBMCs - a lymphocyte-monocyte mixture isolated on a Ficoll gradient, a B lymphoblastoid cell line (BLCL, and two leukemia cell lines, namely K562 and Jurkat. An oxidation peak at about 363 mV vs. SCE for the PMA (phorbol ester activated primary cells, with a notable absence of a reduction peak was observed. Oxidation peaks were not observed for the BLCL, K562 or Jurkat cell lines. HPLC confirmed the release of serotonin (5-HT from the PMA activated primary cells. It is believed that serotonin, among other biochemical species released by the activated cells, contributes to the observed BFC currents.

THE PERIPHERAL BLOOD LEUKOCYTES SUSCEPTIBILITY TO INTERFERON-α2 IN VITRO CHANGE AMONGST CHILDREN WITH INFECTIOUS MONONUCLEOSIS

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L. M. Kurtasova

2017-01-01

Full Text Available Objective: to measure cell susceptibility of peripheral blood leukocytes to interferon-α2 in vitro at children during the acute period in 1 and 6 months after infectious mononucleosis caused by the Epstein–Barr virus. 47 children aged 4–6 years with moderately severe form of the disease in the acute period of infectious mononucleosis caused by the Epstein–Barr virus (EBV were examined, as well as in 1 month (n = 17 and 6 months (n = 11 after the disease. The focus group consisted of 36 nearly healthy children. The cell susceptibility to interferon-α2 in vitro was determined by the method of Kurtasova L.M. et al. (2007. Chemiluminescence of blood leukocytes was studied by De Sole et al. (1983. The susceptibility change of peripheral blood leukocytes to interferon-α2 in vitro at children with infectious mononucleosis caused by Epstein–Barr virus in dynamics of the disease has been revealed. The expansion of the range of cell susceptibility to interferon-α2 in 6 months after the disease has been enclosed. The dependence of the susceptibility of peripheral blood leukocytes to interferon-α2 in vitro on the dose and the period of the disease has been fixed.

Optimisation of the CT h4S bioassay for detection of human interleukin-4 secreted by mononuclear cells stimulated by phytohaemaglutinin or by human leukocyte antigen mismatched mixed lymphocyte culture

DEFF Research Database (Denmark)

Petersen, Søren Lykke; Russell, Charlotte Astrid; Bendtzen, Klaus

2002-01-01

S bioassay detects 5 pg/ml of human recombinant IL-4 with no detection of IL-2 in concentrations below 500 pg/ml. We have found 72 h of culture optimal for detection of IL-2 and IL-4 produced by human mononuclear cells (MNC) in response to stimulation with phytohaemaglutinin and for detection of IL......-2 in human leukocyte antigen (HLA)-mismatched mixed leukocyte culture (MLC). An interindividual variation in cytokine accumulation was demonstrated for IL-4 but not for IL-2. With the use of 5x10(4) responder cells/well no IL-4 could be detected in HLA-mismatched MLC between days 1 and 16. The lack...

Feeder-cell-independent culture of the pig-embryonic-stem-cell-derived exocrine pancreatic cell line, PICM-31

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The adaptation to feeder-independent growth of a pig embryonic stem cell-derived pancreatic cell line is described. The parental PICM-31 cell line, previously characterized as an exocrine pancreas cell line, was colony-cloned two times in succession resulting in the subclonal cell line, PICM-31A1. P...

Application of DNA fingerprints for cell-line individualization.

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Gilbert, D A; Reid, Y A; Gail, M H; Pee, D; White, C; Hay, R J; O'Brien, S J

1990-09-01

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children.

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Genoveva Keustermans

Full Text Available Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D and cardiovascular disease (CVD. The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children.13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2 and leptin receptor expression on peripheral blood mononuclear cell subsets was performed.Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI. The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also

Phosphatidylcholine Reverses Ethanol-Induced Increase in Transepithelial Endotoxin Permeability and Abolishes Transepithelial Leukocyte Activation

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Mitzscherling, Katja; Volynets, Valentina; Parlesak, Alexandr

2009-01-01

Chronic alcohol abuse increases both intestinal bacterial overgrowth and intestinal permeability to macromolecules. Intestinal permeability of endotoxin, a component of the outer cell membrane of Gram-negative bacteria, plays a crucial role in the development of alcohol-induced liver disease (ALD......). As impaired bile flow leads to endotoxemia and the bile component phosphatidylcholine (PC) is therapeutically active in ALD, we tested the hypothesis that conjugated primary bile salts (CPBS) and PC inhibit ethanol-enhanced transepithelial permeability of endotoxin and the subsequent transepithelial...... activation of human leukocytes. For this purpose, we used a model in which intestinal epithelial cells (Caco-2) were basolaterally cocultivated with mononuclear leukocytes. Cells were challenged apically with endotoxin from Escherichia coli K12 and were incubated with or without the addition of CPBS (1.5 m...

Natural killer cells for immunotherapy – Advantages of cell lines over blood NK cells

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Hans eKlingemann

2016-03-01

Full Text Available Natural killer cells are potent cytotoxic effector cells for cancer therapy and potentially for severe viral infections. However, there are technical challenges to obtain sufficient numbers of functionally active NK cells form a patient’s blood since they represent only 10% of the lymphocytes. Especially, cancer patients are known to have dysfunctional NK cells. The alternative is to obtain cells from a healthy donor, which requires depletion of the allogeneic T-cells. Establishing cell lines from donor blood NK cells have not been successful, in contrast to blood NK cells obtained from patients with a clonal NK cell lymphoma. Those cells can be expanded in culture in the presence of IL-2. However, except for the NK-92 cell line none of the other six known cell lines has consistent and reproducibly high anti-tumor cytotoxicity, nor can they be easily genetically manipulated to recognize specific tumor antigens or to augment monoclonal antibody activity through ADCC. NK-92 is also the only cell line product that has been widely given to patients with advanced cancer with demonstrated efficiency and minimal side effects.

Susceptibility testing of fish cell lines for virus isolation

DEFF Research Database (Denmark)

Ariel, Ellen; Skall, Helle Frank; Olesen, Niels Jørgen

2009-01-01

and laboratories, but also between lineages of the same cell line. To minimise the occurrence of false negatives in a cell culture based surveillance system, we have investigated methods, to select cell lineages that are relatively superior in their susceptibility to a panel of virus isolates. The procedures...... cell lineages, we increased the number of isolates of each virus, propagated stocks in a given cell line and tested all lineages of that line in use in the laboratory. Testing of relative cell line susceptibility between laboratories is carried out annually via the Inter-laboratory Proficiency Test...... sensitivity for surveillance purposes within a cell line and between laboratories.In terms of economic and practical considerations as well as attempting to approach a realistic test system, we suggest the optimal procedure for susceptibility testing of fish cell lines for virus isolation to be a combination...

Low pH immobilizes and kills human leukocytes and prevents transmission of cell-associated HIV in a mouse model

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Markham Richard B

2005-09-01

Full Text Available Abstract Background Both cell-associated and cell-free HIV virions are present in semen and cervical secretions of HIV-infected individuals. Thus, topical microbicides may need to inactivate both cell-associated and cell-free HIV to prevent sexual transmission of HIV/AIDS. To determine if the mild acidity of the healthy vagina and acid buffering microbicides would prevent transmission by HIV-infected leukocytes, we measured the effect of pH on leukocyte motility, viability and intracellular pH and tested the ability of an acidic buffering microbicide (BufferGel® to prevent the transmission of cell-associated HIV in a HuPBL-SCID mouse model. Methods Human lymphocyte, monocyte, and macrophage motilities were measured as a function of time and pH using various acidifying agents. Lymphocyte and macrophage motilities were measured using video microscopy. Monocyte motility was measured using video microscopy and chemotactic chambers. Peripheral blood mononuclear cell (PBMC viability and intracellular pH were determined as a function of time and pH using fluorescent dyes. HuPBL-SCID mice were pretreated with BufferGel, saline, or a control gel and challenged with HIV-1-infected human PBMCs. Results Progressive motility was completely abolished in all cell types between pH 5.5 and 6.0. Concomitantly, at and below pH 5.5, the intracellular pH of PBMCs dropped precipitously to match the extracellular medium and did not recover. After acidification with hydrochloric acid to pH 4.5 for 60 min, although completely immotile, 58% of PBMCs excluded ethidium homodimer-1 (dead-cell dye. In contrast, when acidified to this pH with BufferGel, a microbicide designed to maintain vaginal acidity in the presence of semen, only 4% excluded dye at 10 min and none excluded dye after 30 min. BufferGel significantly reduced transmission of HIV-1 in HuPBL-SCID mice (1 of 12 infected compared to saline (12 of 12 infected and a control gel (5 of 7 infected. Conclusion These

Leukocyte telomere dynamics in the elderly

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Steenstrup, Troels; Hjelmborg, Jacob V B; Mortensen, Laust H

2013-01-01

Limited data suggest that leukocytes of the elderly display ultra-short telomeres. It was reported that in some elderly persons leukocyte telomere length (LTL) shows age-dependent elongation. Using cross-sectional and longitudinal models, we characterized LTL dynamics in participants...

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

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Qin, J.-Z.; Xin, H. [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States); Nickoloff, B.J., E-mail: bnickol@lumc.edu [Oncology Institute, Cardinal Bernardin Cancer Center, Loyola University of Chicago Medical Center (United States)

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines.

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Qin, J-Z; Xin, H; Nickoloff, B J

2010-05-28

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma. Copyright (c) 2010 Elsevier Inc. All rights reserved.

3-Bromopyruvate induces necrotic cell death in sensitive melanoma cell lines

International Nuclear Information System (INIS)

Qin, J.-Z.; Xin, H.; Nickoloff, B.J.

2010-01-01

Clinicians successfully utilize high uptake of radiolabeled glucose via PET scanning to localize metastases in melanoma patients. To take advantage of this altered metabolome, 3-bromopyruvate (BrPA) was used to overcome the notorious resistance of melanoma to cell death. Using four melanoma cell lines, BrPA triggered caspase independent necrosis in two lines, whilst the other two lines were resistant to killing. Mechanistically, sensitive cells differed from resistant cells by; constitutively lower levels of glutathione, reduction of glutathione by BrPA only in sensitive cells; increased superoxide anion reactive oxygen species, loss of outer mitochondrial membrane permeability, and rapid ATP depletion. Sensitive cell killing was blocked by N-acetylcysteine or glutathione. When glutathione levels were reduced in resistant cell lines, they became sensitive to killing by BrPA. Taken together, these results identify a metabolic-based Achilles' heel in melanoma cells to be exploited by use of BrPA. Future pre-clinical and clinical trials are warranted to translate these results into improved patient care for individuals suffering from metastatic melanoma.

Tuft (caveolated) cells in two human colon carcinoma cell lines.

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Barkla, D H; Whitehead, R H; Foster, H; Tutton, P J

1988-09-01

The presence of an unusual cell type in two human colon carcinoma cell lines is reported. The cells show the same morphology as "tuft" (caveolated) cells present in normal gastrointestinal epithelium. Tuft cells were seen in cell line LIM 1863 growing in vitro and in human colon carcinoma cell line LIM 2210 growing as subcutaneous solid tumour xenografts in nude mice. Characteristic morphologic features of tuft cells included a wide base, narrow apex and a tuft of long microvilli projecting from the apical surface. The microvilli are attached by a core of long microfilaments passing deep into the apical cytoplasm. Between the microvilli are parallel arrays of vesicles (caveoli) containing flocculent material. Two different but not mutually exclusive explanations for the presence of tuft cells are proposed. The first explanation is that tuft cells came from the resected tumour and have survived by mitotic division during subsequent passages. The second explanation suggests that tuft cells are the progeny of undifferentiated tumour cells. Descriptions of tuft cells in colon carcinomas are uncommon and possible reasons for this are presented. The morphology of tuft cells is consistent with that of a highly differentiated cell specialised for absorption, and these new models provide an opportunity to further investigate the structure and function of tuft cells.

A tritherapy combination of inactivated allogeneic leukocytes infusion and cell vaccine with cyclophosphamide in a sequential regimen enhances antitumor immunity

OpenAIRE

Yishu Tang; Wenbo Ma; Chunxia Zhou; Dongmei Wang; Shuren Zhang

2018-01-01

Background: Tumor-induced immunosuppression can impede tumor-specific immune responses and limit the effects of cancer immunotherapy. The aim of this study was to investigate the possible effects of sequential chemoimmunotherapeutic strategies to enhance antitumor immune responses. Methods: Using the E7-expressing tumor TC-1 as the tumor model, the treatment groups were divided into the following groups: (1) inactivated allogeneic leukocyte infusion (ALI), (2) ALI + MMC-inactivated TC-1 cell ...

Total hip and knee replacement surgery results in changes in leukocyte and endothelial markers

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Maclean Kirsty M

2010-01-01

Full Text Available Abstract Background It is estimated that over 8 million people in the United Kingdom suffer from osteoarthritis. These patients may require orthopaedic surgical intervention to help alleviate their clinical condition. Investigations presented here was to test the hypothesis that total hip replacement (THR and total knee replacement (TKR orthopaedic surgery result in changes to leukocyte and endothelial markers thus increasing inflammatory reactions postoperatively. Methods During this 'pilot study', ten test subjects were all scheduled for THR or TKR elective surgery due to osteoarthritis. Leukocyte concentrations were measured using an automated full blood count analyser. Leukocyte CD11b (Mac-1 and CD62L cell surface expression, intracellular production of H2O2 and elastase were measured as markers of leukocyte function. Von Willebrand factor (vWF and soluble intercellular adhesion molecule-1 (sICAM-1 were measured as markers of endothelial activation. Results The results obtained during this study demonstrate that THR and TKR orthopaedic surgery result in similar changes of leukocyte and endothelial markers, suggestive of increased inflammatory reactions postoperatively. Specifically, THR and TKR surgery resulted in a leukocytosis, this being demonstrated by an increase in the total leukocyte concentration following surgery. Evidence of leukocyte activation was demonstrated by a decrease in CD62L expression and an increase in CD11b expression by neutrophils and monocytes respectively. An increase in the intracellular H2O2 production by neutrophils and monocytes and in the leukocyte elastase concentrations was also evident of leukocyte activation following orthopaedic surgery. With respect to endothelial activation, increases in vWF and sICAM-1 concentrations were demonstrated following surgery. Conclusion In general it appeared that most of the leukocyte and endothelial markers measured during these studies peaked between days 1

Mitochondrial DNA levels in Huntington disease leukocytes and dermal fibroblasts.

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Jędrak, Paulina; Krygier, Magdalena; Tońska, Katarzyna; Drozd, Małgorzata; Kaliszewska, Magdalena; Bartnik, Ewa; Sołtan, Witold; Sitek, Emilia J; Stanisławska-Sachadyn, Anna; Limon, Janusz; Sławek, Jarosław; Węgrzyn, Grzegorz; Barańska, Sylwia

2017-08-01

Huntington disease (HD) is an inherited neurodegenerative disorder caused by mutations in the huntingtin gene. Involvement of mitochondrial dysfunctions in, and especially influence of the level of mitochondrial DNA (mtDNA) on, development of this disease is unclear. Here, samples of blood from 84 HD patients and 79 controls, and dermal fibroblasts from 10 HD patients and 9 controls were analysed for mtDNA levels. Although the type of mitochondrial haplogroup had no influence on the mtDNA level, and there was no correlation between mtDNA level in leukocytes in HD patients and various parameters of HD severity, some considerable differences between HD patients and controls were identified. The average mtDNA/nDNA relative copy number was significantly higher in leukocytes, but lower in fibroblasts, of symptomatic HD patients relative to the control group. Moreover, HD women displayed higher mtDNA levels in leukocytes than HD men. Because this is the largest population analysed to date, these results might contribute to explanation of discrepancies between previously published studies concerning levels of mtDNA in cells of HD patients. We suggest that the size of the investigated population and type of cells from which DNA is isolated could significantly affect results of mtDNA copy number estimation in HD. Hence, these parameters should be taken into consideration in studies on mtDNA in HD, and perhaps also in other diseases where mitochondrial dysfunction occurs.

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

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Lacroix, Romaric; Plawinski, Laurent; Robert, Stéphane; Doeuvre, Loïc; Sabatier, Florence; Martinez de Lizarrondo, Sara; Mezzapesa, Anna; Anfosso, Francine; Leroyer, Aurelie S.; Poullin, Pascale; Jourde, Noémie; Njock, Makon-Sébastien; Boulanger, Chantal M.; Anglés-Cano, Eduardo; Dignat-George, Françoise

2012-01-01

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium. PMID:22733025

Effect of mixed γ-plus neutron-radiation on permeability to taurine of peripheral blood leukocyte membranes

International Nuclear Information System (INIS)

Dokshina, G.A.; Naumenko, L.A.

1980-01-01

A study was made of permeability to taurine of cellular membranes of peripheral blood leukocytes in vitro under normal conditions and 24 k following mixed γ-plus neutron-irradiation in a dose of 3.5 Gy. It was established that radiation increases the taurine content of cells. The protein content of leukocytes also increases probably due to a better sorption of serum proteins of blood

Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice

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Akinori Okumura

2017-12-01

Full Text Available Leukocyte cell-derived chemotaxin 2 (LECT2 is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.

Intracellular lipid dysregulation interferes with leukocyte function in the ovaries of meat-type hens under unrestricted feed intake.

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Liu, Zu-Chen; Su, Chia-Ming; Xie, Yi-Lun; Chang, Chai-Ju; Chen, Jiang-Young; Wu, Shu-Wei; Chen, Yu-Hui; Walzem, Rosemary L; Huang, San-Yuan; Chen, Shuen-Ei

2016-04-01

Meat-type Red-feather country hens fed ad libitum (AD-hens) exhibit obesity-associated morbidities and a number of ovarian irregularities. Leukocyte participations in ovarian activities are unstudied in AD-hens. In contrast to feed-restricted hens (R-hens), ovulatory process of the F1 follicle appeared delayed in AD-hens in association with reduced F1 follicle progesterone content, gelatinase A (MMP-2) and collagenase-3 (MMP-13) activities coincident with elevated IL-1β and no production (Pcultures of granulosa cells with increasing numbers of leukocytes from either AD-hens or R-hens exhibited dose dependent reductions in progesterone production and increases in cell death. AD-hen leukocytes were less proapoptotic than their R counterparts (Pcultures with heterophils or monocytes in a dose-dependent manner (Pcultures than their respective counterparts (P<0.05). Both basal and LPS-induced IL-1β secretion and MMP-22 or MMP-2 activities in freshly isolated AD-hen leukocytes were reduced (P<0.05). Exposure of AD or R leukocytes to 0.5mM palmitate impaired IL-1β secretion and MMP-22 or MMP-2 activity. Inhibition of ceramide synthesis with FB1 and ROS production with n-MPG scavenging rescued MMP activity and IL-1β production in palmitate treated heterophils, but exacerbated monocyte suppression. These latter findings suggest that intracellular lipid dysregulation in leukocytes contributes to ovarian dysfunction in AD-hens. Copyright © 2016 Elsevier B.V. All rights reserved.

Dendritic cells take up and present antigens from viable and apoptotic polymorphonuclear leukocytes.

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Carlos Alfaro

Full Text Available Dendritic cells (DC are endowed with the ability to cross-present antigens from other cell types to cognate T cells. DC are poised to meet polymorphonuclear leukocytes (PMNs as a result of being co-attracted by interleukin-8 (IL-8, for instance as produced by tumor cells or infected tissue. Human monocyte-derived and mouse bone marrow-derived DC can readily internalize viable or UV-irradiated PMNs. Such internalization was abrogated at 4°C and partly inhibited by anti-CD18 mAb. In mice, DC which had internalized PMNs containing electroporated ovalbumin (OVA protein, were able to cross-present the antigen to CD8 (OT-1 and CD4 (OT-2 TCR-transgenic T cells. Moreover, in humans, tumor cell debris is internalized by PMNs and the tumor-cell material can be subsequently taken up from the immunomagnetically re-isolated PMNs by DC. Importantly, if human neutrophils had endocytosed bacteria, they were able to trigger the maturation program of the DC. Moreover, when mouse PMNs with E. coli in their interior are co-injected in the foot pad with DC, many DC loaded with fluorescent material from the PMNs reach draining lymph nodes. Using CT26 (H-2(d mouse tumor cells, it was observed that if tumor cells are intracellularly loaded with OVA protein and UV-irradiated, they become phagocytic prey of H-2(d PMNs. If such PMNs, that cannot present antigens to OT-1 T cells, are immunomagnetically re-isolated and phagocytosed by H-2(b DC, such DC productively cross-present OVA antigen determinants to OT-1 T cells. Cross-presentation to adoptively transferred OT-1 lymphocytes at draining lymph nodes also take place when OVA-loaded PMNs (H-2(d are coinjected in the footpad of mice with autologous DC (H-2(b. In summary, our results indicate that antigens phagocytosed by short-lived PMNs can be in turn internalized and productively cross-presented by DC.

In-111-labeled leukocyte scintigraphy in postoperative joint infection

International Nuclear Information System (INIS)

Ogawa, Yoji; Uetani, Masataka; Aziz, A.; Hayashi, Kuniaki

2000-01-01

To evaluate the role of In-111-labeled leukocyte scintigraphy in the patients with suspected postoperative joint infection, 41 scintigraphic examinations were performed in 24 patients. Scintigrams were interpreted by the degree of accumulation of labeled leukocytes, and were classified into 3 groups: positive, intermediate, and negative. In the cases of positive leukocyte scans, definite diagnosis of infection was made in all cases except one. In the cases of negative scans, there was no evidence of infection. In 13 cases, leukocyte scintigrams were interpreted in conjunction with bone scintigrams. Definite diagnosis of infection was made in all of the cases with positive combined leukocyte/bone scan, and there was no evidence of infection in cases with negative combined leukocyte/bone scan. This study demonstrates that In-111-labeled leukocyte scintigraphy is a useful method in diagnosis of postoperative joint infection, and accuracy of the examination improves when combined with bone scintigraphy. (author)

Expression of the Grb2-related protein of the lymphoid system in B cell subsets enhances B cell antigen receptor signaling through mitogen-activated protein kinase pathways.

Science.gov (United States)

Yankee, Thomas M; Solow, Sasha A; Draves, Kevin D; Clark, Edward A

2003-01-01

Adapter proteins play a critical role in regulating signals triggered by Ag receptor cross-linking. These small molecules link receptor proximal events with downstream signaling pathways. In this study, we explore the expression and function of the Grb2-related protein of the lymphoid system (GrpL)/Grb2-related adaptor downstream of Shc adapter protein in human B cells. GrpL is expressed in naive B cells and is down-regulated following B cell Ag receptor ligation. By contrast, germinal center and memory B cells express little or no GrpL. Using human B cell lines, we detected constitutive interactions between GrpL and B cell linker protein, Src homology (SH)2 domain-containing leukocyte protein of 76 kDa, hemopoietic progenitor kinase 1, and c-Cbl. The N-terminal SH3 domain of GrpL binds c-Cbl while the C-terminal SH3 domain binds B cell linker protein and SH2 domain-containing leukocyte protein of 76 kDa. Exogenous expression of GrpL in a GrpL-negative B cell line leads to enhanced Ag receptor-induced extracellular signal-related kinase and p38 mitogen-activated protein kinase phosphorylation. Thus, GrpL expression in human B cell subsets appears to regulate Ag receptor-mediated signaling events.

Accurate detection of carcinoma cells by use of a cell microarray chip.

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Shohei Yamamura

Full Text Available BACKGROUND: Accurate detection and analysis of circulating tumor cells plays an important role in the diagnosis and treatment of metastatic cancer treatment. METHODS AND FINDINGS: A cell microarray chip was used to detect spiked carcinoma cells among leukocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth, was made from polystyrene; and the formation of monolayers of leukocytes in the microchambers was observed. Cultured human T lymphoblastoid leukemia (CCRF-CEM cells were used to examine the potential of the cell microarray chip for the detection of spiked carcinoma cells. A T lymphoblastoid leukemia suspension was dispersed on the chip surface, followed by 15 min standing to allow the leukocytes to settle down into the microchambers. Approximately 29 leukocytes were found in each microchamber when about 600,000 leukocytes in total were dispersed onto a cell microarray chip. Similarly, when leukocytes isolated from human whole blood were used, approximately 89 leukocytes entered each microchamber when about 1,800,000 leukocytes in total were placed onto the cell microarray chip. After washing the chip surface, PE-labeled anti-cytokeratin monoclonal antibody and APC-labeled anti-CD326 (EpCAM monoclonal antibody solution were dispersed onto the chip surface and allowed to react for 15 min; and then a microarray scanner was employed to detect any fluorescence-positive cells within 20 min. In the experiments using spiked carcinoma cells (NCI-H1650, 0.01 to 0.0001%, accurate detection of carcinoma cells was achieved with PE-labeled anti-cytokeratin monoclonal antibody. Furthermore, verification of carcinoma cells in the microchambers was performed by double staining with the above monoclonal antibodies. CONCLUSION: The potential application of the cell microarray chip for the detection of CTCs was shown, thus demonstrating accurate detection by double staining for cytokeratin and EpCAM at the single carcinoma cell level.

Characterization of cloned cells from an immortalized fetal pulmonary type II cell line

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Henderson, R.F.; Waide, J.J.; Lechner, J.F.

1995-12-01

A cultured cell line that maintained expression of pulmonary type II cell markers of differentiation would be advantageous to generate a large number of homogenous cells in which to study the biochemical functions of type II cells. Type II epithelial cells are the source of pulmonary surfactant and a cell of origin for pulmonary adenomas. Last year our laboratory reported the induction of expression of two phenotypic markers of pulmonary type II cells (alkaline phosphatase activity and surfactant lipid synthesis) in cultured fetal rat lung epithelial (FRLE) cells, a spontaneously immortalized cell line of fetal rat lung type II cell origin. Subsequently, the induction of the ability to synthesize surfactant lipid became difficult to repeat. We hypothesized that the cell line was heterogenuous and some cells were more like type II cells than others. The purpose of this study was to test this hypothesis and to obtain a cultured cell line with type II cell phenotypic markers by cloning several FRLE cells and characterizing them for phenotypic markers of type II cells (alkaline phosphatase activity and presence of surfactant lipids). Thirty cloned cell lines were analyzed for induced alkaline phosphatase activity (on x-axis) and for percent of phospholipids that were disaturated (i.e., surfactant).

Change of cell cycle arrest of tumor cell lines after 60Co γ-irradiation

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Tang Yi; Liu Wenli; Zhou Jianfeng; Gao Qinglei; Wu Jianhong

2003-01-01

Objective: To observe the cell cycle arrest changes in peripheral blood mononuclear cells (PBMNCs) of normal persons and several kinds of tumor cell lines after 60 Co γ-irradiation. Methods: PBMNCs of normal persons, HL-60, K562, SiHA and 113 tumor cell lines were irradiated with 60 Co γ-rays at the absorbed doses of 6, 10,15 Gy. Cell cycles changes were checked 6, 12, 24, 48 and 60 h after the irradiation. Results: A stasis state was observed in normal person PBMNCs, 95 percents of which were in G 1 phase, and they still remained stasis after the irradiation. Except the 113 cell line manifesting G 1 phase arrest, all other tumor cell lines showed G 2 /M phase arrest after irradiation. The radiation sensitivity of HL-60 was higher than that of SiHA cell line. Conclusion: Different cell lines have different cell cycle arrest reaction to radiation and their radiation sensitivity are also different

Neutrophils are not less sensitive than other blood leukocytes to the genomic effects of glucocorticoids.

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Gaelle Hirsch

Full Text Available Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out.We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes.Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL alone or combined with hydrocortisone, prednisolone or dexamethasone (10(-8 M and 10(-6 M. IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations.We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils.Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some

Neutrophils Are Not Less Sensitive Than Other Blood Leukocytes to the Genomic Effects of Glucocorticoids

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Hirsch, Gaelle; Lavoie-Lamoureux, Anouk; Beauchamp, Guy; Lavoie, Jean-Pierre

2012-01-01

Background Neutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out. Objective We assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes. Methods Blood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10−8 M and 10−6 M). IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations. Results We found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils. Conclusions Glucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to

Altered mitochondrial function and oxidative stress in leukocytes of anorexia nervosa patients.

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Victor, Victor M; Rovira-Llopis, Susana; Saiz-Alarcon, Vanessa; Sangüesa, Maria C; Rojo-Bofill, Luis; Bañuls, Celia; Falcón, Rosa; Castelló, Raquel; Rojo, Luis; Rocha, Milagros; Hernández-Mijares, Antonio

2014-01-01

Anorexia nervosa is a common illness among adolescents and is characterised by oxidative stress. The effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated. A multi-centre, cross-sectional case-control study was performed. Our study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women. Anthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells. Mitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (Panorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

Leukocyte changes across menstruation, ovulation, and mid-luteal phase and association with sex hormone variation.

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Nowak, Judyta; Borkowska, Barbara; Pawlowski, Boguslaw

2016-09-10

Total leukocyte count (white blood cells-WBC) and the count of each subpopulation vary across the menstrual cycle, but results of studies examining the time and direction of these changes are inconsistent and methodologically flawed. Besides, no previous study focused on leukocyte count on the day of ovulation. Blood samples were obtained from 37 healthy and regularly cycling women aged 19.8-36.1 years. Samples were taken three times: during menstruation (M), ovulation (O), and in the mid-luteal phase (ML). WBC, neutrophils, lymphocytes, mixed cells, progesterone (P,) and estradiol (E) were measured in each of the three target phases of the cycle. Compared to menstruation, WBC (P = 0.002) and neutrophils (P < 0.001) increased around ovulation and remained stable in the mid-luteal phase, whereas lymphocyte and mixed cell counts did not change throughout the menstrual cycle. There were some correlations of sex hormone variation with leukocyte changes between M and O (positive for E and WBC, negative for P and WBC and for P and neutrophil count; P < 0.05), but not between O and ML. Peripheral leukocyte changes taking place in the second half of the cycle are already observable on the day of ovulation and they are associated with sex hormone variation. We speculate that these changes may lead to increased immune protection against pathogens at a time when fertilization and implantation typically occur. Am. J. Hum. Biol. 28:721-728, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Effect of human polymorphonuclear and mononuclear leukocytes on chromosomal and plasmid DNA of Escherichia coli. Role of acid DNase

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Rozenberg-Arska, M.; van Strijp, J.A.; Hoekstra, W.P.; Verhoef, J.

1984-01-01

Phagocytosis and killing by polymorphonuclear and mononuclear leukocytes are important host resistance factors against invading microorganisms. Evidence showing that killing is rapidly followed by degradation of bacterial components is limited. Therefore, we studied the fate of Escherichia coli DNA following phagocytosis of E. coli by polymorphonuclear and mononuclear leukocytes. [ 3 H]Thymidine-labeled, unencapsulated E. coli PC2166 and E. coli 048K1 were incubated in serum, washed, and added to leukocytes. Uptake and killing of the bacteria and degradation of DNA were measured. Although phagocytosis and killing by mononuclear leukocytes was less efficient than that by polymorphonuclear leukocytes, only mononuclear leukocytes were able to degrade E. coli PC2166 DNA. Within 2 h, 60% of the radioactivity added to mononuclear leukocytes was released into the supernate, of which 40% was acid soluble. DNA of E. coli 048K1 was not degraded. To further analyze the capacity of mononuclear leukocytes to degrade E. coli DNA, chromosomal and plasmid DNA was isolated from ingested bacteria and subjected to agarose gel-electrophoresis. Only chromosomal DNA was degraded after phagocytosis. Plasmid DNA of E. coli carrying a gene coding for ampicillin resistance remained intact for a 2-h period after ingestion, and was still able to transform recipient E. coli cells after this period. Although we observed no DNA degradation during phagocytosis by polymorphonuclear leukocytes, lysates of both polymorphonuclear and mononuclear leukocytes contained acid-DNase activity with a pH optimum of 4.9. However, the DNase activity of mononuclear leukocytes was 20 times higher than that of polymorphonuclear leukocytes. No difference was observed between DNase activity from polymorphonuclear and mononuclear leukocytes from a chronic granulomatous disease patient with DNase activity from control polymorphonuclear and mononuclear leukocytes

Effect of streptavidin-biotin on endothelial vasoregulation and leukocyte adhesion.

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Chan, Bernard P; Reichert, William M; Truskey, George A

2004-08-01

The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.

Leukocyte counts and lymphocyte subsets in relation to pregnancy and HIV infection in Malawian women.

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Mandala, Wilson L; Gondwe, Esther N; Molyneux, Malcolm E; MacLennan, Jenny M; MacLennan, Calman A

2017-09-01

We investigated leukocyte and lymphocyte subsets in HIV-infected or HIV-uninfected, pregnant or non-pregnant Malawian women to explore whether HIV infection and pregnancy may act synergistically to impair cellular immunity. We recruited 54 pregnant and 48 non-pregnant HIV-uninfected women and 24 pregnant and 20 non-pregnant HIV-infected Malawian women. We compared peripheral blood leukocyte and lymphocyte subsets between women in the four groups. Parturient HIV-infected and HIV-uninfected women had more neutrophils (each PHIV-uninfected parturient women had fewer CD4 + and γδ T cells, B and NK cells (each Ppregnancy. Malawian women at parturition have an increased total white cell count due to neutrophilia and an HIV-unrelated pan-lymphopenia. © 2017 The Author. American Journal of Reproductive Immunology Published by John Wiley & Sons Ltd.

TGF-b2 induction regulates invasiveness of Theileria-transformed leukocytes and disease susceptibility.

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Marie Chaussepied

2010-11-01

Full Text Available Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva, or tropical theileriosis (T. annulata. Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK. We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence.

Reference values for maternal total and differential leukocyte counts in different trimesters of pregnancy and the initial postpartum period in western Turkey.

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Sanci, Muzaffer; Töz, Emrah; Ince, Onur; Özcan, Aykut; Polater, Kevser; Inan, Abdurrahman Hamdi; Beyan, Emrah; Akkaya, Emrah

2017-07-01

The aim of this study was to investigate alterations in the leukocyte and differential leukocyte counts in different trimesters of pregnancy and the initial postpartum period. The study population consisted of 40,325 pregnant women. A full blood count and automated differential leukocyte count were performed and all the haemogram results in the different trimesters of pregnancy were recorded. Percentiles were calculated using statistical software. A total of 82,786 complete blood count evaluations were performed in 40,325 subjects from the 6th to 41st week of pregnancy and in the initial postpartum period. The leukocyte counts increased from the 1st to the 3rd trimester and peaked in the initial postpartum period. Our reference values for the total and differential leukocyte counts may assist clinicians in distinguishing between leukocytosis and pathological elevation of the white blood cell count during pregnancy and the initial postpartum period. Impact statement Pregnancy requires profound adaptation by multiple systems to accommodate the demands of the developing foetus. Similar to all other systems, many haematological changes occur during pregnancy. Studies of normal variation in leukocyte counts were insufficient to distinguish normal from abnormal leukocyte counts during pregnancy and in the initial postpartum period, due to small numbers of patients and a lack of differential leukocyte counts. Without reference leukocyte levels, infections may be more difficult to assess during pregnancy and in the postpartum period. In this study, we report the 3rd, 5th, 10th, 50th, 95th and 99th percentile values for the total and differential leukocyte counts according to trimester in normal pregnancy and the initial postpartum period. Our reference values for the total and differential leukocyte counts in each trimester and the initial postpartum period may assist clinicians in distinguishing between normal leukocytosis and pathological elevation of the white blood

Mutagenicity assessment of two herbal medicines, Urtan and Carmint in human leukocytes by single cell gel electrophoresis

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Jalili, M.; Hatami, A.; Kalantari, H.; Kalantar, E.

2006-01-01

The use of herbal medicine is an old and still widespread particle, which makes studies their genotoxicity essential. Urtan and Carmint are examples of herbal medicines used in Iran which used for the treatment of hyperplasia, diuretic, urinary diffusion and antispasmodic action, carminative gastrointestinal disfunction respectively. The aim of this study was to evaluate the mutagenicity of these herbal medicines in human leukocytes by Single Cell Gel Electrophoresis (SCGE). Both the herbal medicines were used at four concentrations (0.046, 0.092, 0.138 and 0.184/ 100 ml). The results were compared against positive (Cr VI) and negative (no mutagen) control groups. Fluorescence microscope was used to observe the DNA damage in randomly selected cells, which were stained with ethidium bromide. Microscopic observation of the affected cells due to Urtan and Carmint was encouraging as compared to previous studies using SCGE. Both Urtan and Carmint exhibited considerable DNA damage to the blood cells. For example, Urtan at o.184 g/100ml concentration had almost 23% mutagenic effect and as the concentration increased the mutagenic effect also increased. Similarly Carmint exhibited considerable DNA damage on blood cells. Therefore, both the herbal medicines may have some mutagenic effect. (author)

Pseudomonas aeruginosa recognizes and responds aggressively to the presence of polymorphonuclear leukocytes

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Alhede, Morten; Bjarnsholt, T.; Jensen, P.O.

2009-01-01

Polymorphonuclear neutrophilic leukocytes (PMNs) play a central role in innate immunity, where they dominate the response to infections, in particular in the cystic fibrosis lung. PMNs are phagocytic cells that produce a wide range of antimicrobial agents aimed at killing invading bacteria. Howev...

Mild episodes of tourniquet-induced forearm ischaemia-reperfusion injury results in leukocyte activation and changes in inflammatory and coagulation markers

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Bastawrous Salah S

2007-05-01

Full Text Available Abstract Background Monocytes and neutrophils are examples of phagocytic leukocytes, with neutrophils being considered as the 'chief' phagocytic leukocyte. Both monocytes and neutrophils have been implicated to play a key role in the development of ischaemia-reperfusion injury, where they are intrinsically involved in leukocyte-endothelial cell interactions. In this pilot study we hypothesised that mild episodes of tourniquet induced forearm ischaemia-reperfusion injury results in leukocyte activation and changes in inflammatory and coagulation markers. Methods Ten healthy human volunteers were recruited after informed consent. None had any history of cardiovascular disease with each subject volunteer participating in the study for a 24 hour period. Six venous blood samples were collected from each subject volunteer at baseline, 10 minutes ischaemia, 5, 15, 30, 60 minutes and 24 hours reperfusion, by means of a cannula from the ante-cubital fossa. Monocyte and neutrophil leukocyte sub-populations were isolated by density gradient centrifugation techniques. Leukocyte trapping was investigated by measuring the concentration of leukocytes in venous blood leaving the arm. The cell surface expression of CD62L (L-selectin, CD11b and the intracellular production of hydrogen peroxide (H2O2 were measured via flow cytometry. C-reactive protein (CRP was measured using a clinical chemistry analyser. Plasma concentrations of D-dimer and von Willebrand factor (vWF were measured using enzyme-linked fluorescent assays (ELFA. Results During ischaemia-reperfusion injury, there was a decrease in CD62L and an increase in CD11b cell surface expression for both monocytes and neutrophils, with changes in the measured parameters reaching statistical significance (p =2O2 production by leukocyte sub-populations, which was measured as a marker of leukocyte activation. Intracellular production of H2O2 in monocytes during ischaemia-reperfusion injury reached statistical

Peripheral blood film review. The demise of the eyecount leukocyte differential.

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Pierre, Robert V

2002-03-01

initiate blood film review based on findings of the CBC and automated differential is a more sensitive and accurate method of detecting patients with blood film abnormalities than routine blood film review of all specimens by technologists. Clinicians need to recognize that daily differential results or differentials at intervals of less than a week are not medically necessary in most patients. The laboratory, however, must provide opportunities for the clinician to request differentials at any time for specific medical reasons. The laboratory must establish the validity of screening criteria for detection of distribution and morphologic abnormalities of leukocytes by clinical correlation studies or adopt criteria established by laboratories with the same instrumentation and which have conducted clinical evaluations. A final observation on the eyecount differential is that it was the only way to identify cell types and their relative proportion for nearly 100 years. Cells were identified by their shape, intracellular structures, and staining characteristics. Many studies were able eventually to correlate some aspect of each cell type's function with their morphologic appearance. It has also been learned that the bone marrow is the source of production of most circulating cells and a great deal of the controls of cell production and release into the peripheral blood have been learned. But leukocytes have many functions, almost none of which are performed in the peripheral blood. The peripheral blood is mainly a conduit from the bone marrow to the tissues where the leukocytes perform their function in the case of the neutrophils and monocytes. It is mainly a recirculation and redistribution system for lymphocytes that usually receive their instructions from antigen processing cells in the tissues and allow these modified cells to home to sites where their functions occur. Cellular morphology and staining characteristics tell little about the maturation stage and functional

Derivation and characterization of a pig embryonic stem cell-derived exocrine pancreatic cell line

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The establishment and initial characterization of a pig embryonic stem cell-derived pancreatic cell line, PICM-31, and a colony-cloned derivative cell line, PICM-31A, is described. The cell lines were propagated for several months at split ratios of 1:3 or 1:5 at each passage on STO feeder cells af...

Radiation-induced apoptosis and cell cycle checkpoints in human colorectal tumour cell lines

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Playle, L.C.

2001-03-01

The p53 tumour suppressor gene is mutated in 75% of colorectal carcinomas and is critical for DNA damage-induced G1 cell cycle arrest. Data presented in this thesis demonstrate that after treatment with Ionizing Radiation (IR), colorectal tumour cell lines with mutant p53 are unable to arrest at G1 and undergo cell cycle arrest at G2. The staurosporine derivative, UCN-01, was shown to abrogate the IR-induced G2 checkpoint in colorectal tumour cell lines. Furthermore, in some cell lines, abrogation of the G2 checkpoint was associated with radiosensitisation. Data presented in this study demonstrate that 2 out of 5 cell lines with mutant p53 were sensitised to IR by UCN-01. In order to determine whether radiosensitisation correlated with lack of functional p53, transfected derivatives of an adenoma-derived cell line were studied, in which endogenous wild type p53 was disrupted by expression of a dominant negative p53 mutant protein (and with a vector control). In both these cell lines UCN-01 abrogated the G2 arrest however this was not associated with radiosensitisation, indicating that radiosensitisation is a cell type-specific phenomenon. Although 2 colorectal carcinoma cell lines, with mutant p53, were sensitised to IR by UCN-01, the mechanisms of p53-independent IR-induced apoptosis in the colon are essentially unknown. The mitogen-activated protein kinase (MAPK) pathways (that is the JNK, p38 and ERK pathways) have been implicated in apoptosis in a range of cell systems and in IR-induced apoptosis in some cell types. Data presented in this study show that, although the MAPKs can be activated by the known activator anisomycin, there is no evidence of a role for MAPKs in IR-induced apoptosis in colorectal tumour cell lines, regardless of p53 status. In summary, some colorectal tumour cell lines with mutant p53 can be sensitised to IR-induced cell death by G2 checkpoint abrogation and this may be an important treatment strategy, however mechanisms of IR-induced p53

Polymer encapsulated dopaminergic cell lines as "alternative neural grafts".

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Jaeger, C B; Greene, L A; Tresco, P A; Winn, S R; Aebischer, P

1990-01-01

Our preliminary findings (Jaeger et al., 1988; Aebischer et al., 1989; Tresco et al., 1989) and the studies in progress show that encapsulated dopaminergic cell lines survive enclosure within a semi-permeable membrane. The encapsulated cells remained viable for extended time periods when maintained in vitro. Moreover, encapsulated PC12 and T28 cells have the potential to survive following their implantation into the forebrain of rats. Cell lines are essentially "immortal" because they continue to divide indefinitely. This property allows perpetual "self-renewal" of a given cell population. However, the capacity of continuous uncontrolled cell division may also lead to tumor formation. This in fact is the case for unencapsulated PC12 cell implants placed into the brain of young Sprague Dawley rats (Jaeger, 1985). Cell line encapsulation has the potential to prevent tumor growth (Jaeger et al., 1988). Survival for 6 months in vitro suggests that encapsulation does not preclude long-term maintenance of an homogeneous cell line like PC12 cells. The presence of mitotic figures in the capsules further supports the likelihood of propagation and self renewal of the encapsulated population. Another significant property of cell lines is that they consist of a single, genetically homogeneous cell type. They do not require specific synaptic interactions for their survival. In the case of PC12 and T28 lines, the cells synthesize and release neurotransmitters. Our data show that PC12 and T28 cells continue to release dopamine spontaneously and to express specific transmitters and enzymes following encapsulation. Thus, cell lines such as these may constitute relatively simple "neural implants" exerting their function via humoral release.(ABSTRACT TRUNCATED AT 250 WORDS)

Discovery of HeLa Cell Contamination in HES Cells: Call for Cell Line Authentication in Reproductive Biology Research.

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Kniss, Douglas A; Summerfield, Taryn L

2014-08-01

Continuous cell lines are used frequently in reproductive biology research to study problems in early pregnancy events and parturition. It has been recognized for 50 years that many mammalian cell lines contain inter- or intraspecies contaminations with other cells. However, most investigators do not routinely test their culture systems for cross-contamination. The most frequent contributor to cross-contamination of cell lines is the HeLa cell isolated from an aggressive cervical adenocarcinoma. We report on the discovery of HeLa cell contamination of the human endometrial epithelial cell line HES isolated in our laboratory. Short tandem repeat analysis of 9 unique genetic loci demonstrated molecular identity between HES and HeLa cells. In addition, we verified that WISH cells, isolated originally from human amnion epithelium, were also contaminated with HeLa cells. Inasmuch as our laboratory did not culture HeLa cells at the time of HES cell derivations, the source of contamination was the WISH cell line. These data highlight the need for continued diligence in authenticating cell lines used in reproductive biology research. © The Author(s) 2014.

Peripheral blood and milk leukocytes subsets of lactating Sarda ewes

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Piero Bonelli

2013-05-01

Full Text Available Leukocytes subpopulations in blood and milk of lactating Sarda ewes were investigated. Animals characterized by a SSC level <500×103cells/mL and a negative bacteriological examination were sampled in early, mid and late lactation. Milk differential cell count evidenced that macrophage represented the main population (42.8%±3.5 followed by lymphocytes (40.2%±3.4 and neutrophils (8,6%±2.1. Flow cytometry analysis showed that lymphocytes subsets in milk were quite different from blood. High CD8+ and low CD4+ lymphocytes percentages determined a CD4/CD8 ratio inversion in milk compared to blood (0.3%±0.03 vs 1.8%±0.08. CD8+ decreased while, conversely, CD4+ increased in late lactation. γδ T cells were more represented in milk (12.6%±1.3 than in blood (6.8%±0.3 and their proportions appeared similar throughout lactation in both compartments. IL-2 receptor was mainly expressed in milk on T cytotoxic lymphocytes. Data obtained in uninfected mammary glands could allow an early discrimination between physiological and pathological changes occurring in ewe milk. Further phenotypical and functional studies on milk leukocytes subsets might help to understand defense mechanisms of the ovine mammary gland against IMI.

Impact of sex, MHC, and age of recipients on the therapeutic effect of transferred leukocytes from cancer-resistant SR/CR mice

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Adams Jonathan M

2009-09-01

Full Text Available Abstract Background Spontaneous Regression/Complete Resistant (SR/CR mice are resistant to cancer through a mechanism that is mediated entirely by leukocytes of innate immunity. Transfer of leukocytes from SR/CR mice can confer cancer resistance in wild-type (WT recipients in both preventative and therapeutic settings. In the current studies, we investigated factors that may impact the efficacy and functionality of SR/CR donor leukocytes in recipients. Results In sex-mismatched transfers, functionality of female donor leukocytes was not affected in male recipients. In contrast, male donor leukocytes were greatly affected in the female recipients. In MHC-mismatches, recipients of different MHC backgrounds, or mice of different strains, showed a greater negative impact on donor leukocytes than sex-mismatches. The negative effects of sex-mismatch and MHC-mismatch on donor leukocytes were additive. Old donor leukocytes performed worse than young donor leukocytes in all settings including in young recipients. Young recipients were not able to revive the declining function of old donor leukocytes. However, the function of young donor leukocytes declined gradually in old recipients, suggesting that an aged environment may contain factors that are deleterious to cellular functions. The irradiation of donor leukocytes prior to transfers had a profound suppressive effect on donor leukocyte functions, possibly as a result of impaired transcription. The cryopreserving of donor leukocytes in liquid nitrogen had no apparent effect on donor leukocyte functions, except for a small loss of cell number after revival from freezing. Conclusion Despite the functional suppression of donor leukocytes in sex- and MHC-mismatched recipients, as well as old recipients, there was a therapeutic time period during the initial few weeks during which donor leukocytes were functional before their eventual rejection or functional decline. The eventual rejection of donor

Biomimetic carriers mimicking leukocyte plasma membrane to increase tumor vasculature permeability

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Palomba, R.; Parodi, A.; Evangelopoulos, M.; Acciardo, S.; Corbo, C.; De Rosa, E.; Yazdi, I. K.; Scaria, S.; Molinaro, R.; Furman, N. E. Toledano; You, J.; Ferrari, M.; Salvatore, F.; Tasciotti, E.

2016-10-01

Recent advances in the field of nanomedicine have demonstrated that biomimicry can further improve targeting properties of current nanotechnologies while simultaneously enable carriers with a biological identity to better interact with the biological environment. Immune cells for example employ membrane proteins to target inflamed vasculature, locally increase vascular permeability, and extravasate across inflamed endothelium. Inspired by the physiology of immune cells, we recently developed a procedure to transfer leukocyte membranes onto nanoporous silicon particles (NPS), yielding Leukolike Vectors (LLV). LLV are composed of a surface coating containing multiple receptors that are critical in the cross-talk with the endothelium, mediating cellular accumulation in the tumor microenvironment while decreasing vascular barrier function. We previously demonstrated that lymphocyte function-associated antigen (LFA-1) transferred onto LLV was able to trigger the clustering of intercellular adhesion molecule 1 (ICAM-1) on endothelial cells. Herein, we provide a more comprehensive analysis of the working mechanism of LLV in vitro in activating this pathway and in vivo in enhancing vascular permeability. Our results suggest the biological activity of the leukocyte membrane can be retained upon transplant onto NPS and is critical in providing the particles with complex biological functions towards tumor vasculature.

THP-1 cell line: an in vitro cell model for immune modulation approach.

Science.gov (United States)

Chanput, Wasaporn; Mes, Jurriaan J; Wichers, Harry J

2014-11-01

THP-1 is a human leukemia monocytic cell line, which has been extensively used to study monocyte/macrophage functions, mechanisms, signaling pathways, and nutrient and drug transport. This cell line has become a common model to estimate modulation of monocyte and macrophage activities. This review attempts to summarize and discuss recent publications related to the THP-1 cell model. An overview on the biological similarities and dissimilarities between the THP-1 cell line and human peripheral blood mononuclear cell (PBMC) derived-monocytes and macrophages, as well as the advantages and disadvantages of the use of THP-1 cell line, is included. The review summarizes different published co-cultivation studies of THP-1 cells with other cell types, for instance, intestinal cells, adipocytes, T-lymphocytes, platelets, and vascular smooth muscle cells, which can be an option to study cell-cell interaction in vitro and can be an approach to better mimic in vivo conditions. Macrophage polarization is a relatively new topic which gains interest for which the THP-1 cell line also may be relevant. Besides that an overview of newly released commercial THP-1 engineered-reporter cells and THP-1 inflammasome test-cells is also given. Evaluation of recent papers leads to the conclusion that the THP-1 cell line has unique characteristics as a model to investigate/estimate immune-modulating effects of compounds in both activated and resting conditions of the cells. Although the THP-1 response can hint to potential responses that might occur ex vivo or in vivo, these should be, however, validated by in vivo studies to draw more definite conclusions. Copyright © 2013. Published by Elsevier B.V.

Indium-111 leukocyte imaging in patients with rheumatoid arthritis

International Nuclear Information System (INIS)

Uno, K.; Matsui, N.; Nohira, K.

1986-01-01

This study evaluates the usefulness of labeled leukocyte imaging in patients with rheumatoid arthritis. In 33 patients, the incidence of pain and swelling in 66 wrist joints and 66 knee joints was compared with the accumulation of [ 111 In]leukocytes. No accumulation of [ 111 In]leukocytes was seen in any of the patients' wrists (0/12) or knee joints (0/14) when both pain and swelling were absent. In contrast, 93% (25/27) of wrist joints and 80% (24/30) of knee joints with both pain and swelling were positive by [ 111 In]leukocyte scintigraphy. There was little correlation between the stage of the disease, as determined by radiography, and [ 111 In]leukocyte accumulation. This study suggests that [ 111 In]leukocyte imaging may be a reliable procedure for monitoring the activity of rheumatoid arthritis, especially for confirming the lack of an ongoing inflammatory response

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

International Nuclear Information System (INIS)

Ristic, Biljana; Bosnjak, Mihajlo; Arsikin, Katarina; Mircic, Aleksandar; Suzin-Zivkovic, Violeta; Bogdanovic, Andrija; Perovic, Vladimir; Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir; Trajkovic, Vladimir; Harhaji-Trajkovic, Ljubica

2014-01-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Idarubicin induces mTOR-dependent cytotoxic autophagy in leukemic cells

Energy Technology Data Exchange (ETDEWEB)

Ristic, Biljana [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Bosnjak, Mihajlo [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Arsikin, Katarina [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Mircic, Aleksandar; Suzin-Zivkovic, Violeta [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Bogdanovic, Andrija [Clinic for Hematology, Clinical Centre of Serbia, School of Medicine, University of Belgrade, Belgrade (Serbia); Perovic, Vladimir [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Martinovic, Tamara; Kravic-Stevovic, Tamara; Bumbasirevic, Vladimir [Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade (Serbia); Trajkovic, Vladimir, E-mail: vtrajkovic@med.bg.ac.rs [Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade (Serbia); Harhaji-Trajkovic, Ljubica, E-mail: buajk@yahoo.com [Institute for Biological Research, University of Belgrade, Belgrade, Despot Stefan Blvd. 142, 11000 Belgrade (Serbia)

2014-08-01

We investigated if the antileukemic drug idarubicin induces autophagy, a process of programmed cellular self-digestion, in leukemic cell lines and primary leukemic cells. Transmission electron microscopy and acridine orange staining demonstrated the presence of autophagic vesicles and intracellular acidification, respectively, in idarubicin-treated REH leukemic cell line. Idarubicin increased punctuation/aggregation of microtubule-associated light chain 3B (LC3B), enhanced the conversion of LC3B-I to autophagosome-associated LC3B-II in the presence of proteolysis inhibitors, and promoted the degradation of the selective autophagic target p62, thus indicating the increase in autophagic flux. Idarubicin inhibited the phosphorylation of the main autophagy repressor mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase. The treatment with the mTOR activator leucine prevented idarubicin-mediated autophagy induction. Idarubicin-induced mTOR repression was associated with the activation of the mTOR inhibitor AMP-activated protein kinase and down-regulation of the mTOR activator Akt. The suppression of autophagy by pharmacological inhibitors or LC3B and beclin-1 genetic knockdown rescued REH cells from idarubicin-mediated oxidative stress, mitochondrial depolarization, caspase activation and apoptotic DNA fragmentation. Idarubicin also caused mTOR inhibition and cytotoxic autophagy in K562 leukemic cell line and leukocytes from chronic myeloid leukemia patients, but not healthy controls. By demonstrating mTOR-dependent cytotoxic autophagy in idarubicin-treated leukemic cells, our results warrant caution when considering combining idarubicin with autophagy inhibitors in leukemia therapy. - Highlights: • Idarubicin induces autophagy in leukemic cell lines and primary leukemic cells. • Idarubicin induces autophagy by inhibiting mTOR in leukemic cells. • mTOR suppression by idarubicin is associated with AMPK activation and Akt blockade. �

Induction of expression of iNOS by N-nitrosodimethylamine (NDMA) in human leukocytes.

Science.gov (United States)

Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Jablonski, Jakub; Marcinczyk, Magdalena

2009-01-01

The aim of this study was to assess the influence of N-nitrosodimethylamine (NDMA) on expression of inducible nitric oxide synthase (iNOS), as well as production of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) by human neutrophils (PMN) and peripheral blood mononuclear cells (PBMC), and the participation of the p38 MAPK kinase in this process. Furthermore, the ability of neutrophils to release superoxide anion was determined. The influence of N-nitrosodimethylamine on iNOS expression was determined in isolated PMN and PBMC cells from peripheral blood of healthy individuals. The mononuclear cells showed higher sensitivity to NDMA. Moreover, cytotoxic effect of NDMA can be influenced in some way by the impact of this xenobiotic on nitric oxide and superoxide anion release from human leukocytes. Furthermore, increased generation of these radicals by human leukocytes suggest that neutrophils and mononuclear cells that are exposed to NDMA activity can play a key role in endogenous NDMA generation. However the relationship between iNOS expression and phospho-p38 MAPK in neutrophils and mononuclear cells shows that p38 MAPK pathway participates in induction of iNOS expression in the presence of NDMA.

Differential heat shock response of primary human cell cultures and established cell lines

DEFF Research Database (Denmark)

Richter, W W; Issinger, O G

1986-01-01

degrees C treatment, whereas in immortalized cell lines usually 90% of the cells were found in suspension. Enhanced expression of the major heat shock protein (hsp 70) was found in all heat-treated cells. In contrast to the primary cell cultures, established and transformed cell lines synthesized...

Radiation response of haematopoietic cell lines of human origin

International Nuclear Information System (INIS)

Lehnert, S.; Rybka, W.B.; Suissa, S.; Giambattisto, D.

1986-01-01

Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radio-sensitivity of the cells varied widely, the D 0 s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivities. (author)

Human rhabdomyosarcoma cell lines for rhabdomyosarcoma research: Utility and pitfalls

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Ashley R.P. Hinson

2013-07-01

Full Text Available Rhabdomyosarcoma (RMS is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.

Human Rhabdomyosarcoma Cell Lines for Rhabdomyosarcoma Research: Utility and Pitfalls

Science.gov (United States)

Hinson, Ashley R. P.; Jones, Rosanne; Crose, Lisa E. S.; Belyea, Brian C.; Barr, Frederic G.; Linardic, Corinne M.

2013-01-01

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis. PMID:23882450

Radiation sensitivity of human lung cancer cell lines

International Nuclear Information System (INIS)

Carmichael, J.; Degraff, W.G.; Gamson, J.; Russo, G.; Mitchell, J.B.; Gazdar, A.F.; Minna, J.D.; Levitt, M.L.

1989-01-01

X-Ray survival curves were determined using a panel of 17 human lung cancer cell lines, with emphasis on non-small cell lung cancer (NSCLC). In contrast to classic small cell lung cancer (SCLC) cell lines, NSCLC cell lines were generally less sensitive to radiation as evidenced by higher radiation survival curve extrapolation numbers, surviving fraction values following a 2Gy dose (SF2) and the mean inactivation dose values (D) values. The spectrum of in vitro radiation responses observed was similar to that expected in clinical practice, although mesothelioma was unexpectedly sensitive in vitro. Differences in radiosensitivity were best distinguished by comparison of SF2 values. Some NSCLC lines were relatively sensitive, and in view of this demonstrable variability in radiation sensitivity, the SF2 value may be useful for in vitro predictive assay testing of clinical specimens. (author)

Generation of genome-modified Drosophila cell lines using SwAP.

Science.gov (United States)

Franz, Alexandra; Brunner, Erich; Basler, Konrad

2017-10-02

The ease of generating genetically modified animals and cell lines has been markedly increased by the recent development of the versatile CRISPR/Cas9 tool. However, while the isolation of isogenic cell populations is usually straightforward for mammalian cell lines, the generation of clonal Drosophila cell lines has remained a longstanding challenge, hampered by the difficulty of getting Drosophila cells to grow at low densities. Here, we describe a highly efficient workflow to generate clonal Cas9-engineered Drosophila cell lines using a combination of cell pools, limiting dilution in conditioned medium and PCR with allele-specific primers, enabling the efficient selection of a clonal cell line with a suitable mutation profile. We validate the protocol by documenting the isolation, selection and verification of eight independently Cas9-edited armadillo mutant Drosophila cell lines. Our method provides a powerful and simple workflow that improves the utility of Drosophila cells for genetic studies with CRISPR/Cas9.

Peptidomic analysis of human cell lines

Science.gov (United States)

Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

2011-01-01

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

The transcriptional diversity of 25 Drosophila cell lines

Energy Technology Data Exchange (ETDEWEB)

Cherbas, Lucy [Indiana Univ., Bloomington, IN (United States); Willingham, Aarron [Affymetrix Inc., Santa Clara, CA (United States); Zhang, Dayu [Indiana Univ., Bloomington, IN (United States); Yang, Li [University of Connecticut Health Center, Farmington, Connecticut (United States); Zou, Yi [Indiana Univ., Bloomington, IN (United States); Eads, Brian D. [Indiana Univ., Bloomington, IN (United States); Carlson, Joseph W. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Landolin, Jane M. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kapranov, Philipp [Affymetrix Inc., Santa Clara, CA (United States); Dumais, Jacqueline [Affymetrix Inc., Santa Clara, CA (United States); Samsonova, Anastasia [Harvard Medical School, Boston, MA (United States); Choi, Jeong-Hyeon [Indiana Univ., Bloomington, IN (United States); Roberts, Johnny [Indiana Univ., Bloomington, IN (United States); Davis, Carrie A. [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Tang, Haixu [Indiana Univ., Bloomington, IN (United States); van Baren, Marijke J. [Washington Univ., St. Louis, MO (United States); Ghosh, Srinka [Affymetrix Inc., Santa Clara, CA (United States); Dobin, Alexander [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Bell, Kim [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Lin, Wei [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Langton, Laura [Washington Univ., St. Louis, MO (United States); Duff, Michael O. [University of Connecticut Health Center, Farmington, Connecticut (United States); Tenney, Aaron E. [Washington Univ., St. Louis, MO (United States); Zaleski, Chris [Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Brent, Michael R. [Washington Univ., St. Louis, MO (United States); Hoskins, Roger A. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Kaufman, Thomas C. [Indiana University, Bloomington, Indiana (United States); Andrews, Justen [Indiana University, Bloomington, Indiana (United States); Graveley, Brenton R. [University of Connecticut Health Center, Farmington, Connecticut (United States); Perrimon, Norbert [Harvard Medical School, Boston, MA (United States); Howard Hughes Medical Institute, Boston, MA (United States); Celniker, Susan E. [Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Gingeras, Thomas R. [Affymetrix Inc., Santa Clara, CA (United States); Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (United States); Cherbas, Peter [Indiana Univ., Bloomington, IN (United States)

2010-12-22

Drosophila melanogaster cell lines are important resources for cell biologists. In this article, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are ‘‘off’’ and survival/growth pathways ‘‘on.’’ Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common ‘‘cell line‘‘ gene expression pattern. We report the transcriptional profiles of 25 Drosophila melanogaster cell lines, principally by whole-genome tiling microarray analysis of total RNA, carried out as part of the modENCODE project. The data produced in this study add to our knowledge of the cell lines and of the Drosophila transcriptome in several ways. We summarize the expression of previously annotated genes in each of the 25

Investigation of the selenium metabolism in cancer cell lines

DEFF Research Database (Denmark)

Lunøe, Kristoffer; Gabel-Jensen, Charlotte; Stürup, Stefan

2011-01-01

The aim of this work was to compare different selenium species for their ability to induce cell death in different cancer cell lines, while investigating the underlying chemistry by speciation analysis. A prostate cancer cell line (PC-3), a colon cancer cell line (HT-29) and a leukaemia cell line...... (Jurkat E6-1) were incubated with five selenium compounds representing inorganic as well as organic Se compounds in different oxidation states. Selenomethionine (SeMet), Se-methylselenocysteine (MeSeCys), methylseleninic acid (MeSeA), selenite and selenate in the concentration range 5-100 mu M were...... incubated with cells for 24 h and the induction of cell death was measured using flow cytometry. The amounts of total selenium in cell medium, cell lysate and the insoluble fractions was determined by ICP-MS. Speciation analysis of cellular fractions was performed by reversed phase, anion exchange and size...

Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma.

Science.gov (United States)

Drexler, H G; Matsuo, Y

2000-05-01

Malignancies involving natural killer (NK) cells are rare disorders. The complexity of NK cell-involving disorders has only recently been appreciated. Modern classifications discern immature (precursor) from mature NK cell leukemias-lymphomas. Continuous NK leukemia-lymphoma cell lines represent important model systems to study these neoplasms. While there are a number of putative NK cell lines which are, however, either not characterized, not immortalized, non-malignant, non-NK, or plain false cell lines, six bona fide malignant NK cell lines have been established and are sufficiently well characterized: HANK1, KHYG-1, NK-92, NKL, NK-YS and YT. Except for YT which was derived from a not further defined acute lymphoblastic lymphoma, these cell lines were established from patients with various NK cell malignancies. Five of the six cell lines are constitutively interleukin-2-dependent. Their immunoprofile is remarkably similar: CD1-, CD2+, surface CD3 (but cytoplasmic CD3epsilon+), CD4-, CD5-, CD7+, CD8-, CD16-, CD56+, CD57-, TCRalphabeta-, TCRgammadelta-, negative for B cell and myelomonocytic markers. The immunoglobulin heavy chain and T cell receptor genes are all in germline configuration. All six lines show complex chromosomal alterations, with both numerical and structural aberrations, attesting to their malignant and monoclonal nature. Functionally, these cells which contain azurophilic granules in their cytoplasm are nearly universally positive in NK activity assays. Three of five cell lines are Epstein-Barr virus-positive (type II latency). The composite data on these six cell lines allow for the operational definition of a typical malignant NK cell line profile. NK leukemia-lymphoma cell lines will prove invaluable for studies of normal and malignant NK cell biology.

Menadione inhibits MIBG uptake in two neuroendocrine cell lines

NARCIS (Netherlands)

Cornelissen, J.; Tytgat, G. A.; van den Brug, M.; van Kuilenburg, A. B.; Voûte, P. A.; van Gennip, A. H.

1997-01-01

In this paper we report on our studies of the effect of menadione on the uptake of MIBG in the neuroendocrine cell lines PC12 and SK-N-SH. Menadione inhibits the uptake of MIBG in both cell lines in a dose-dependent manner. Inhibition of MIBG uptake is most pronounced in the PC12 cell line.

Antiproliferative effect of Tualang honey on oral squamous cell carcinoma and osteosarcoma cell lines

Directory of Open Access Journals (Sweden)

Ismail Noorliza M

2010-09-01

Full Text Available Abstract Background The treatment of oral squamous cell carcinomas (OSCC and human osteosarcoma (HOS includes surgery and/or radiotherapy which often lead to reduced quality of life. This study was aimed to study the antiproliferative activity of local honey (Tualang on OSCC and HOS cell lines. Methods Several concentrations of Tualang honey (1% - 20% were applied on OSCC and HOS cell lines for 3, 6, 12, 24, 48 and 72 hours. Morphological characteristics were observed under light and fluorescent microscope. Cell viability was assessed using MTT assay and the optical density for absorbance values in each experiment was measured at 570 nm by an ELISA reader. Detection of cellular apoptosis was done using the Annexin V-FITC Apoptosis Detection Kit. Results Morphological appearance showed apoptotic cellular changes like becoming rounded, reduction in cell number, blebbed membrane and apoptotic nuclear changes like nuclear shrinkage, chromatin condensation and fragmented nucleus on OSCC and HOS cell lines. Cell viability assay showed a time and dose-dependent inhibitory effect of honey on both cell lines. The 50% inhibitory concentration (IC50 for OSCC and HOS cell lines was found to be 4% and 3.5% respectively. The maximum inhibition of cell growth of ≥80% was obtained at 15% for both cell lines. Early apoptosis was evident by flow cytometry where percentage of early apoptotic cells increased in dose and time dependent manner. Conclusion Tualang honey showed antiproliferative effect on OSCC and HOS cell lines by inducing early apoptosis.

Aberrant leukocyte telomere length in Birdshot Uveitis.

Directory of Open Access Journals (Sweden)

Nadia Vazirpanah

Full Text Available Birdshot Uveitis (BU is an archetypical chronic inflammatory eye disease, with poor visual prognosis, that provides an excellent model for studying chronic inflammation. BU typically affects patients in the fifth decade of life. This suggests that it may represent an age-related chronic inflammatory disease, which has been linked to increased erosion of telomere length of leukocytes.To study this in detail, we exploited a sensitive standardized quantitative real-time polymerase chain reaction to determine the peripheral blood leukocyte telomere length (LTL in 91 genotyped Dutch BU patients and 150 unaffected Dutch controls.Although LTL erosion rates were very similar between BU patients and healthy controls, we observed that BU patients displayed longer LTL, with a median of log (LTL = 4.87 (= 74131 base pair compared to 4.31 (= 20417 base pair in unaffected controls (P<0.0001. The cause underpinning the difference in LTL could not be explained by clinical parameters, immune cell-subtype distribution, nor genetic predisposition based upon the computed weighted genetic risk score of genotyped validated variants in TERC, TERT, NAF1, OBFC1 and RTEL1.These findings suggest that BU is accompanied by significantly longer LTL.

Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients.

Science.gov (United States)

Maggi, Laura; Margheri, Francesca; Luciani, Cristina; Capone, Manuela; Rossi, Maria Caterina; Chillà, Anastasia; Santarlasci, Veronica; Mazzoni, Alessio; Cimaz, Rolando; Liotta, Francesco; Maggi, Enrico; Cosmi, Lorenzo; Del Rosso, Mario; Annunziato, Francesco

2016-01-01

This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.

Establishment and characterization of rat portal myofibroblast cell lines.

Directory of Open Access Journals (Sweden)

Michel Fausther

Full Text Available The major sources of scar-forming myofibroblasts during liver fibrosis are activated hepatic stellate cells (HSC and portal fibroblasts (PF. In contrast to well-characterized HSC, PF remain understudied and poorly defined. This is largely due to the facts that isolation of rodent PF for functional studies is technically challenging and that PF cell lines had not been established. To address this, we have generated two polyclonal portal myofibroblast cell lines, RGF and RGF-N2. RGF and RGF-N2 were established from primary PF isolated from adult rat livers that underwent culture activation and subsequent SV40-mediated immortalization. Specifically, Ntpdase2/Cd39l1-sorted primary PF were used to generate the RGF-N2 cell line. Both cell lines were functionally characterized by RT-PCR, immunofluorescence, immunoblot and bromodeoxyuridine-based proliferation assay. First, immortalized RGF and RGF-N2 cells are positive for phenotypic myofibroblast markers alpha smooth muscle actin, type I collagen alpha-1, tissue inhibitor of metalloproteinases-1, PF-specific markers elastin, type XV collagen alpha-1 and Ntpdase2/Cd39l1, and mesenchymal cell marker ecto-5'-nucleotidase/Cd73, while negative for HSC-specific markers desmin and lecithin retinol acyltransferase. Second, both RGF and RGF-N2 cell lines are readily transfectable using standard methods. Finally, RGF and RGF-N2 cells attenuate the growth of Mz-ChA-1 cholangiocarcinoma cells in co-culture, as previously demonstrated for primary PF. Immortalized rat portal myofibroblast RGF and RGF-N2 cell lines express typical markers of activated PF-derived myofibroblasts, are suitable for DNA transfection, and can effectively inhibit cholangiocyte proliferation. Both RGF and RGF-N2 cell lines represent novel in vitro cellular models for the functional studies of portal (myofibroblasts and their contribution to the progression of liver fibrosis.

Leukocytic Response and Peripheral Venous Blood Lymphocyte Apoptosis as a Marker of Tissue Ischemia in Acute Massive Blood Loss

Directory of Open Access Journals (Sweden)

N. V. Borovkova

2013-01-01

Full Text Available Objective: to estimate the level of peripheral venous blood lymphocyte apoptosis and intraoperative hypoxia in victims with acute massive blood loss. Subjects and methods. Twenty-two patients with open and close chest and abdominal traumas complicated by acute massive blood loss were examined. All the patients were emergently operated on to stop bleeding. Tissue metabolism was evaluated from gases, acid-base parameters, and plasma lactate, glucose, potassium, and sodium levels. Apoptosis of mononuclear cells was studied and dead leukocytes were counted using flow cytometry. Results. Preoperatively, the victims were found to have venous hypoxemia, hyperlactatemia, hyperglycemia, moderate leukocytosis, and higher dead leukocyte counts. There were also raised counts of lymphocytes coming into the process of apoptosis. A significant relationship was found between monocyte counts and hypoxia values. At the end of surgery, oxygen balance values became stable and exerted an effect on the count of leukocytes, the relative level of granulocytes, the relative and absolute counts of dead and damaged leukocytes, and the concentration of lymphocytes in the victims’ venous blood during the early stages of apoptosis, as evidenced by nonlinear regression models. Conclusion. The indicators of immunocompetent cell apoptosis and the count of venous blood dead leukocytes along with lactate levels and venous hypoxemia parameters reflect the degree of tissue hypoxia and may be used as specific markers.

Casein gene expression in mouse mammary epithelial cell lines: Dependence upon extracellular matrix and cell type

International Nuclear Information System (INIS)

Medina, D.; Oborn, C.J.; Li, M.L.; Bissell, M.J.

1987-01-01

The COMMA-D mammary cell line exhibits mammary-specific functional differentiation under appropriate conditions in cell culture. The cytologically heterogeneous COMMA-D parental line and the clonal lines DB-1, TA-5, and FA-1 derived from the COMMA-D parent were examined for similar properties of functional differentiation. In monolayer cell culture, the cell lines DB-1, TA-5, FA-1, and MA-4 were examined for expression of mammary-specific and epithelial-specific proteins by an indirect immunofluorescence assay. The clonal cell lines were relatively homogeneous in their respective staining properties and seemed to represent three subpopulations found in the heterogeneous parental COMMA-D lines. None of the four clonal lines appeared to represent myoepithelial cells. The cell lines were examined for expression of β-casein mRNA in the presence or absence of prolactin. The inducibility of β-casein in the COMMA-D cell line was further enhanced by a reconstituted basement membrane preparation enriched in laminin, collagen IV, and proteoglycans. These results support the hypothesis that the functional response of inducible mammary cell populations is a result of interaction among hormones, multiple extracellular matrix components, and specific cell types

Leukocyte telomere length in major depression: correlations with chronicity, inflammation and oxidative stress--preliminary findings.

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Owen M Wolkowitz

2011-03-01

Full Text Available Depression is associated with an unusually high rate of aging-related illnesses and early mortality. One aspect of "accelerated aging" in depression may be shortened leukocyte telomeres. When telomeres critically shorten, as often occurs with repeated mitoses or in response to oxidation and inflammation, cells may die. Indeed, leukocyte telomere shortening predicts early mortality and medical illnesses in non-depressed populations. We sought to determine if leukocyte telomeres are shortened in Major Depressive Disorder (MDD, whether this is a function of lifetime depression exposure and whether this is related to putative mediators, oxidation and inflammation.Leukocyte telomere length was compared between 18 unmedicated MDD subjects and 17 controls and was correlated with lifetime depression chronicity and peripheral markers of oxidation (F2-isoprostane/Vitamin C ratio and inflammation (IL-6. Analyses were controlled for age and sex.The depressed group, as a whole, did not differ from the controls in telomere length. However, telomere length was significantly inversely correlated with lifetime depression exposure, even after controlling for age (p<0.05. Average telomere length in the depressed subjects who were above the median of lifetime depression exposure (≥9.2 years' cumulative duration was 281 base pairs shorter than that in controls (p<0.05, corresponding to approximately seven years of "accelerated cell aging." Telomere length was inversely correlated with oxidative stress in the depressed subjects (p<0.01 and in the controls (p<0.05 and with inflammation in the depressed subjects (p<0.05.These preliminary data indicate that accelerated aging at the level of leukocyte telomeres is proportional to lifetime exposure to MDD. This might be related to cumulative exposure to oxidative stress and inflammation in MDD. This suggest that telomere shortening does not antedate depression and is not an intrinsic feature. Rather, telomere shortening

The Effect of Hemiscorpius lepturus (Scorpionida: Hemiscorpiidae Venom on Leukocytes and the Leukocyte Subgroups in Peripheral Blood of Rat

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Mehri Ghafourian

2016-01-01

Full Text Available Background: The aim of this study was to investigate the effect of Hemiscorpius lepturus venom on leukocytes and the leukocyte subgroups in peripheral blood of rat.Methods: In this experimental study, sixty N-Mari rats were divided into three groups of 20 rats. Then the rats in each group were divided into four subgroups based on the blood sampling time that was 2, 6, 24 and 48 hours after the venom injection, respectively. The control group did not receive anything, however, the first and the second ex­perimental groups received 0.1 and 0.01mg/kg of venom, subcutaneously. In accordance with a designated four sam­pling times, the blood sampling was carried out in three groups. After RBC lysis, the leukocytes and leukocyte sub­populations were determined and counted using appropriate hematological standard methods.Results: The leukocyte and the neutrophil count at two (P<0.05, six (P<0.01 and 24 (P<0.05 hours after the venom injection showed a significant decline compared with the control group, this decrease was significant at the dose of 0.1 mg/kg until 48 hours after the venom injection (P<0.05. The lymphocyte count showed a significant decline throughout the all hours of the experiment, compared with the control group (P<0.05.Conclusion: Leukocytes are probably affected by the cytotoxicity effect of the H. lepturus venom in a dose-dependent manner. This could be a wakeup call for the medical staff to perform quick and accurate treatment in the least time possible.

VEGF-A isoforms differentially regulate ATF-2-dependent VCAM-1 gene expression and endothelial-leukocyte interactions.

Science.gov (United States)

Fearnley, Gareth W; Odell, Adam F; Latham, Antony M; Mughal, Nadeem A; Bruns, Alexander F; Burgoyne, Nicholas J; Homer-Vanniasinkam, Shervanthi; Zachary, Ian C; Hollstein, Monica C; Wheatcroft, Stephen B; Ponnambalam, Sreenivasan

2014-08-15

Vascular endothelial growth factor A (VEGF-A) regulates many aspects of vascular physiology. VEGF-A stimulates signal transduction pathways that modulate endothelial outputs such as cell migration, proliferation, tubulogenesis, and cell-cell interactions. Multiple VEGF-A isoforms exist, but the biological significance of this is unclear. Here we analyzed VEGF-A isoform-specific stimulation of VCAM-1 gene expression, which controls endothelial-leukocyte interactions, and show that this is dependent on both ERK1/2 and activating transcription factor-2 (ATF-2). VEGF-A isoforms showed differential ERK1/2 and p38 MAPK phosphorylation kinetics. A key feature of VEGF-A isoform-specific ERK1/2 activation and nuclear translocation was increased phosphorylation of ATF-2 on threonine residue 71 (T71). Using reverse genetics, we showed ATF-2 to be functionally required for VEGF-A-stimulated endothelial VCAM-1 gene expression. ATF-2 knockdown blocked VEGF-A-stimulated VCAM-1 expression and endothelial-leukocyte interactions. ATF-2 was also required for other endothelial cell outputs, such as cell migration and tubulogenesis. In contrast, VCAM-1 was essential only for promoting endothelial-leukocyte interactions. This work presents a new paradigm for understanding how soluble growth factor isoforms program complex cellular outputs and responses by modulating signal transduction pathways. © 2014 Fearnley et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Altered mitochondrial function and oxidative stress in leukocytes of anorexia nervosa patients.

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Victor M Victor

Full Text Available CONTEXT: Anorexia nervosa is a common illness among adolescents and is characterised by oxidative stress. OBJECTIVE: The effects of anorexia on mitochondrial function and redox state in leukocytes from anorexic subjects were evaluated. DESIGN AND SETTING: A multi-centre, cross-sectional case-control study was performed. PATIENTS: Our study population consisted of 20 anorexic patients and 20 age-matched controls, all of which were Caucasian women. MAIN OUTCOME MEASURES: Anthropometric and metabolic parameters were evaluated in the study population. To assess whether anorexia nervosa affects mitochondrial function and redox state in leukocytes of anorexic patients, we measured mitochondrial oxygen consumption, membrane potential, reactive oxygen species production, glutathione levels, mitochondrial mass, and complex I and III activity in polymorphonuclear cells. RESULTS: Mitochondrial function was impaired in the leukocytes of the anorexic patients. This was evident in a decrease in mitochondrial O2 consumption (P<0.05, mitochondrial membrane potential (P<0.01 and GSH levels (P<0.05, and an increase in ROS production (P<0.05 with respect to control subjects. Furthermore, a reduction of mitochondrial mass was detected in leukocytes of the anorexic patients (P<0.05, while the activity of mitochondrial complex I (P<0.001, but not that of complex III, was found to be inhibited in the same population. CONCLUSIONS: Oxidative stress is produced in the leukocytes of anorexic patients and is closely related to mitochondrial dysfunction. Our results lead us to propose that the oxidative stress that occurs in anorexia takes place at mitochondrial complex I. Future research concerning mitochondrial dysfunction and oxidative stress should aim to determine the physiological mechanism involved in this effect and the physiological impact of anorexia.

Leukocyte counts in urine reflect the risk of concomitant sepsis in bacteriuric infants: A retrospective cohort study

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Harper Marvin B

2007-06-01

Full Text Available Abstract Background When urine infections are missed in febrile young infants with normal urinalysis, clinicians may worry about the risk – hitherto unverified – of concomitant invasion of blood and cerebrospinal fluid by uropathogens. In this study, we determine the extent of this risk. Methods In a retrospective cohort study of febrile 0–89 day old infants evaluated for sepsis in an urban academic pediatric emergency department (1993–1999, we estimated rates of bacteriuric sepsis (urinary tract infections complicated by sepsis after stratifying infants by urine leukocyte counts higher, or lower than 10 cells/hpf. We compared the global accuracy of leukocytes in urine, leukocytes in peripheral blood, body temperature, and age for predicting bacteruric sepsis. The global accuracy of each test was estimated by calculating the area under its receiver operating characteristic curve (AUC. Chi-square and Fisher exact tests compared count data. Medians for data not normally distributed were compared by the Kruskal-Wallis test. Results Two thousand two hundred forty-nine young infants had a normal screening dipstick. None of these developed bacteremia or meningitis despite positive urine culture in 41 (1.8%. Of 1516 additional urine specimens sent for formal urinalysis, 1279 had 0–9 leukocytes/hpf. Urine pathogens were isolated less commonly (6% vs. 76% and at lower concentrations in infants with few, compared to many urine leukocytes. Urine leukocytes (AUC: 0.94 were the most accurate predictors of bacteruric sepsis. Infants with urinary leukocytes Conclusion In young infants evaluated for fever, leukocytes in urine reflect the likelihood of bacteriuric sepsis. Infants with urinary tract infections missed because of few leukocytes in urine are at relatively low risk of invasive bacterial sepsis by pathogens isolated from urine.

9-β-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

International Nuclear Information System (INIS)

Heaton, D.

1992-06-01

The effect of 9-β-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D 0 values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D 0 values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 μM) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 μM were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo

9-{beta}-arabinofuranosyladenine preferentially sensitizes radioresistant squamous cell carcinoma cell lines to x-rays

Energy Technology Data Exchange (ETDEWEB)

Heaton, D. [Rush Univ. Medical Center, Chicago, IL (United States). Therapeutic Radiology; Mustafi, R. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology; Schwartz, J.L. [Chicago Univ., IL (United States). Dept. of Radiation and Cellular Oncology]|[Argonne National Lab., IL (United States)

1992-06-01

The effect of 9-{beta}-arabinofuranosyladenine (ara-A) on sensitivity to the deleterious effects of x-rays was studied in six squamous cell carcinoma cell lines. Three lines were relatively radioresistant, having D{sub 0} values of 2.31 to 2.89 Gy, and the other three lines were relatively radiosensitive, having D{sub 0} values of between 1.07 and 1.45 Gy. Ara-A (50 or 500 {mu}M) was added to cultures 30 min prior to irradiation and removed 30 min after irradiation, and sensitivity was measured in terms of cell survival. The radiosensitizing effect of ara-A was very dependent on the inherent radiosensitivity of the tumor cell line. Fifty micromolar concentrations of ara-A sensitized only the two most radioresistant lines, SCC-12B.2 and JSQ-3. Five hundred micromolar concentrations of ara-A sensitized the more sensitive cell lines, SQ-20B and SQ-9G, but failed to have any effect on the radiation response of the two most sensitive cell lines, SQ-38 and SCC-61. Concentrations of ara-A as low as 10 {mu}M were equally efficient in inhibiting DNA synthesis in all six cell lines. These results suggest that the target for the radiosensitizing effect of ara-A is probably related to the factor controlling the inherent radiosensitivity of human tumor cells. Therefore, ara-A might be useful in overcoming radiation resistance in vivo.

In vitro culture of human osteosarcoma cell lines: a comparison of functional characteristics for cell lines cultured in medium without and with fetal calf serum.

Science.gov (United States)

Bruserud, Oystein; Tronstad, Karl Johan; Berge, Rolf

2005-06-01

Experimental in vitro models including well-characterised cell lines can be used to identify possible new therapeutic targets for the treatment of osteosarcoma. Culture media including inactivated serum is often recommended for in vitro culture of osteosarcoma cells, but the serum component then represents a nonstandardised parameter including a wide range of unidentified mediators. To improve the standardisation we have investigated whether serum-free culture media can be used in experimental in vitro studies of osteosarcoma cell lines. The seven osteosarcoma cell lines Cal72, SJSA-1, Saos-2, SK-ES-1, U2OS, 143.98.2, and KHOS-32IH were cultured in vitro in various serum-free media and media supplemented with 10% heat-inactivated fetal calf serum (FCS). Although proliferation often was relatively low in serum-free media (X-vivo 10, X-vivo 15, X-vivo 20, Stem Span SFEM), some cell lines (Cal72, KHOS-32IH, Saos-2) showed proliferation comparable with the recommended FCS-containing media even when using serum-free conditions. The optimal serum-free medium then varied between cell lines. We also compared 6 different FCS-containing media (including Stem Span with 10% FCS) and the optimal FCS-containing medium varied between cell lines. However, all cell lines proliferated well in Stem Span with FCS, and this medium was regarded as optimal for four of the lines. FCS could not be replaced by fatty acids or low density lipoprotein when testing the Stem Span medium. The release of a wide range of soluble mediators showed only minor differences when using serum-free and FCS-containing media (including Stem Span with and without FCS), and serum-free Stem Span could also be used for in vitro studies of mitogen-stimulated T cell activation in the presence of accessory osteosarcoma cells. The use of Stem Span with 10% FCS allowed the release of a wide range of chemokines by osteosarcoma cell lines (Cal72, SJSA-1), and the chemokine release profile was very similar to the

Clonogenic cell line survival of a human liver cancer cell line SMMC-7721 after carbon ion irradiation with different LET

International Nuclear Information System (INIS)

Lei Suwen; Su Xu; Wang Jifang; Li Wenjian

2003-01-01

Objective: To investigate the survival fraction of a human liver cancer cell line SMMC-7721 following irradiation with carbon ions with different LET. Methods: cells of the human liver cancer cell line SMMC-7721 were irradiated with carbon ions (LET=30 and 70 keV/μm). The survival fraction was determined with clonogenic assay after 9 days incubation in a 5% CO 2 incubator at 37 degree C. Results: When the survival fractions of 70 keV/μm were D s = 0.1 and D s=0.01 absorption dose were 2.94 and 5.88 Gy respectively, and those of 30 keV/μm were 4.00 and 8.00 Gy respectively. Conclusion: For the SMMC-7721 cell line, 70 keV/μm is more effective for cell killing than 30 keV/μm

Production of antibodies which recognize opiate receptors on murine leukocytes

Energy Technology Data Exchange (ETDEWEB)

Carr, D.J.J.; Bost, K.L.; Blalock, J.E.

1988-01-01

An antibody has been developed which recognizes opiate receptors on cells of the immune system. This antibody blocks specific binding of the radiolabeled opiate receptor ligand, /sup 3/H-dihydromorphine, to receptors on murine splenocytes. Additionally, the anti-receptor antibody competes with ..beta..-endorphin, meta-enkephalin, and naloxone for the same binding site on the leukocytes. Moreover, the anti-receptor antibody possesses agonist activity similar to ..beta..-endorphin in suppressing cAMP production by lymphocytes. These results suggest the development of an antibody which recognizes classical opiate receptors on cells of the immune system.

Maternal circulating leukocytes display early chemotactic responsiveness during late gestation

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Gomez-Lopez Nardhy

2013-01-01

Full Text Available Abstract Background Parturition has been widely described as an immunological response; however, it is unknown how this is triggered. We hypothesized that an early event in parturition is an increased responsiveness of peripheral leukocytes to chemotactic stimuli expressed by reproductive tissues, and this precedes expression of tissue chemotactic activity, uterine activation and the systemic progesterone/estradiol shift. Methods Tissues and blood were collected from pregnant Long-Evans rats on gestational days (GD 17, 20 and 22 (term gestation. We employed a validated Boyden chamber assay, flow cytometry, quantitative real time-polymerase chain reaction, and enzyme-linked immunosorbent assays. Results We found that GD20 maternal peripheral leukocytes migrated more than those from GD17 when these were tested with GD22 uterus and cervix extracts. Leukocytes on GD20 also displayed a significant increase in chemokine (C-C motif ligand 2 (Ccl2 gene expression and this correlated with an increase in peripheral granulocyte proportions and a decrease in B cell and monocyte proportions. Tissue chemotactic activity and specific chemokines (CCL2, chemokine (C-X-C motif ligand 1/CXCL1, and CXCL10 were mostly unchanged from GD17 to GD20 and increased only on GD22. CXCL10 peaked on GD20 in cervical tissues. As expected, prostaglandin F2α receptor and oxytocin receptor gene expression increased dramatically between GD20 and 22. Progesterone concentrations fell and estradiol-17β concentrations increased in peripheral serum, cervical and uterine tissue extracts between GD20 and 22. Conclusion Maternal circulating leukocytes display early chemotactic responsiveness, which leads to their infiltration into the uterus where they may participate in the process of parturition.

21 CFR 864.7675 - Leukocyte peroxidase test.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leukocyte peroxidase test. 864.7675 Section 864.7675 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7675 Leukocyte...

Flow cytometry as a tool in the evaluation of blood leukocyte function in Chelonia mydas (Linnaeus, 1758 (Testudines, Cheloniidae

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S. Rossi

Full Text Available Chelonia mydas is a sea turtle that feeds and nests on the Brazilian coast and a disease called fibropapillomatosis is a threat to this species. Because of this, it is extremely necessary to determine a methodology that would enable the analysis of blood leukocyte function in these sea turtles. In order to achieve this aim, blood samples were collected from C. mydas with or without fibropapillomas captured on the São Paulo north coast. Blood samples were placed in tubes containing sodium heparin and were transported under refrigeration to the laboratory in sterile RPMI 1640 cell culture medium. Leukocytes were separated by density gradient using Ficoll-PaqueTM Plus, Amershan Biociences®. The following stimuli were applied in the assessment of leukocyte function: Phorbol Miristate-Acetate (PMA for oxidative burst activity evaluation and Zymosan A (Saccharomyces cerevisiae Bio Particles®, Alexa Fluor® 594 conjugate for phagocytosis evaluation. Three cell populations were identified: heterophils, monocytes and lymphocytes. Monocytes were the cells responsible for phagocytosis and oxidative burst.

Scintigraphy with /sup 111/In-labeled leukocytes. Simplified procedure for labeling

Energy Technology Data Exchange (ETDEWEB)

Terada, Hitoshi; Shiire, Yasushi; Koizumi, Kiyoshi; Aburano, Tamio; Tonami, Norihisa; Hisada, Kin-ichi

1987-12-01

To utilize /sup 111/In leukocytes in a routine work, simplified procedure for sterile leukocytes preparation and labeling with water soluble oxine sulfate was performed. Viability and chemotaxis of leukocytes were maintained during separation and labeling. Chelated rate of /sup 111/In with oxine sulfate was 93.5 %. Labeling efficiency of /sup 111/In leukocytes was 93.8 %. Obvious blood pool images due to remaind erythrocytes were not observed. /sup 111/In labeled leukocytes showed good migration into inflammatory focci.

In vitro invasion of small-cell lung cancer cell lines correlates with expression of epidermal growth factor receptor

DEFF Research Database (Denmark)

Damstrup, L; Rude Voldborg, B; Spang-Thomsen, M

1998-01-01

receptor (EGFR) in a panel of 21 small-cell lung cancer (SCLC) cell lines. We have previously reported that ten of these cell lines expressed EGFR protein detected by radioreceptor and affinity labelling assays. In 11 small-cell lung cancer (SCLC) cell lines, EGFR mRNA was detected by Northern blot...... analysis. In vitro invasion in a Boyden chamber assay was found in all EGFR-positive cell lines, whereas no invasion was detected in the EGFR-negative cell lines. Quantification of the in vitro invasion in 12 selected SCLC cell lines demonstrated that, in the EGFR-positive cell lines, between 5% and 16......-PCR). However, in vitro invasive SCLC cell lines could not be distinguished from non-invasive cell lines based on the expression pattern of these molecules. In six SCLC cell lines, in vitro invasion was also determined in the presence of the EGFR-neutralizing monoclonal antibody mAb528. The addition...

Piscirickettsia salmonis Imbalances the Innate Immune Response to Succeed in a Productive Infection in a Salmonid Cell Line Model.

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Claudio A Álvarez

Full Text Available Piscirickettsia salmonis is a facultative intracellular bacterium that causes the disease called "salmon rickettsial syndrome". Attempts to control this disease have been unsuccessful, because existing vaccines have not achieved the expected effectiveness and the antibiotics used fail to completely eradicate the pathogen. This is in part the product of lack of scientific information that still lacks on the mechanisms used by this bacterium to overcome infected-cell responses and survive to induce a productive infection in macrophages. For that, this work was focused in determining if P. salmonis is able to modify the expression and the imbalance of IL-12 and IL-10 using an in vitro model. Additionally, we also evaluated the role the antimicrobial peptide hepcidin had in the control of this pathogen in infected cells. Therefore, the expression of IL-10 and IL-12 was evaluated at earlier stages of infection in the RTS11 cell line derived from Oncorhynchus mykiss macrophages. Simultaneously, the hepcidin expression and location was analyzed in the macrophages infected with the pathogen. Our results suggest that IL-10 is clearly induced at early stages of infection with values peaking at 36 hours post infection. Furthermore, infective P. salmonis downregulates the expression of antimicrobial peptide hepcidin and vesicles containing this peptide were unable to merge with the infective bacteria. Our results suggest that P. salmonis is able to manipulate the behavior of host cytokines and likely might constitute a virulence mechanism that promotes intracellular bacterial replication in leukocytes cells lines of trout and salmon. This mechanism involves the generation of an optimum environment for the microorganism and the downregulation of antimicrobial effectors like hepcidin.

Inhibition of time-dependent enhancement of amino acid transport by leukemic leukocytes: a possible index of the sensitivity of cells to drugs

Energy Technology Data Exchange (ETDEWEB)

Frengley, P A; Peck, W A; Lichtman, M A

1975-01-01

Leukemic leukocytes increase their rates of alpha aminoisobutyric acid (AIB) accumulation when incubated for prolonged periods in amino acid deficient media. The time-dependent increase was prevented by concurrent exposure of cells to cycloheximide or actinomycin D in vitro. In addition, the increase in AIB uptake was not present in leukemic blasts studied in vitro when the cells were obtained from subjects with acute myeloblastic leukemia who had received antileukemic therapy. Cortisol added to cell suspensions in vitro inhibited the development of time-dependent increases in AIB uptake in lymphoid cells, but accentuated the process slightly in myeloblasts. Cortisol administered to a subject with CLL by infusion reduced the time-dependent increase in AIB uptake by CLL cells subsequently studied in vitro. These data indicate that the time-dependent increase in AIB uptake may be a means of testing the sensitivity of leukemic cells to drugs.

In-111-labeled leukocytes in the diagnosis of rejection and cytomegalovirus infection in renal transplant patients

International Nuclear Information System (INIS)

Forstrom, L.A.; Loken, M.K.; Cook, A.; Chandler, R.; McCullough, J.

1981-01-01

Indium-111-labeled (In-111) leukocytes have been shown to be useful in the localization of inflammatory processes, including renal transplant rejection. Using previously reported labeling methods, 63 studies with this agent have been performed in 53 renal transplant patients. Indications for study included suspected rejection or cytomegalovirus (CMV) infection. Studies were performed in 33 men and 20 women, with ages ranging from 6 to 68 years. Autologous cells were normally used for labeling, although leukocytes obtained from ABO-compatible donors were used in three subjects. Rectilinear scanner and/or scintillation camera images were obtained at 24 hours after intravenous administration of 0.1 to 0.6 mCi of In-111-leukocytes. There was abnormal uptake of In-111-leukocytes in the transplanted kidney in 11 of 15 cases of rejection. In three additional cases of increased transplant uptake, CMV infection was present in two. Abnormal lung uptake was present in 13 of 14 patients with CMV infection. In four additional cases, increased lung uptake was associated with other pulmonary inflammatory disease. Increased lung activity was not seen in patients with uncomplicated transplant rejection. These results suggest that In-111-leukocyte imaging may be useful in the differential diagnosis of rejection versus CMV infection in renal transplant patients

In-111-labeled leukocytes in the diagnosis of rejection and cytomegalovirus infection in renal transplant patients

International Nuclear Information System (INIS)

Forstrom, L.A.; Loken, M.K.; Cook, A.; Chandler, R.; McCullough, J.

1981-01-01

Indium-111-labelled (In-111) leukocytes have been shown to be useful in the localization of inflammatory processes, including renal transplant rejection. Using previously reported labelling methods, 63 studies with this agent have been performed in 53 renal transplant patients. Indications for study included suspected rejection or cytomegalovirus (CMV) infection. Studies were performed in 33 men and 20 women, with ages ranging from 6 to 68 years. Autologous cells were normally used for labeling, although leukocytes obtained from ABO-compatible donors were used in three subjects. Rectilinear scanner and/or scintillation camera images were obtained at 24 hours after intravenous administration of 0.1 to 0.6 mCi of In-111 leukocytes. There was abnormal uptake of In-111-leukocytes in the transplanted kidney in 11 of 15 cases of rejection. In three additional cases of increased transplant uptake, CMV infection was present in two. Abnormal lung uptake was present in 13 of 14 patients with CMV infection. In four additional cases, increased lung uptake was associated with other pulmonary inflammatory disease. Increased lung activity was not seen in patients with uncomplicated transplant rejection. These results suggest that In-111-leukocyte imaging may be useful in the differential diagnosis of rejection versus CMV infection in renal transplant patients

Identification of a novel rhabdovirus in Spodoptera frugiperda cell lines.

Science.gov (United States)

Ma, Hailun; Galvin, Teresa A; Glasner, Dustin R; Shaheduzzaman, Syed; Khan, Arifa S

2014-06-01

The Sf9 cell line, derived from Spodoptera frugiperda, is used as a cell substrate for biological products, and no viruses have been reported in this cell line after extensive testing. We used degenerate PCR assays and massively parallel sequencing (MPS) to identify a novel RNA virus belonging to the order Mononegavirales in Sf9 cells. Sequence analysis of the assembled virus genome showed the presence of five open reading frames (ORFs) corresponding to the genes for the N, P, M, G, and L proteins in other rhabdoviruses and an unknown ORF of 111 amino acids located between the G- and L-protein genes. BLAST searches indicated that the S. frugiperda rhabdovirus (Sf-rhabdovirus) was related in a limited region of the L-protein gene to Taastrup virus, a newly discovered member of the Mononegavirales from a leafhopper (Hemiptera), and also to plant rhabdoviruses, particularly in the genus Cytorhabdovirus. Phylogenetic analysis of sequences in the L-protein gene indicated that Sf-rhabdovirus is a novel virus that branched with Taastrup virus. Rhabdovirus morphology was confirmed by transmission electron microscopy of filtered supernatant samples from Sf9 cells. Infectivity studies indicated potential transient infection by Sf-rhabdovirus in other insect cell lines, but there was no evidence of entry or virus replication in human cell lines. Sf-rhabdovirus sequences were also found in the Sf21 parental cell line of Sf9 cells but not in other insect cell lines, such as BT1-TN-5B1-4 (Tn5; High Five) cells and Schneider's Drosophila line 2 [D.Mel.(2); SL2] cells, indicating a species-specific infection. The results indicate that conventional methods may be complemented by state-of-the-art technologies with extensive bioinformatics analysis for identification of novel viruses. The Spodoptera frugiperda Sf9 cell line is used as a cell substrate for the development and manufacture of biological products. Extensive testing has not previously identified any viruses in this cell

Flow cytofluorometric monitoring of leukocyte apoptosis in experimental cholera

Science.gov (United States)

Lotsmanova, Ekaterina Y.; Kravtsov, Alexander L.; Livanova, Ludmila F.; Kobkova, Irina M.; Kuznetsov, Oleg S.; Shchukovskaya, Tatyana N.; Smirnova, Nina I.; Kutyrev, Vladimir V.

2003-10-01

Flow cytofluorometric DNA analysis was applied to determine of the relative contents of proliferative (more then 2C DNA per cell) and apoptotic (less then 2C DNA per cell) leukocytes in blood of adult rabbits, challenged with 10,000 times the 50 % effective dose of Vibrio cholerae virulent strain by the RITARD technique. It has been shown that irreversible increase the percentage of cells carrying DNA in the degradation stage brings to disbalance between the genetically controlled cell proliferation and apoptosis that leads to animal death from the cholera infection. Such fatal changes were not observed in challenging of immunized animals that were not died. Thus received data show that the flow cytofluorometric measurements may be used for detection of transgressions in homeostasis during acute infection diseases, for outlet prognosis of the cholera infection.

Variable leukocyte composition of red blood cell concentrates prepared in top-bottom systems: possible implications for pre-transplant blood transfusion

NARCIS (Netherlands)

Weinreich, S. S.; Remmerswaal, E. B.; Laport, R.; van Rhenen, D. J.; Rombout-Sastrienkova, E.; Smit Sibinga, C. T.; Vrielink, H.; Boog, C. J.

2000-01-01

BACKGROUND AND OBJECTIVES: The beneficial effect of blood transfusion on kidney graft survival requires the presence of leukocytes in the transfusate, but a minimal dose has not been defined, nor has the role of individual leukocyte subsets been investigated. In the Netherlands, a standard

Isolation of two chloroethylnitrosourea-sensitive Chinese hamster cell lines

International Nuclear Information System (INIS)

Hata, H.; Numata, M.; Tohda, H.; Yasui, A.; Oikawa, A.

1991-01-01

1-[(4-Amino-2-methylpyrimidin-5-yl)methyl]-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU), a cancer chemotherapeutic bifunctional alkylating agent, causes chloroethylation of DNA and subsequent DNA strand cross-linking through an ethylene bridge. We isolated and characterized two ACNU-sensitive mutants from mutagenized Chinese hamster ovary cells and found them to be new drug-sensitive recessive Chinese hamster mutants. Both mutants were sensitive to various monofunctional alkylating agents in a way similar to that of the parental cell lines CHO9. One mutant (UVS1) was cross-sensitive to UV and complemented the UV sensitivity of all Chinese hamster cell lines of 7 established complementation groups. Since UV-induced unscheduled DNA synthesis was very low, a new locus related to excision repair is thought to be defective in this cell line. Another ACNU-sensitive mutant, CNU1, was slightly more sensitive to UV than the parent cell line. CNU1 was cross-sensitive to 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea and slightly more sensitive to mitomycin C. No increased accumulation of ACNU and a low level of UV-induced unscheduled DNA synthesis in this cell as compared with the parental cell line suggest that there is abnormality in a repair response of this mutant cell to some types of DNA cross-links

Lining cells on normal human vertebral bone surfaces

International Nuclear Information System (INIS)

Henning, C.B.; Lloyd, E.L.

1982-01-01

Thoracic vertebrae from two individuals with no bone disease were studied with the electron microscope to determine cell morphology in relation to bone mineral. The work was undertaken to determine if cell morphology or spatial relationships between the bone lining cells and bone mineral could account for the relative infrequency of bone tumors which arise at this site following radium intake, when compared with other sites, such as the head of the femur. Cells lining the vertebral mineral were found to be generally rounded in appearance with varied numbers of cytoplasmic granules, and they appeared to have a high density per unit of surface area. These features contrasted with the single layer of flattened cells characteristic of the bone lining cells of the femur. A tentative discussion of the reasons for the relative infrequency of tumors in the vertebrae following radium acquisition is presented

Susceptibility of various cell lines to Neospora caninum tachyzoites cultivation

Directory of Open Access Journals (Sweden)

Khordadmehr, M.,

2014-05-01

Full Text Available Neospora caninum is a coccidian protozoan parasite which is a major cause of bovine abortions and neonatal mortality in cattle, sheep, goat and horse. Occasionally, cultured cells are used for isolation and multiplication of the agent in vitro with several purposes. In this study the tachyzoite yields of N. caninum were compared in various cell cultures as the host cell lines. Among the cell cultures tested, two presented good susceptibility to the agent: cell lines Vero and MA-104. SW742 and TLI (in vitro suspension culture of lymphoid cells infected with Theileria lestoquardi showed moderate sensitivity. No viable tachyzoite were detected in the culture of MDCK and McCoy cell lines. These results demonstrate that MA-104 and SW742 cells present adequate susceptibility to N. caninum compared to Vero cells, which have been largely used to multiply the parasite in vitro. Moreover, these have easy manipulation, fast multiplication and relatively low nutritional requirements. In addition, the result of this study showed that TLI cell line as a suspension cell culture is susceptible to Nc-1 tachyzoites infection and could be used as an alternative host cell line for tachyzoites culture in vitro studies.

3H-histamine release from human leukocytes

International Nuclear Information System (INIS)

Stahl Skov, P.; Norn, S.; Weeke, B.

1979-01-01

A rapid, simple, and inexpensive method for large scale screening of patients suspected of type I allergy has been developed. The method is based on in vitro incorporation of 3 H-histamine in the leukocytes of the patient, whereafter release of labelled histamine is measured after provocation of the cells with the suspected allergen. The new method was compared with the conventional basophil histamine release technique by in vitro provocation of six asthmatic patients under suspicion of type I allergy against animal dander, house dust, and mite, and an almost identical release of histamine was observed in both assays. (author)

Molecular characterization of breast cancer cell lines through multiple omic approaches.

Science.gov (United States)

Smith, Shari E; Mellor, Paul; Ward, Alison K; Kendall, Stephanie; McDonald, Megan; Vizeacoumar, Frederick S; Vizeacoumar, Franco J; Napper, Scott; Anderson, Deborah H

2017-06-05

Breast cancer cell lines are frequently used as model systems to study the cellular properties and biology of breast cancer. Our objective was to characterize a large, commonly employed panel of breast cancer cell lines obtained from the American Type Culture Collection (ATCC 30-4500 K) to enable researchers to make more informed decisions in selecting cell lines for specific studies. Information about these cell lines was obtained from a wide variety of sources. In addition, new information about cellular pathways that are activated within each cell line was generated. We determined key protein expression data using immunoblot analyses. In addition, two analyses on serum-starved cells were carried out to identify cellular proteins and pathways that are activated in these cells. These analyses were performed using a commercial PathScan array and a novel and more extensive phosphopeptide-based kinome analysis that queries 1290 phosphorylation events in major signaling pathways. Data about this panel of breast cancer cell lines was also accessed from several online sources, compiled and summarized for the following areas: molecular classification, mRNA expression, mutational status of key proteins and other possible cancer-associated mutations, and the tumorigenic and metastatic capacity in mouse xenograft models of breast cancer. The cell lines that were characterized included 10 estrogen receptor (ER)-positive, 12 human epidermal growth factor receptor 2 (HER2)-amplified and 18 triple negative breast cancer cell lines, in addition to 4 non-tumorigenic breast cell lines. Within each subtype, there was significant genetic heterogeneity that could impact both the selection of model cell lines and the interpretation of the results obtained. To capture the net activation of key signaling pathways as a result of these mutational combinations, profiled pathway activation status was examined. This provided further clarity for which cell lines were particularly deregulated

Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

Science.gov (United States)

Branham-O'Connor, Melissa; Robichaux, William G; Zhang, Xian-Kui; Cho, Hyeseon; Kehrl, John H; Lanier, Stephen M; Blumer, Joe B

2014-04-11

Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

In vitro comparison of HMPAO and gentisic acid for labelling leukocytes with 99mTc

International Nuclear Information System (INIS)

Ecclestone, M.; Proulx, A.; Ballinger, J.R.; Gerson, B.; Reid, R.H.; Gulenchyn, K.Y.

1990-01-01

Leukocytes can be labelled with 99m Tc using HMPAO and gentisic acid methods. We compared the two methods with respect to labelling efficiency on mixed leukocytes and isolated polymorphonuclear (PMN) and mononuclear (MN) cells, and the in vitro stability of the label. HMPAO produced approximately 70% labelling efficiency on mixed or PMN cells and the label was stable in saline or plasma. Labelling efficiency on MN was only 14% and was less stable. Gentisic acid produced a labelling efficiency of 52% on PMN and 35% on MN; both were stable in saline but less stable in plasma. In conclusion, HMPAO produces higher labelling efficiency and the label shows greater in vitro stability in plasma. However, gentistic acid is much less expensive to use, allows labelling of MN cells, and should result in more favourable microdosimetry. Preliminary clinical results suggest that gentistic acid is equivalent to HMPAO but has the advantage of being much cheaper. (orig.)

Oral warfarin affects peripheral blood leukocyte IL-6 and TNFα production in rats.

Science.gov (United States)

Popov, Aleksandra; Belij, Sandra; Subota, Vesna; Zolotarevski, Lidija; Mirkov, Ivana; Kataranovski, Dragan; Kataranovski, Milena

2013-01-01

Warfarin is a Vitamin K (VK) antagonist that affects Vitamin K-dependent (VKD) processes, including blood coagulation, as well as processes unrelated to hemostasis such as bone growth, calcification, and growth of some cell types. In addition, warfarin exerts influence on some non-VKD-related activities, including anti-tumor and immunomodulating activity. With respect to the latter, both immune stimulating and suppressive effects have been noted in different experimental systems. To explore the in vivo immunomodulatory potential of warfarin on one type of activity (i.e., cytokine production) in two different immune cell populations (i.e., mononuclear or polymorphonuclear cells), effects of subchronic oral warfarin intake in rats on pro-inflammatory cytokine (i.e., TNFα, IL-6) production by peripheral blood mononuclear and polymorphonuclear cells (granulocytes) was examined. Differential effects of warfarin intake on TNFα and IL-6 were noted, depending on the type of peripheral blood leukocytes and on the cytokine examined. Specifically, a lack of effect on TNFα and a priming of IL-6 production by mononuclear cells along with a decrease in TNFα and a lack of effect on IL-6 in polymorphonuclear cells were seen in warfarin-exposed hosts. The cell- and cytokine-dependent effects from subchronic oral warfarin intake on peripheral blood leukocytes demonstrated in this study could, possibly, differentially affect reactions mediated by these cells. Ultimately, the observed effects in rats might have implications for those humans who are on long-term/prolonged warfarin therapy.

Assessment of citalopram and escitalopram on neuroblastoma cell lines: Cell toxicity and gene modulation

Science.gov (United States)

Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-01-01

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram. PMID:28467792

Assessment of citalopram and escitalopram on neuroblastoma cell lines. Cell toxicity and gene modulation.

Science.gov (United States)

Sakka, Laurent; Delétage, Nathalie; Chalus, Maryse; Aissouni, Youssef; Sylvain-Vidal, Valérie; Gobron, Stéphane; Coll, Guillaume

2017-06-27

Selective serotonin reuptake inhibitors (SSRI) are common antidepressants which cytotoxicity has been assessed in cancers notably colorectal carcinomas and glioma cell lines. We assessed and compared the cytotoxicity of 2 SSRI, citalopram and escitalopram, on neuroblastoma cell lines. The study was performed on 2 non-MYCN amplified cell lines (rat B104 and human SH-SY5Y) and 2 human MYCN amplified cell lines (IMR32 and Kelly). Citalopram and escitalopram showed concentration-dependent cytotoxicity on all cell lines. Citalopram was more cytotoxic than escitalopram. IMR32 was the most sensitive cell line. The absence of toxicity on human primary Schwann cells demonstrated the safety of both molecules for myelin. The mechanisms of cytotoxicity were explored using gene-expression profiles and quantitative real-time PCR (qPCR). Citalopram modulated 1 502 genes and escitalopram 1 164 genes with a fold change ≥ 2. 1 021 genes were modulated by both citalopram and escitalopram; 481 genes were regulated only by citalopram while 143 genes were regulated only by escitalopram. Citalopram modulated 69 pathways (KEGG) and escitalopram 42. Ten pathways were differently modulated by citalopram and escitalopram. Citalopram drastically decreased the expression of MYBL2, BIRC5 and BARD1 poor prognosis factors of neuroblastoma with fold-changes of -107 (pescitalopram.

Establishment and conventional cytogenetic characterization of three gastric cancer cell lines.

Science.gov (United States)

Leal, Mariana Ferreira; Martins do Nascimento, José Luiz; da Silva, Carla Elvira Araújo; Vita Lamarão, Maria Fernanda; Calcagno, Danielle Queiroz; Khayat, André Salim; Assumpção, Paulo Pimentel; Cabral, Isabel Rosa; de Arruda Cardoso Smith, Marília; Burbano, Rommel Rodríguez

2009-11-01

Gastric cancer is the fourth most frequent type of cancer and the second most frequent cause of cancer mortality worldwide. Only a modest number of gastric carcinoma cell lines have been isolated thus far. Here we describe the establishment and cytogenetic characterization of three new gastric cancer cell lines obtained from primary gastric adenocarcinoma (ACP02 and ACP03) and cancerous ascitic fluid (AGP01) of individuals from northern Brazil. ACP02, ACP03, and AGP01 cell lines are presently in the 60th passage. The cell lines grew in a disorganized single layer with some agglomerations and heterogeneous divisions (bipolar and multipolar). All cell lines exhibited a composite karyotype with several clonal chromosome alterations. Trisomy 8 was the most frequent alteration. Chromosome 8 aneusomy was confirmed by fluorescence in situ hybridization. All cell lines also exhibited trisomy 7 and deletion of chromosome arm 17p. These results suggest that, although frequent chromosome alterations are commonly observed due to culture process, the ACP02, ACP03, and AGP01 cell lines and primary gastric cancer from individuals of northern Brazil share genetic alterations, supporting use of these cell lines as a model of gastric carcinogenesis in this population.

DNA fingerprinting of the NCI-60 cell line panel.

Science.gov (United States)

Lorenzi, Philip L; Reinhold, William C; Varma, Sudhir; Hutchinson, Amy A; Pommier, Yves; Chanock, Stephen J; Weinstein, John N

2009-04-01

The National Cancer Institute's NCI-60 cell line panel, the most extensively characterized set of cells in existence and a public resource, is frequently used as a screening tool for drug discovery. Because many laboratories around the world rely on data from the NCI-60 cells, confirmation of their genetic identities represents an essential step in validating results from them. Given the consequences of cell line contamination or misidentification, quality control measures should routinely include DNA fingerprinting. We have, therefore, used standard DNA microsatellite short tandem repeats to profile the NCI-60, and the resulting DNA fingerprints are provided here as a reference. Consistent with previous reports, the fingerprints suggest that several NCI-60 lines have common origins: the melanoma lines MDA-MB-435, MDA-N, and M14; the central nervous system lines U251 and SNB-19; the ovarian lines OVCAR-8 and OVCAR-8/ADR (also called NCI/ADR); and the prostate lines DU-145, DU-145 (ATCC), and RC0.1. Those lines also show that the ability to connect two fingerprints to the same origin is not affected by stable transfection or by the development of multidrug resistance. As expected, DNA fingerprints were not able to distinguish different tissues-of-origin. The fingerprints serve principally as a barcodes.

The perivascular phagocyte of the mouse pineal gland: An antigen-presenting cell

DEFF Research Database (Denmark)

Møller, Morten; Rath, Martin F; Klein, David C

2006-01-01

The perivascular space of the rat pineal gland is known to contain phagocytic cells that are immunoreactive for leukocyte antigens, and thus they appear to belong to the macrophage/microglial cell line. These cells also contain MHC class II proteins. We investigated this cell type in the pineal g...... for MHC class II protein and for CD68, a marker of monocytes/phagocytes. This study verifies that perivascular phagocytes with antigen-presenting properties are present in the mouse pineal gland....

Studies on ADCC (antibody-dependent cell-mediated cytotoxicity) using sheep red blood cells as target cells, 2

International Nuclear Information System (INIS)

Ichikawa, Yukinobu; Takaya, Masatoshi; Arimori, Shigeru

1979-01-01

A non-specific cytotoxic mediator from effector cells (human peripheral blood leukocytes) was investigated in the ADCC (antibody-dependent cell-mediated cytotoxicity) system using antibody-coated sheep red blood cells (SRBC) as target cells. 51 Cr-labelled homologous (sheep) or heterologous (human) red blood cells were used as adjacent cells. Either crude lymphocyte fraction, phagocyte depleted fraction or granulocyte rich fraction separated from human peripheral leukocytes showed moderate cytotoxic effect on homologous adjacent cells, however no cytotoxic activity on heterologous adjacent cells was demonstrated in any leukocyte fraction. This suggests that the cytotoxic effects on homologous adjacent cells were resulted from the translocation of antibody molecules to adjacent cells from antibody-coated target cells. We concluded that the cytotoxic mechanism in this ADCC system was not mediated by non-specific soluble factors released from either human peripheral lymphocytes, monocytes or granulocytes. (author)

Peroxisomal abnormalities in the immortalized human hepatocyte (IHH) cell line.

Science.gov (United States)

Klouwer, Femke C C; Koster, Janet; Ferdinandusse, Sacha; Waterham, Hans R

2017-04-01

The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

Dose-response relationships in gene expression profiles in a harbor seal B lymphoma cell line exposed to 17α-ethinyl estradiol

Directory of Open Access Journals (Sweden)

Christine Kleinert

2017-05-01

Full Text Available The determination of changes in gene expression profiles with xenobiotic dose will allow identifying biomarkers and modes of toxicant action. The harbor seal (Phoca vitulina 11B7501 B lymphoma cell line was exposed to 1, 10, 100, 1000, 10,000, or 25,000 μg/L 17α-ethinyl estradiol (EE2, the active compound of the contraceptive pill for 24 h. Following exposure, RNA was extracted and transformed into cDNA. Transcript expression in exposed vs. control lymphocytes was analyzed via RT-qPCR to identify genes with altered expression. Our analysis indicates that gene expression for all but the reference gene varied with dose, suggesting that different doses induce distinct physiological responses. These findings demonstrate that RT-qPCR could be used to identify immunotoxicity and relative dose in harbor seal leukocytes.

Cellular radiosensitivity of small-cell lung cancer cell lines

DEFF Research Database (Denmark)

Krarup, M; Poulsen, H S; Spang-Thomsen, M

1997-01-01

PURPOSE: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based...

Improving the efficiency of CHO cell line generation using glutamine synthetase gene knockout cells.

Science.gov (United States)

Fan, Lianchun; Kadura, Ibrahim; Krebs, Lara E; Hatfield, Christopher C; Shaw, Margaret M; Frye, Christopher C

2012-04-01

Although Chinese hamster ovary (CHO) cells, with their unique characteristics, have become a major workhorse for the manufacture of therapeutic recombinant proteins, one of the major challenges in CHO cell line generation (CLG) is how to efficiently identify those rare, high-producing clones among a large population of low- and non-productive clones. It is not unusual that several hundred individual clones need to be screened for the identification of a commercial clonal cell line with acceptable productivity and growth profile making the cell line appropriate for commercial application. This inefficiency makes the process of CLG both time consuming and laborious. Currently, there are two main CHO expression systems, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection and glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection, that have been in wide industrial use. Since selection of recombinant cell lines in the GS-CHO system is based on the balance between the expression of the GS gene introduced by the expression plasmid and the addition of the GS inhibitor, L-MSX, the expression of GS from the endogenous GS gene in parental CHOK1SV cells will likely interfere with the selection process. To study endogenous GS expression's potential impact on selection efficiency, GS-knockout CHOK1SV cell lines were generated using the zinc finger nuclease (ZFN) technology designed to specifically target the endogenous CHO GS gene. The high efficiency (∼2%) of bi-allelic modification on the CHO GS gene supports the unique advantages of the ZFN technology, especially in CHO cells. GS enzyme function disruption was confirmed by the observation of glutamine-dependent growth of all GS-knockout cell lines. Full evaluation of the GS-knockout cell lines in a standard industrial cell culture process was performed. Bulk culture productivity improved two- to three-fold through the use of GS-knockout cells as parent cells. The selection stringency was

Derivation and Osmotolerance Characterization of Three Immortalized Tilapia (Oreochromis mossambicus) Cell Lines

Science.gov (United States)

Gardell, Alison M.; Qin, Qin; Rice, Robert H.; Li, Johnathan; Kültz, Dietmar

2014-01-01

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish. PMID:24797371

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

Science.gov (United States)

Hamilton, Gerhard

2014-01-01

Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4) inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC) cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS) and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose)-Polymerase 1 (PARP1) inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS. PMID:24608973

MODERATE CYTOTOXICITY OF PROANTHOCYANIDINS TO HUMAN TUMOR-CELL LINES

NARCIS (Netherlands)

KOLODZIEJ, H; HABERLAND, C; WOERDENBAG, HJ; KONINGS, AWT

In the present study the cytotoxicity of 16 proanthocyanidins was evaluated in GLC(4), a human small cell lung carcinoma cell line, and in COLO 320, a human colorectal cancer cell line, using the microculture tetrazolium (MTT) assay. With IC50 values ranging from 18 to >200 mu m following continuous

Anti-leukemic activity of bortezomib and carfilzomib on B-cell precursor ALL cell lines.

Directory of Open Access Journals (Sweden)

Kazuya Takahashi

Full Text Available Prognosis of childhood acute lymphoblastic leukemia (ALL has been dramatically improved. However, prognosis of the cases refractory to primary therapy is still poor. Recent phase 2 study on the efficacy of combination chemotherapy with bortezomib (BTZ, a proteasome inhibitor, for refractory childhood ALL demonstrated favorable clinical outcomes. However, septic death was observed in over 10% of patients, indicating the necessity of biomarkers that could predict BTZ sensitivity. We investigated in vitro BTZ sensitivity in a large panel of ALL cell lines that acted as a model system for refractory ALL, and found that Philadelphia chromosome-positive (Ph+ ALL, IKZF1 deletion, and biallelic loss of CDKN2A were associated with favorable response. Even in Ph-negative ALL cell lines, IKZF1 deletion and bilallelic loss of CDKN2A were independently associated with higher BTZ sensitivity. BTZ showed only marginal cross-resistance to four representative chemotherapeutic agents (vincristine, dexamethasone, l-asparaginase, and daunorubicin in B-cell precursor-ALL cell lines. To improve the efficacy and safety of proteasome inhibitor combination chemotherapy, we also analyzed the anti-leukemic activity of carfilzomib (CFZ, a second-generation proteasome inhibitor, as a substitute for BTZ. CFZ showed significantly higher activity than BTZ in the majority of ALL cell lines except for the P-glycoprotein-positive t(17;19 ALL cell lines, and IKZF1 deletion was also associated with a favorable response to CFZ treatment. P-glycoprotein inhibitors effectively restored the sensitivity to CFZ, but not BTZ, in P-glycoprotein-positive t(17;19 ALL cell lines. P-glycoprotein overexpressing ALL cell line showed a CFZ-specific resistance, while knockout of P-glycoprotein by genome editing with a CRISPR/Cas9 system sensitized P-glycoprotein-positive t(17;19 ALL cell line to CFZ. These observations suggested that IKZF1 deletion could be a useful biomarker to predict good

Characteristics of an autologous leukocyte and platelet-rich fibrin patch intended for the treatment of recalcitrant wounds

DEFF Research Database (Denmark)

Lundquist, Rasmus; Holmstrøm, Kim; Clausen, Christian

2012-01-01

We have investigated the physical, biochemical, and cellular properties of an autologous leukocyte and platelet-rich fibrin patch. This was generated in an automated device from a sample of a patient's blood at the point of care. Using microscopy, cell counting, enzyme-linked immunosorbent assay...... of chronic wound fluid. By comparison with traditional platelet-rich plasma, differences in immune components were found. The relevance of these findings was assessed by showing a mitogenic and migratory effect on cultured human dermal fibroblasts. Further, we showed that fibrocytes, a cell type important......, antibody arrays, and cell culture assays, we show that the patch is a three-layered membrane comprising a fibrin sheet, a layer of platelets, and a layer of leukocytes. Mean recovery of platelets from the donated blood was 98% (±95%CI 0.8%). Mean levels of platelet-derived growth factor AB, human...

Cellular radiosensitivity of small-cell lung cancer cell lines

International Nuclear Information System (INIS)

Krarup, Marianne; Poulsen, Hans Skovgaard; Spang-Thomsen, Mogens

1997-01-01

Purpose: The objective of this study was to determine the radiobiological characteristics of a panel of small-cell lung cancer (SCLC) cell lines by use of a clonogenic assay. In addition, we tested whether comparable results could be obtained by employing a growth extrapolation method based on the construction of continuous exponential growth curves. Methods and Materials: Fifteen SCLC cell lines were studied, applying a slightly modified clonogenic assay and a growth extrapolation method. A dose-survival curve was obtained for each experiment and used for calculating several survival parameters. The multitarget single hit model was applied to calculate the cellular radiosensitivity (D 0 ), the capacity for sublethal damage repair (D q ), and the extrapolation number (n). Values for α and β were determined from best-fit curves according to the linear-quadratic model and these values were applied to calculate the surviving fraction after 2-Gy irradiation (SF 2 ). Results: In our investigation, the extrapolation method proved to be inappropriate for the study of in vitro cellular radiosensitivity due to lack of reproducibility. The results obtained by the clonogenic assay showed that the cell lines studied were radiobiologically heterogeneous with no discrete features of the examined parameters including the repair capacity. Conclusion: The results indicate that SCLC tumors per se are not generally candidates for hyperfractionated radiotherapy

Fraction against Human Cancer Cell Lines

African Journals Online (AJOL)

fraction of A. sieberi against seven cancer cell lines (Colo20, HCT116, DLD, MCF7, Jurkat, HepG2 and ... The morphology of the HepG2 cell nucleus was investigated by Hoechst 33342, ..... Gong F, Liang Y, Xie P, Chau F. Information theory.

A novel cell growth-promoting factor identified in a B cell leukemia cell line, BALL-1

International Nuclear Information System (INIS)

Dao, T.; Holan, V.; Minowada, J.

1993-01-01

A novel leukemia cell growth-promoting activity has been identified in the culture supernatant from a human B cell leukemia cell line, BALL-1. The supernatant from unstimulated cultures of the BALL-1 cells significantly promoted the growth of 16 out of 24 leukemia/lymphoma cell lines of different lineages (T, B and non-lymphoid) in a minimal concentration of fetal bovine serum (FBS), and 5 out of 12 cases of fresh leukemia cells in FBS-free medium. The growth-promoting sieve filtration and dialysis. The MW of the factor was less than 10 kDa. The growth-promoting activity was heat and acid stable and resistant to trypsin treatment. The factor isolated from the BALL-1 supernatant was distinct from known polypeptide growth factors with MW below 10 kDa, such as epidermal growth factor, transforming growth factor α, insulin-like growth factor I (IGF-I), IGF-II and insulin, as determine by specific antibodies and by cell-growth-promoting tests. The factor is the BALL-1 supernatant did not promote the proliferation of normal human fresh peripheral blood lymphocytes or mouse fibroblast cell line, BALB/C 3T3. In addition to the BALL-1 supernatant, a similar growth-promoting activity was found in the culture supernatant from 13 of 17 leukemia/lymphoma cell lines tested. The activity in these culture supernatant promoted the growth of leukemia/lymphoma cell lines in autocrine and/or paracrine fashions. These observations suggest that the low MW cell growth-promoting activity found in the BALL-1 culture supernatant is mediated by a novel factor which may be responsible for the clonal expansion of particular leukemic clones. (author)

Microbicidal properties of Leukocyte- and Platelet-Rich Plasma/Fibrin (L-PRP/L-PRF): new perspectives.

Science.gov (United States)

Cieslik-Bielecka, A; Dohan Ehrenfest, D M; Lubkowska, A; Bielecki, T

2012-01-01

Platelets, as main actors of the first stage of the healing process, play an important role in tissue repair. Their granules contain many active substances, particularly over 30 growth factors with significant effects on the resident cells at the site of injury, such as mesenchymal stem cells, chondrocytes, fibroblasts, osteoblasts. This potential may be increased by the concentration of the platelets, using platelet-rich plasma/fibrin products. In the four families of platelet concentrates, 2 families contain also significant concentrations of leukocytes: L-PRP (Leukocyte- and Platelet-Rich Plasma) and L-PRF (Leukocyte- and Platelet-Rich Fibrin). Inductive properties of platelet concentrates were widely described. However, they present also antimicrobial effects. The antibacterial effects of L-PRP were highlighted in only a few in vitro studies. Strong activity comparable to gentamicin and oxacillin for L-PRP against methicillin susceptible Staphylococcus aureus (MSSA) was already demonstrated. L-PRP also inhibited the growth of methicillin resistant Staphylococcus aureus (MRSA) and Escherichia coli. Some authors also reported clinical observations about the reduction of infections and the induction of healing processes after the use of platelet concentrates in cardiac, orthopaedic, oral and maxillofacial surgery. However, very little is yet known about the antibacterial effects of these concentrates. In this manuscript, the current data about the antimicrobial agents and cells present in the platelet-rich plasma/fibrin are highlighted and discussed, in order to introduce this new key chapter of the platelet concentrate technology history.

Granulocytes: New Members of the Antigen-Presenting Cell Family

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Ang Lin

2017-12-01

Full Text Available Granulocytes, the most abundant types of leukocytes, are the first line of defense against pathogen invasion. However, the plasticity and diversity of granulocytes have been increasingly revealed, especially with regard to their versatile functions in orchestrating adaptive immune responses. A substantial body of recent evidence demonstrates that granulocytes can acquire the function as antigen-presenting cells under pathological or inflammatory conditions. In addition, they can acquire surface expression of MHC class II and costimulatory molecules as well as T cell stimulatory behavior when cultured with selected cytokines. The classic view of granulocytes as terminally differentiated, short-lived phagocytes is therefore changing to phenotypically and functionally heterogeneous cells that are engaged in cross-talk with other leukocyte populations and provide an additional link between innate and adaptive immunity. In this brief review, we summarize the current knowledge on the antigen-presenting capacity of granulocyte subsets (neutrophils, eosinophils, and basophils. Underlying mechanisms, relevant physiological significance and potential controversies are also discussed.

Myeloid derived suppressor cells (MDSCs are increased and exert immunosuppressive activity together with polymorphonuclear leukocytes (PMNs in chronic myeloid leukemia patients.

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Cesarina Giallongo

Full Text Available Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs, able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1 that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.

Cell lines derived from the squash bug, Anasa tristis (Coreidae: Hemiptera).

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Goodman, Cynthia L; Ringbauer, Joseph A; Li, Yao-Fa; Lincoln, Tamra Reall; Stanley, David

2017-05-01

The squash bug, Anasa tristis, is a pest of cucurbits that exerts direct damage on crops and is a vector of plant pathogens. We established cell lines from this insect to serve as tools for basic biology, including virology and immunology, as well as applied studies, such as insecticide development programs. We initiated 15 cell cultures, using nine media or combinations of media. The media yielding the best results were a modification of Kimura's medium and a combination of two commercially available cell culture media (EX-CELL 420 and L15). We designated the two cell lines as BCIRL-AtE-CLG11 and BCIRL-AtE-CLG15. From the AtE-CLG15 line, we isolated two sub-lines, A and B. Of these, the most consistently replicating line was AtE-CLG15A. We determined the doubling time of this line (190 h) and its mean cell diameter (14.5 ± 0.7 μm). We characterized the AtE-CLG15A line using DAF-PCR. The BCIRL-AtE-CLG15A cell line is now available for researchers world-wide.

Bifenthrin activates homotypic aggregation in human T-cell lines.

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Hoffman, Nataly; Tran, Van; Daniyan, Anthony; Ojugbele, Olutosin; Pryor, Stephen C; Bonventre, Josephine A; Flynn, Katherine; Weeks, Benjamin S

2006-03-01

Here, we addressed the concern that, despite the lack of overt toxicity, exposure to low levels of the common household pyrethroid pesticide, bifenthrin, could cause harm to the immune system. To do this, we measure the effect of bifenthrin on phytohemagglutinin (PHA) activation of homotypic aggregation in human T-cell lines. The human CD4+ H9, and Jurkat cell lines and the human promonocyte U937 cell line, were exposed to varying concentrations of bifenthrin. Cell viability was determined using the AlmarBlue Toxicity Assay. Concentrations of bifenthrin which did not reduce cell viability were determined and these concentrations were tested for the effect of bifenthrin on PHA-mediated homotypic aggregation. Blocking antibodies to ICAM and LFA-1 were used to disrupt aggregation and a nonspecific IgG was used as a control. Bifenthrin was found to be nontoxic at concentrations ranging from 10(-4) to 10(-13) M. Bifenthrin did not inhibit PHA induced cell aggregation in all cell lines tested. However, at 10(-4) M, bifenthrin to form aggregates stimulated homotypic aggregation in the H9 and Jurkat T-cell lines. The bifenthrin-induced aggregate formation, like that seen with PHA, was blocked by treating the cells with antibodies to either LFA-1 or ICAM. The results here show that bifenthrin activates T-cell function by stimulating ICAM/LFA-1 mediated homotypic aggregation. This data suggests that exposure to bifenthrin, even at "acceptable" limits, can increase the risk for and frequency of inflammatory responses and diseases such as asthma.

Establishment, immortalisation and characterisation of pteropid bat cell lines.

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Gary Crameri

Full Text Available BACKGROUND: Bats are the suspected natural reservoir hosts for a number of new and emerging zoonotic viruses including Nipah virus, Hendra virus, severe acute respiratory syndrome coronavirus and Ebola virus. Since the discovery of SARS-like coronaviruses in Chinese horseshoe bats, attempts to isolate a SL-CoV from bats have failed and attempts to isolate other bat-borne viruses in various mammalian cell lines have been similarly unsuccessful. New stable bat cell lines are needed to help with these investigations and as tools to assist in the study of bat immunology and virus-host interactions. METHODOLOGY/FINDINGS: Black flying foxes (Pteropus alecto were captured from the wild and transported live to the laboratory for primary cell culture preparation using a variety of different methods and culture media. Primary cells were successfully cultured from 20 different organs. Cell immortalisation can occur spontaneously, however we used a retroviral system to immortalise cells via the transfer and stable production of the Simian virus 40 Large T antigen and the human telomerase reverse transcriptase protein. Initial infection experiments with both cloned and uncloned cell lines using Hendra and Nipah viruses demonstrated varying degrees of infection efficiency between the different cell lines, although it was possible to infect cells in all tissue types. CONCLUSIONS/SIGNIFICANCE: The approaches developed and optimised in this study should be applicable to bats of other species. We are in the process of generating further cell lines from a number of different bat species using the methodology established in this study.

Incorrect strain information for mouse cell lines: sequential influence of misidentification on sublines.

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Uchio-Yamada, Kozue; Kasai, Fumio; Ozawa, Midori; Kohara, Arihiro

2017-03-01

Misidentification or cross-contamination of cell lines can cause serious issues. Human cell lines have been authenticated by short tandem repeat profiling; however, mouse cell lines have not been adequately assessed. In this study, mouse cell lines registered with the JCRB cell bank were examined by simple sequence length polymorphism (SSLP) analysis to identify their strains. Based on comparisons with 7 major inbred strains, our results revealed their strains in 80 of 90 cell lines. However, 12 of the 80 cell lines (15%) were found to differ from registered information. Of them, 4 cell lines originated from the same mouse, which had been generated through mating between two different inbred strains. The genotype of the mouse sample had not been examined after the backcross, leading to strain misidentification in those cell lines. Although 8 other cell lines had been established as sublines of a BALB/c cell line, their SSLP profiles are similar to a Swiss cell line. This affects differences in genotypes between inbred and outbred strains. Because the use of inbred samples and interbreeding between strains are not involved in human materials, our results suggest that the cause and influence of misidentification in mouse cell lines are different from those in human.

Effect of leukocyte alteration on treatment outcomes following preoperative chemoradiotherapy in patients with rectal cancer

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Kim, Tae Gyu; Park, Won; Choi, Doo Ho [Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul (Korea, Republic of); and others

2017-09-15

Hematotoxicity following anti-cancer treatment is known to be related to treatment efficacy in several malignancies. The purpose of this study was to examine the hematologic parameters related to the tumor response and survival in patients treated with curative surgery following preoperative chemoradiotherapy (CRT) for rectal cancer. Four hundred eighteen patients with rectal cancer who underwent preoperative CRT and curative surgery were analyzed, retrospectively. The main clinical factors and blood cell counts before and after CRT were investigated with respect to their relationships with tumor downstaging and patient survival. The post-CRT leukocyte count was significantly different between the tumor downstaging group and the nondownstaging group (median, 4740/uL vs. 5130/uL; p = 0.013). Multivariate analysis showed that histological grade, circumferential extent, and post-CRT leukocyte count were related to tumor downstaging. In addition, histological grade, post-CRT leukocyte count, and tumor downstaging were related to disease-free survival. The 5-year disease-free survival and overall survival in patients with post-CRT leukocyte count ≤3730/uL, which is the cut-off value derived from the receiver operation characteristic (ROC) curve analysis, were significantly higher than those with higher counts (88.0% vs. 71.6%, p = 0.001; 94.4% vs. 84.1%, p = 0.024). Post-CRT leukocyte count of ≤3730/uL could be regarded as a good prognostic factor for tumor response and survival in rectal cancer patients treated with preoperative CRT.

Relationships between leukocytes and Hepatozoon spp. In green frogs, Rana clamitans.

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Shutler, Dave; Smith, Todd G; Robinson, Stephen R

2009-01-01

There are few published data on amphibian leukocyte profiles, and relationships between amphibian leukocytes and parasites are even less well known. Using counts from 35 pairs of blood smears taken 2 days apart, we tested for correlations between leukocyte proportions and infection intensities of Hepatozoon spp. (either Hepatozoon catesbianae or Hepatozoon clamatae) in green frogs (Rana clamitans). On average (SE), we counted 65.4 (1.7) lymphocytes, 14.0 (1.3) neutrophils, 19.3 (1.6) eosinophils, 0.9 (0.1) monocytes, and 0.4 (0.1) basophils per 100 leukocytes. All frogs harbored Hepatozoon spp. (median seven parasites per 100 leukocytes; range 1-250). Significant relationships were not observed between numbers of leukocytes and infection intensities of Hepatozoon spp. Among the possible explanations for these null results are that Hepatozoon spp. is benign, that Hepatozoon spp. is able to evade detection by the immune system, that Hepatozoon spp. is able to manipulate leukocyte investment, or that other unmeasured or undetected parasites were more important in affecting immune response.

Characteristics of bovine inner cell mass-derived cell lines and their fate in chimeric conceptuses.

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Furusawa, Tadashi; Ohkoshi, Katsuhiro; Kimura, Koji; Matsuyama, Shuichi; Akagi, Satoshi; Kaneda, Masahiro; Ikeda, Mitsumi; Hosoe, Misa; Kizaki, Keiichiro; Tokunaga, Tomoyuki

2013-08-01

Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

Leukocyte counts in urine reflect the risk of concomitant sepsis in bacteriuric infants: a retrospective cohort study.

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Bonsu, Bema K; Harper, Marvin B

2007-06-13

When urine infections are missed in febrile young infants with normal urinalysis, clinicians may worry about the risk--hitherto unverified--of concomitant invasion of blood and cerebrospinal fluid by uropathogens. In this study, we determine the extent of this risk. In a retrospective cohort study of febrile 0-89 day old infants evaluated for sepsis in an urban academic pediatric emergency department (1993-1999), we estimated rates of bacteriuric sepsis (urinary tract infections complicated by sepsis) after stratifying infants by urine leukocyte counts higher, or lower than 10 cells/hpf. We compared the global accuracy of leukocytes in urine, leukocytes in peripheral blood, body temperature, and age for predicting bacteruric sepsis. The global accuracy of each test was estimated by calculating the area under its receiver operating characteristic curve (AUC). Chi-square and Fisher exact tests compared count data. Medians for data not normally distributed were compared by the Kruskal-Wallis test. Two thousand two hundred forty-nine young infants had a normal screening dipstick. None of these developed bacteremia or meningitis despite positive urine culture in 41 (1.8%). Of 1516 additional urine specimens sent for formal urinalysis, 1279 had 0-9 leukocytes/hpf. Urine pathogens were isolated less commonly (6% vs. 76%) and at lower concentrations in infants with few, compared to many urine leukocytes. Urine leukocytes (AUC: 0.94) were the most accurate predictors of bacteruric sepsis. Infants with urinary leukocytes urinary tract infections complicated by bacteremia (N = 11) or bacterial meningitis (N = 1)--relative risk, 0 (CI:0-0.06) [RR, 0 (CI: 0-0.02), when including infants with negative dipstick]. Bands in peripheral blood had modest value for detecting bacteriuric sepsis (AUC: 0.78). Cases of sepsis without concomitant bacteriuria were comparatively rare (0.8%) and equally common in febrile young infants with low and high concentrations of urine leukocytes. In

Enhancement in ex vivo phagocytic capacity of peritoneal leukocytes in mice by oral delivery of various lactic-acid-producing bacteria.

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Lee, Yeonhee; Lee, Taik-Soo

2005-01-01

Lactic-acid-producing bacteria (LABs) are known to have immunomodulating activity. In the current study, various LABs were tested for their immunity-enhancing activity, especially the phagocytic activity of leukocytes. Viable but not heat-killed cells of Weissella kimchii strain PL9001, Lactobacillus fermentum strain PL9005, and L. plantarum strain PL9011 significantly increased the ex vivo phagocytic capacity of mouse peritoneal leukocytes to ingest fluorescein isothiocyanate (FITC)-labeled Escherichia coli in a strain-dependent manner. Results of this and previous studies suggest these LABs as candidates for new probiotics. This is the first report of the enhancement of peritoneal leukocyte activity of these species.

LET effects on normal and radiosensitive cell lines

International Nuclear Information System (INIS)

Geard, C.R.; Travisano, M.

1986-01-01

Charged particles in the track segment mode were produced by the RARAF Van de Graaff accelerator and used to irradiate two CHO cell lines, a radiosensitive hypermutable line EM9 and its normal parent AA8. Asynchronous cells were irradiated attached to 6 micrometer thick Mylar with protons, deuterons and helium-3 particles at LETs ranging from 10 to 150 keV per micrometer. A 50 kVp x-ray tube integrated into the track segment facility provided a low LET comparison. Following irradiation cells were monitored for clonogenicity, and in a separate series of experiments frequencies of sister chromatid exchanges. Up to 9 experiments were carried out at each LET, with a total of 8 radiations of different LETs being compared. The optimally effective LET for cell survival was between 80 and 120 keV per micrometer, with the 150 keV per micrometer particles indicating energy wastage. The differential between the normal and radiosensitive cell lines was maintained at all LETs

Effects of radiolabelled monoclonal antibody infusion on blood leukocytes in cancer patients

International Nuclear Information System (INIS)

Gridley, D.S.; Slater, J.M.; Stickney, D.R.

1990-01-01

This study was undertaken to investigate the effects of a single infusion of radiolabelled murine monoclonal antibody (MAb) on peripheral blood leukocytes in cancer patients. Eleven patients with disseminated colon cancer, malignant melanoma, or lung adenocarcinoma were infused with 111In-labelled anti-ZCE 025, anti-p97 type 96.5c, or LA 20207 MAb, respectively. Blood samples were obtained before infusion, immediately after infusion (1 hr), and at 4 and 7 days postinfusion. Flow cytometry analysis of CD3+, CD4+, CD8+, CD16+, and CD19+ lymphocytes showed increasing CD4:CD8 ratios in seven patients after infusion. This phenomenon was not restricted to antibody subclass or to type of cancer. Two of the remaining patients exhibited a marked post-infusion increase in CD8+ cells. In all three patients with malignant melanoma, decreasing levels of CD16+ lymphocytes were noted after infusion and natural killer cell cytotoxicity showed fluctuations which paralleled the changes in the CD16+ subpopulation. Oxygen radical production by phagocytic cells was markedly affected in three subjects. These results suggest that a single infusion of radiolabelled murine MAb may alter the balance of critical lymphocyte subpopulations and modulate other leukocyte responses in cancer patients

Radiation of different human melanoma cell lines increased expression of RHOB. Level of this tumor suppressor gene in different cell lines

International Nuclear Information System (INIS)

Notcovich, C.; Molinari, B.; Duran, H.; Delgado González, D.; Sánchez Crespo, R.

2013-01-01

Previous results of our group show that a correlation exists between intrinsic radiosensitivity of human melanoma cells and cell death by apoptosis. RhoB is a small GTPase that regulates cytoskeletal organization. Besides, is related to the process of apoptosis in cells exposed to DNA damage as radiation. Also, RhoB levels decrease in a wide variety of tumors with the tumor stage, being considered a tumor suppressor gene due to its antiproliferative and proapoptotic effect. The aim of this study was to analyze the expression of RhoB in different human melanoma cell lines in relation to melanocytes, and evaluate the effect of gamma radiation on the expression of RhoB. We used the A375, SB2 and Meljcell lines, and the derived from melanocytes Pig1. It was found for all three tumor lines RhoB expression levels significantly lower than those of Pig1 (p <0.05), as assessed by semiquantitative RT-PCR . When tumor cells were irradiated to a dose of 2Gyinduction was observed at 3 hours RhoB irradiation. RhoB expression increased in all lines relative to non-irradiated control, showing a greater induction ( p< 0.05) for the more radiosensitive line SB2, consistent with apoptosis in response to radiation. The results allow for the first time in melanoma demonstrate that RhoB, as well as in other tumor types, has a lower expression in tumor cells than their normal counterparts. Moreover, induction in the expression of RhoB in irradiated cells may be associated with the process of radiation-induced apoptosis. The modulation of RhoB could be a new tool to sensitize radioresistant melanoma. (author)

Induction of apoptosis by opium in some tumor cell lines.

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Khaleghi, M; Farsinejad, A; Dabiri, S; Asadikaram, G

2016-09-30

The current study is aimed at investigation of the opium effects on the apoptosis of different cell lines in culture medium and compares such effects with one another. The study is carried out on over 8 cell lines (AA8, AGS, Hela, HepG2, MCF7, N2a, PC12, WEHI). A 2.86 x 10-4 g/ml opium concentration was prepared and added to the culture medium of the cell lines for 48 hours. Cytotoxicity was tested by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic effect of opium on the cell lines was analyzed by Annexin-PI test. Opium with concentration of 2.86 x 10-4 g/ml in 48 hours significantly induces apoptosis in certain cell lines (i.e. AA8, N2a, WEHI), apoptosis and necrosis in some others (i.e. Hela, HepG2, MCF7, and PC12), and also solely necrosis in the AGS cell line. One could infer that the usage of opium with different levels in different tissues leads to certain disorders in some tissues and may have therapeutic effects under distinctive conditions (i.e. unchecked growth of cells) as confirmed by the results.

Radiosensitivity evaluation of Human tumor cell lines by single cell gel electrophoresis

International Nuclear Information System (INIS)

Zhang Yipei; Cao Jia; Wang Yan; Du Liqing; Li Jin; Wang Qin; Fan Feiyue; Liu Qiang

2011-01-01

Objective: To explore the feasibility of determining radiosensitivity of human tumor cell lines in vitro using single cell gel electrophoresis (SCGE). Methods: Three human tumor cell lines were selected in this study, HepG 2 , EC-9706 and MCF-7. The surviving fraction (SF) and DNA damage were detected by MTT assay, nested PCR technique and comet assay respectively. Results: MTT assay: The SF of HepG 2 and EC-9706 after irradiated by 2, 4 and 8 Gy was lower significantly than that of MCF-7, which showed that the radiosensitivity of HepG 2 and EC-9706 was higher than that of MCF-7. But there was no statistical difference of SF between HepG 2 and EC-9706. SCGE: The difference of radiosensitivity among these three tumor cell lines was significant after 8 Gy γ-ray irradiation. Conclusion: The multi-utilization of many biological parameter is hopeful to evaluate the radiosensitivity of tumor cells more objectively and exactly. (authors)

Sensory hair cell regeneration in the zebrafish lateral line.

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Lush, Mark E; Piotrowski, Tatjana

2014-10-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

Science.gov (United States)

Lush, Mark E.; Piotrowski, Tatjana

2014-01-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

Further comparisons of endogenous pyrogens and leukocytic endogenous mediators.

Science.gov (United States)

Kampschmidt, R F; Upchurch, H F; Worthington, M L

1983-07-01

It was recently shown (Murphy et al., Infect. Immun. 34:177-183), that rabbit macrophages produce two biochemically and immunologically distinct endogenous pyrogens. One of these has or copurifies with substances having a molecular weight of 13,000 and a pI of 7.3. This protein was produced by blood monocytes or inflammatory cells elicited in 16-h rabbit peritoneal exudates. These acute peritoneal exudates were produced by the intraperitoneal injection of large volumes of saline containing shellfish glycogen. When the leukocytes in these exudates were washed and incubated at 37 degrees C in saline, they released an endogenous pyrogen. The injection of this pyrogen into rabbits, rats, or mice caused the biological manifestations which have been attributed to leukocytic endogenous mediator. These effects were increases in blood neutrophils, the lowering of plasma iron and zinc levels, and the increased synthesis of the acute-phase proteins. The other rabbit endogenous pyrogen seems to be a family of proteins with isoelectric points between 4.5 and 5.0. These proteins are produced by macrophages in the lung, liver, or in chronic peritoneal exudates. In these experiments, the lower-isoelectric-point endogenous pyrogens were produced by macrophages from the peritoneal cavity of rabbits that had been injected 4 days earlier with 50 ml of light mineral oil. These rabbit pyrogens were found to have leukocytic endogenous mediator activity in mice but to be completely inactive in rats. When injected into rabbits, these proteins produced fever, lowered plasma iron, increased blood neutrophils, but failed to elevate plasma fibrinogen.

Comparison of thermoradiosensitization in two human melanoma cell lines and one fibroblast cell line by concurrent mild hyperthermia and low-dose-rate irradiation

International Nuclear Information System (INIS)

Raaphorst, G.P.; Bussey, A.; Heller, D.P.; Ng, C.E.

1994-01-01

Two human melanoma cell lines, one radioresistant (Sk-MEL-3) and one radiosensitive (HT-144), and a normal human fibroblast line (AG1522) were evaluated for thermoradiosensitization of low-dose-rate irradiation by concurrent mild hyperthermia (39-41 degrees C). None of the cell lines expressed chronic thermotolerance during heating at 39-41 degrees C. The SK-MEL-3 cells were the most heat sensitive, while AG1522 and HT-144 cells had the same sensitivity at 39 and 40 degrees C but HT-144 cells were more sensitive at 41 degrees C. All cell lines expressed thermal enhancement of radiosensitivity with heating during irradiation which increased with heating temperature. The SK-MEL-3 cells, which were the most resistant to radiation and demonstrated the greatest repair of sublethal damage (SLD) during low-dose-rate irradiation, had the greatest thermal enhancement of radiosensitivity, while the HT144 cells, which were the most sensitive and expressed little repair of SLD during low-dose-rate irradiation, had the smallest thermal enhancement of radiosensitivity. These data show that concurrent mild hyperthermia during low-dose-rate irradiation may be most efficacious in radiation-resistant tumor cells which express resistance through an enhanced capacity for repair of SLD. 24 refs., 5 figs., 1 tab

Expression of myc family oncoproteins in small-cell lung-cancer cell lines and xenografts

DEFF Research Database (Denmark)

Rygaard, K; Vindeløv, L L; Spang-Thomsen, M

1993-01-01

A number of genes have altered activity in small-cell lung cancer (SCLC), but especially genes of the myc family (c-myc, L-myc and N-myc) are expressed at high levels in SCLC. Most studies have explored expression at the mRNA level, whereas studies of myc family oncoprotein expression are sparse....... WE examined the expression of myc proto-oncogenes at the mRNA and protein level in 23 cell lines or xenografts. In the cell lines, the doubling time and the cell-cycle distribution, as determined by flow-cytometric DNA analysis, were examined to establish whether the level of myc......-myc. In general, the level of expression of c-myc and N-myc was similar at the mRNA and the protein level. Expression of c-myc was positively correlated with the proliferative index (sum of S and G2+M phases) of cell lines, but not with the population doubling time. In general, L-myc-expressing cell lines had...

Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

International Nuclear Information System (INIS)

Wu Xiaofeng; Fan Jia; Wang Xiaoying; Zhou Jian; Qiu Shuangjian; Yu Yao; Liu Yinkun; Tang Zhaoyou

2007-01-01

CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment

Tumourigenic canine osteosarcoma cell lines associated with frizzled-6 up-regulation and enhanced side population cell frequency.

Science.gov (United States)

de Sá Rodrigues, L C; Holmes, K E; Thompson, V; Newton, M A; Stein, T J

2017-03-01

An increased serum alkaline phosphatase concentration is known to be associated with a negative prognosis in canine and human osteosarcoma. To expand upon previous studies regarding the biological relevance of increased serum alkaline phosphatase as a negative prognostic factor, xenogeneic heterotopic transplants were performed using six canine primary osteosarcoma cell lines generated from patients with differing serum alkaline phosphatase concentrations (three normal and three increased). Three of the six cell lines were capable of generating tumours and tumour formation was independent of the serum alkaline phosphatase status of the cell line. Microarray analysis identified 379 genes as being differentially expressed between the tumourigenic and non-tumourigenic cell lines. Frizzled-6 was upregulated to the greatest extent (7.78-fold) in tumourigenic cell lines compared with non-tumourigenic cell lines. Frizzled-6, a co-receptor for Wnt ligands has been associated with enhanced tumour-initiating cells and poor prognosis for other tumours. The increased expression of frizzled-6 was confirmed by quantitative reverse transcription polymerase chain reaction (QPCR) and Western blot analysis. Additionally, the tumourigenic cell lines also had an increase in the percentage of side population cells compared with non-tumourigenic cell lines (5.89% versus 1.58%, respectively). There were no differences in tumourigenicity, frizzled-6 or percentage of side population cells noted between osteosarcoma cell lines generated from patients of differing serum alkaline phosphatase concentration. However, to our knowledge this is the first study to identified frizzled-6 as a possible marker of osteosarcoma cell populations with enhanced tumourigenicity and side population cells. Future work will focus on defining the role of frizzled-6 in osteosarcoma tumourigenesis and tumour-initiating cells. © 2015 John Wiley & Sons Ltd.

Flow cytometric assay for analysis of cytotoxic effects of potential drugs on human peripheral blood leukocytes

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Nieschke, Kathleen; Mittag, Anja; Golab, Karolina; Bocsi, Jozsef; Pierzchalski, Arkadiusz; Kamysz, Wojciech; Tarnok, Attila

2014-03-01

Toxicity test of new chemicals belongs to the first steps in the drug screening, using different cultured cell lines. However, primary human cells represent the human organism better than cultured tumor derived cell lines. We developed a very gentle toxicity assay for isolation and incubation of human peripheral blood leukocytes (PBL) and tested it using different bioactive oligopeptides (OP). Effects of different PBL isolation methods (red blood cell lysis; Histopaque isolation among others), different incubation tubes (e.g. FACS tubes), anticoagulants and blood sources on PBL viability were tested using propidium iodide-exclusion as viability measure (incubation time: 60 min, 36°C) and flow cytometry. Toxicity concentration and time-depended effects (10-60 min, 36 °C, 0-100 μg /ml of OP) on human PBL were analyzed. Erythrocyte lysis by hypotonic shock (dH2O) was the fastest PBL isolation method with highest viability (>85%) compared to NH4Cl-Lysis (49%). Density gradient centrifugation led to neutrophil granulocyte cell loss. Heparin anticoagulation resulted in higher viability than EDTA. Conical 1.5 mL and 2 mL micro-reaction tubes (both polypropylene (PP)) had the highest viability (99% and 97%) compared to other tubes, i.e. three types of 5.0 mL round-bottom tubes PP (opaque-60%), PP (blue-62%), Polystyrene (PS-64%). Viability of PBL did not differ between venous and capillary blood. A gentle reproducible preparation and analytical toxicity-assay for human PBL was developed and evaluated. Using our assay toxicity, time-course, dose-dependence and aggregate formation by OP could be clearly differentiated and quantified. This novel assay enables for rapid and cost effective multiparametric toxicological screening and pharmacological testing on primary human PBL and can be adapted to high-throughput-screening.°z

Recombinant protein production from stable mammalian cell lines and pools.

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Hacker, David L; Balasubramanian, Sowmya

2016-06-01

We highlight recent developments for the production of recombinant proteins from suspension-adapted mammalian cell lines. We discuss the generation of stable cell lines using transposons and lentivirus vectors (non-targeted transgene integration) and site-specific recombinases (targeted transgene integration). Each of these methods results in the generation of cell lines with protein yields that are generally superior to those achievable through classical plasmid transfection that depends on the integration of the transfected DNA by non-homologous DNA end-joining. This is the main reason why these techniques can also be used for the generation of stable cell pools, heterogenous populations of recombinant cells generated by gene delivery and genetic selection without resorting to single cell cloning. This allows the time line from gene transfer to protein production to be reduced. Copyright © 2016 Elsevier Ltd. All rights reserved.

Selectins mediate small cell lung cancer systemic metastasis.

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Franziska Heidemann

Full Text Available Metastasis formation is the major reason for the extremely poor prognosis in small cell lung cancer (SCLC patients. The molecular interaction partners regulating metastasis formation in SCLC are largely unidentified, however, from other tumor entities it is known that tumor cells use the adhesion molecules of the leukocyte adhesion cascade to attach to the endothelium at the site of the future metastasis. Using the human OH-1 SCLC line as a model, we found that these cells expressed E- and P-selectin binding sites, which could be in part attributed to the selectin binding carbohydrate motif sialyl Lewis A. In addition, protein backbones known to carry these glycotopes in other cell lines including PSGL-1, CD44 and CEA could be detected in in vitro and in vivo grown OH1 SCLC cells. By intravital microscopy of murine mesenterial vasculature we could capture SCLC cells while rolling along vessel walls demonstrating that SCLC cells mimic leukocyte rolling behavior in terms of selectin and selectin ligand interaction in vivo indicating that this mechanism might indeed be important for SCLC cells to seed distant metastases. Accordingly, formation of spontaneous distant metastases was reduced by 50% when OH-1 cells were xenografted into E-/P-selectin-deficient mice compared with wild type mice (p = 0.0181. However, as metastasis formation was not completely abrogated in selectin deficient mice, we concluded that this adhesion cascade is redundant and that other molecules of this cascade mediate metastasis formation as well. Using several of these adhesion molecules as interaction partners presumably make SCLC cells so highly metastatic.

Cytotoxic Effects of Fascaplysin against Small Cell Lung Cancer Cell Lines

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Gerhard Hamilton

2014-03-01

Full Text Available Fascaplysin, the natural product of a marine sponge, exhibits anticancer activity against a broad range of tumor cells, presumably through interaction with DNA, and/or as a highly selective cyclin-dependent kinase 4 (CDK4 inhibitor. In this study, cytotoxic activity of fascaplysin against a panel of small cell lung cancer (SCLC cell lines and putative synergism with chemotherapeutics was investigated. SCLC responds to first-line chemotherapy with platinum-based drugs/etoposide, but relapses early with topotecan remaining as the single approved therapeutic agent. Fascaplysin was found to show high cytotoxicity against SCLC cells and to induce cell cycle arrest in G1/0 at lower and S-phase at higher concentrations, respectively. The compound generated reactive oxygen species (ROS and induced apoptotic cell death in the chemoresistant NCI-H417 SCLC cell line. Furthermore, fascaplysin revealed marked synergism with the topoisomerase I-directed camptothecin and 10-hydroxy-camptothecin. The Poly(ADP-ribose-Polymerase 1 (PARP1 inhibitor BYK 204165 antagonized the cytotoxic activity of fascaplysin, pointing to the involvement of DNA repair in response to the anticancer activity of the drug. In conclusion, fascaplysin seems to be suitable for treatment of SCLC, based on high cytotoxic activity through multiple routes of action, affecting topoisomerase I, integrity of DNA and generation of ROS.

Electrophysiological Characteristics of Embryonic Stem Cell-Derived Cardiomyocytes are Cell Line-Dependent

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Tobias Hannes

2015-01-01

Full Text Available Background: Modelling of cardiac development, physiology and pharmacology by differentiation of embryonic stem cells (ESCs requires comparability of cardiac differentiation between different ESC lines. To investigate whether the outcome of cardiac differentiation is consistent between different ESC lines, we compared electrophysiological properties of ESC-derived cardiomyocytes (ESC-CMs of different murine ESC lines. Methods: Two wild-type (D3 and R1 and two transgenic ESC lines (D3/aPIG44 and CGR8/AMPIGX-7 were differentiated under identical culture conditions. The transgenic cell lines expressed enhanced green fluorescent protein (eGFP and puromycin-N-acetyltransferase under control of the cardiac specific α-myosin heavy chain (αMHC promoter. Action potentials (APs were recorded using sharp electrodes and multielectrode arrays in beating clusters of ESC-CMs. Results: Spontaneous AP frequency and AP duration (APD as well as maximal upstroke velocity differed markedly between unpurified CMs of the four ESC lines. APD heterogeneity was negligible in D3/aPIG44, moderate in D3 and R1 and extensive in CGR8/AMPIGX-7. Interspike intervals calculated from long-term recordings showed a high degree of variability within and between recordings in CGR8/AMPIGX-7, but not in D3/aPIG44. Purification of the αMHC+ population by puromycin treatment posed only minor changes to APD in D3/aPIG44, but significantly shortened APD in CGR8/AMPIGX-7. Conclusion: Electrophysiological properties of ESC-CMs are strongly cell line-dependent and can be influenced by purification of cardiomyocytes by antibiotic selection. Thus, conclusions on cardiac development, physiology and pharmacology derived from single stem cell lines have to be interpreted carefully.

Disintegrins: integrin selective ligands which activate integrin-coupled signaling and modulate leukocyte functions

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Barja-Fidalgo C.

2005-01-01

Full Text Available Extracellular matrix proteins and cell adhesion receptors (integrins play essential roles in the regulation of cell adhesion and migration. Interactions of integrins with the extracellular matrix proteins lead to phosphorylation of several intracellular proteins such as focal adhesion kinase, activating different signaling pathways responsible for the regulation of a variety of cell functions, including cytoskeleton mobilization. Once leukocytes are guided to sites of infection, inflammation, or antigen presentation, integrins can participate in the initiation, maintenance, or termination of the immune and inflammatory responses. The modulation of neutrophil activation through integrin-mediated pathways is important in the homeostatic control of the resolution of inflammatory states. In addition, during recirculation, T lymphocyte movement through distinct microenvironments is mediated by integrins, which are critical for cell cycle, differentiation and gene expression. Disintegrins are a family of low-molecular weight, cysteine-rich peptides first identified in snake venom, usually containing an RGD (Arg-Gly-Asp motif, which confers the ability to selectively bind to integrins, inhibiting integrin-related functions in different cell systems. In this review we show that, depending on the cell type and the microenvironment, disintegrins are able to antagonize the effects of integrins or to act agonistically by activating integrin-mediated signaling. Disintegrins have proven useful as tools to improve the understanding of the molecular events regulated by integrin signaling in leukocytes and prototypes in order to design therapies able to interfere with integrin-mediated effects.

Expression pattern of matrix metalloproteinases in human gynecological cancer cell lines

International Nuclear Information System (INIS)

Schröpfer, Andrea; Kammerer, Ulrike; Kapp, Michaela; Dietl, Johannes; Feix, Sonja; Anacker, Jelena

2010-01-01

Matrix metalloproteinases (MMPs) are involved in the degradation of protein components of the extracellular matrix and thus play an important role in tumor invasion and metastasis. Their expression is related to the progression of gynecological cancers (e.g. endometrial, cervical or ovarian carcinoma). In this study we investigated the expression pattern of the 23 MMPs, currently known in humans, in different gynecological cancer cell lines. In total, cell lines from three endometrium carcinomas (Ishikawa, HEC-1-A, AN3 CA), three cervical carcinomas (HeLa, Caski, SiHa), three chorioncarcinomas (JEG, JAR, BeWo), two ovarian cancers (BG-1, OAW-42) and one teratocarcinoma (PA-1) were examined. The expression of MMPs was analyzed by RT-PCR, Western blot and gelatin zymography. We demonstrated that the cell lines examined can constitutively express a wide variety of MMPs on mRNA and protein level. While MMP-2, -11, -14 and -24 were widely expressed, no expression was seen for MMP-12, -16, -20, -25, -26, -27 in any of the cell lines. A broad range of 16 MMPs could be found in the PA1 cells and thus this cell line could be used as a positive control for general MMP experiments. While the three cervical cancer cell lines expressed 10-14 different MMPs, the median expression in endometrial and choriocarcinoma cells was 7 different enzymes. The two investigated ovarian cancer cell lines showed a distinctive difference in the number of expressed MMPs (2 vs. 10). Ishikawa, Caski, OAW-42 and BeWo cell lines could be the best choice for all future experiments on MMP regulation and their role in endometrial, cervical, ovarian or choriocarcinoma development, whereas the teratocarcinoma cell line PA1 could be used as a positive control for general MMP experiments

Cell line development for biomanufacturing processes: recent advances and an outlook.

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Le, Huong; Vishwanathan, Nandita; Jacob, Nitya M; Gadgil, Mugdha; Hu, Wei-Shou

2015-08-01

At the core of a biomanufacturing process for recombinant proteins is the production cell line. It influences the productivity and product quality. Its characteristics also dictate process development, as the process is optimized to complement the producing cell to achieve the target productivity and quality. Advances in the past decade, from vector design to cell line screening, have greatly expanded our capability to attain producing cell lines with certain desired traits. Increasing availability of genomic and transcriptomic resources for industrially important cell lines coupled with advances in genome editing technology have opened new avenues for cell line development. These developments are poised to help biosimilar manufacturing, which requires targeting pre-defined product quality attributes, e.g., glycoform, to match the innovator's range. This review summarizes recent advances and discusses future possibilities in this area.

Trichloroethylene toxicity in a human hepatoma cell line

Energy Technology Data Exchange (ETDEWEB)

Thevenin, E.; McMillian, J. [Medical Univ. of Charleston South Carolina, SC (United States)

1994-12-31

The experiments conducted in this study were designed to determine the usefullness of hepatocyte cultures and a human hepatoma cell line as model systems for assessing human susceptibility to hepatocellular carcinoma due to exposure to trichloroethylene. The results from these studies will then be analyzed to determine if human cell lines can be used to conduct future experiments of this nature.

Impaired leukocyte influx in cervix of postterm women not responding to prostaglandin priming

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Masironi Britt

2008-09-01

Full Text Available Abstract Background Prolonged pregnancies are associated with increased rate of maternal and fetal complications. Post term women could be divided into at least two subgroups, one where parturition is possible to induce by prostaglandins and one where it is not. Our aim was to study parameters in cervical biopsies in women with spontaneous delivery at term (controls and compare to those that are successfully induced post term (responders, and those that are not induced (non-responders, by local prostaglandin treatment. Methods Stromal parameters examined in this study were the accumulation of leukocytes (CD45, CD68, mRNAs and/or proteins for the extracellular matrix degrading enzymes (matrix metalloproteinase (MMP-2, MMP-8 and MMP-9, their inhibitors (tissue inhibitor of MMP (TIMP-1 and TIMP-2, interleukin-8 (IL-8, the platelet activating factor-receptor (PAF-R, syndecan-1 and estrogen binding receptors (estrogen receptor (ERα, ERβ and G-coupled protein receptor (GPR 30 as well as the proliferation marker Ki-67. Results The influx of leukocytes as assessed by CD45 was strongest in the responders, thereafter in the controls and significantly lower in the non-responders. IL-8, PAF-R and MMP-9, all predominantly expressed in leukocytes, showed significantly reduced immunostaining in the group of non-responders, while ERα and GPR30 were more abundant in the non-responders, as compared to the controls. Conclusion The impaired leukocyte influx, as reflected by the reduced number of CD45 positive cells as well as decreased immunostaining of IL-8, PAF-R and MMP-9 in the non-responders, could be one explanation of the failed ripening of the cervix in post term women. If the decreased leukocyte influx is a primary explanation to absent ripening or secondary, as a result of other factors, is yet to be established.

The differential effects of leukocyte-containing and pure platelet-rich plasma (PRP) on tendon stem/progenitor cells - implications of PRP application for the clinical treatment of tendon injuries.

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Zhou, Yiqin; Zhang, Jianying; Wu, Haishan; Hogan, MaCalus V; Wang, James H-C

2015-09-15

Platelet-rich plasma (PRP) is widely used to treat tendon injuries in clinics. These PRP preparations often contain white blood cells or leukocytes, and the precise cellular effects of leukocyte-rich PRP (L-PRP) on tendons are not well defined. Therefore, in this study, we determined the effects of L-PRP on tendon stem/progenitor cells (TSCs), which play a key role in tendon homeostasis and repair. TSCs isolated from the patellar tendons of rabbits were treated with L-PRP or P-PRP (pure PRP without leukocytes) in vitro, followed by measuring cell proliferation, stem cell marker expression, inflammatory gene expression, and anabolic and catabolic protein expression by using immunostaining, quantitative real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay, respectively. Cell proliferation was induced by both L-PRP and P-PRP in a dose-dependent manner with maximum proliferation at a 10 % PRP dose. Both PRP treatments also induced differentiation of TSCs into active tenocytes. Nevertheless, the two types of PRP largely differed in several effects exerted on TSCs. L-PRP induced predominantly catabolic and inflammatory changes in differentiated tenocytes; its treatment increased the expression of catabolic marker genes, matrix metalloproteinase-1 (MMP-1), MMP-13, interleukin-1beta (IL-1β), IL-6 and tumor necrosis factor-alpha (TNF-α), and their respective protein expression and prostaglandin E2 (PGE 2) production. In contrast, P-PRP mainly induced anabolic changes; that is, P-PRP increased the gene expression of anabolic genes, alpha-smooth muscle actin (α-SMA), collagen types I and III. These findings indicate that, while both L-PRP and P-PRP appear to be "safe" in inducing TSC differentiation into active tenocytes, L-PRP may be detrimental to the healing of injured tendons because it induces catabolic and inflammatory effects on tendon cells and may prolong the effects in healing tendons. On the other hand, when P-PRP is used to

Heparin Interaction with the Primed Polymorphonuclear Leukocyte CD11b Induces Apoptosis and Prevents Cell Activation

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Meital Cohen-Mazor

2015-01-01

Full Text Available Heparin is known to have anti-inflammatory effects, yet the mechanisms are not completely understood. In this study, we tested the hypothesis that heparin has a direct effect on activated polymorphonuclear leukocytes (PMNLs, changing their activation state, and can explain its anti-inflammatory effect. To test our hypothesis, we designed both in vitro and ex vivo studies to elucidate the mechanism by which heparin modulates PMNL functions and therefore the inflammatory response. We specifically tested the hypothesis that priming of PMNLs renders them more susceptible to heparin. Amplified levels of CD11b and increased rate of superoxide release manifested PMNL priming. Increase in cell priming resulted in a dose-dependent increase in heparin binding to PMNLs followed by augmented apoptosis. Blocking antibodies to CD11b inhibited heparin binding and abolished the apoptotic response. Moreover, heparin caused a significant dose-dependent decrease in the rate of superoxide release from PMNLs, which was blunted by blocking antibodies to CD11b. Altogether, this study shows that the interaction of heparin with the PMNL CD11b results in cell apoptosis and explains heparin’s anti-inflammatory effects.

Effectiveness of leukocyte immunotherapy in primary recurrent spontaneous abortion (RSA).

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Gharesi-Fard, Behrouz; Zolghadri, Jaleh; Foroughinia, Leila; Tavazoo, Fahimeh; Samsami Dehaghani, Alamtaj

2007-09-01

Recurrent spontaneous abortion (RSA) is defined as three or more sequential abortions before the twentieth week of gestation. There are evidences to support an allo-immunologic mechanism for RSA. One of the methods for treatment of RSA is leukocyte therapy; however there is still controversy about effectiveness of this method. To evaluate the effectiveness of leukocyte therapy for treatment of RSA. Ninety two non-pregnant women with at least three sequential abortions (60 primary & 32 secondary aborters) recognized as RSA were referred to our Laboratory for immunotherapy. All the cases were immunized by isolated lymphocytes from their husbands. Fifty to 100 million washed and resuspended mononuclear cells were injected by I.V., S.C., and I.D. route. The result of each injection was checked by WBC cross matching between couples after four weeks of injections. Immunization was repeated in fifth week to a maximum of 3 times if needed. Eighty one age-matched non-pregnant RSA women (52 primary and 29 secondary aborters) with at least three sequential abortions were also included in this study as controls. The control group was not immunized. 67 out of 92 (72.8%) immunized cases and 44 out of 81 controls (54.3%) showed a successful outcome of pregnancy (pRSA patients. Despite the current controversy and limitation of leukocyte therapy in RSA, the results of our investigation provide evidence supporting the use of allo-immunization in improving the outcome of pregnancy in primary RSA patients.

Reliability of plant root comet assay in comparison with human leukocyte comet assay for assessment environmental genotoxic agents.

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Reis, Gabriela Barreto Dos; Andrade-Vieira, Larissa Fonseca; Moraes, Isabella de Campos; César, Pedro Henrique Souza; Marcussi, Silvana; Davide, Lisete Chamma

2017-08-01

Comet assay is an efficient test to detect genotoxic compounds based on observation of DNA damage. The aim of this work was to compare the results obtained from the comet assay in two different type of cells extracted from the root tips from Lactuca sativa L. and human blood. For this, Spent Pot Liner (SPL), and its components (aluminum and fluoride) were applied as toxic agents. SPL is a solid waste generated in industry from the aluminum mining and processing with known toxicity. Three concentrations of all tested solutions were applied and the damages observed were compared to negative and positive controls. It was observed an increase in the frequency of DNA damage for human leukocytes and plant cells, in all treatments. On human leukocytes, SPL induced the highest percentage of damage, with an average of 87.68%. For root tips cells of L. sativa the highest percentage of damage was detected for aluminum (93.89%). Considering the arbitrary units (AU), the average of nuclei with high levels of DNA fragmentation was significant for both cells type evaluated. The tested cells demonstrated equal effectiveness for detection of the genotoxicity induced by the SPL and its chemical components, aluminum and fluoride. Further, using a unique method, the comet assay, we proved that cells from root tips of Lactuca sativa represent a reliable model to detect DNA damage induced by genotoxic pollutants is in agreement of those observed in human leukocytes as model. So far, plant cells may be suggested as important system to assess the toxicological risk of environmental agents. Copyright © 2017 Elsevier Inc. All rights reserved.

Generation, isolation, and maintenance of rodent mast cells and mast cell lines

DEFF Research Database (Denmark)

Jensen, Bettina M; Swindle, Emily J; Iwaki, Shoko

2006-01-01

Antigen-mediated mast cell activation, with subsequent mediator release, is a major initiator of the inflammatory allergic response associated with such conditions as asthma. A comprehensive understanding of the principles involved in this process therefore is key to the development of novel...... therapies for the treatment of these disease states. In vitro models of mast cell function have allowed significant progress to be made in the recognition of the fundamental principles of mast cell activation via the high-affinity IgE receptor (FcvarepsilonRI) and, more recently, other receptors expressed...... on mast cells. In addition to human mast cells, the major cell culture systems employed to investigate these responses are rat and mouse peritoneal mast cells, mouse bone-marrow-derived mast cells, the rat basophilic leukemia cell line RBL-2H3, and the mouse MC/9 mast cell line. In this unit, we describe...

Single-cell printing to form three-dimensional lines of olfactory ensheathing cells

International Nuclear Information System (INIS)

Othon, Christina M; Ringeisen, Bradley R; Wu Xingjia; Anders, Juanita J

2008-01-01

Biological laser printing (BioLP(TM)) is a unique tool capable of printing high resolution two- and three-dimensional patterns of living mammalian cells, with greater than 95% viability. These results have been extended to primary cultured olfactory ensheathing cells (OECs), harvested from adult Sprague-Dawley rats. OECs have been found to provide stimulating environments for neurite outgrowth in spinal cord injury models. BioLP is unique in that small load volumes (∼μLs) are required to achieve printing, enabling low numbers of OECs to be harvested, concentrated and printed. BioLP was used to form several 8 mm lines of OECs throughout a multilayer hydrogel scaffold. The line width was as low as 20 μm, with most lines comprising aligned single cells. Fluorescent confocal microscopy was used to determine the functionality of the printed OECs, to monitor interactions between printed OECs, and to determine the extent of cell migration throughout the 3D scaffold. High-resolution printing of low cell count, harvested OECs is an important advancement for in vitro study of cell interactions and functionality. In addition, these cell-printed scaffolds may provide an alternative for spinal cord repair studies, as the single-cell patterns formed here are on relevant size scales for neurite outgrowth

Dynamic properties of blood flow and leukocyte mobilization in infected flaps