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PBMC
Kinds of PBMC Terms modified by PBMC Selected AbstractsReactivity of in vitro activated human T lymphocytes to p -phenylenediamine and related substancesCONTACT DERMATITIS, Issue 4 2008Claudia Skazik Background:, Patch tests to p -phenylenediamine (PPD) and related substances often show concurrent reactions that can be attributed to separate sensitization or cross-reactivity. Objectives:, In order to understand the health risks associated with cross-reactivity, we studied cross-reactivity of eight chemicals in vitro by measurement of T-cell proliferation of peripheral blood mononuclear cells (PBMC), T-cell lines (TCL), and T-cell clones (TCC) of subjects with a positive patch test result to PPD. Patients/Methods:, We studied PBMC from 13 patients and were able to generate TCL from seven and TCC from four patients. Their proliferative responses to the chemicals were estimated. Results:, Concurrent reactions to these compounds on the polyclonal and monoclonal level were found. A restricted T-cell receptor (TCR) V,16-usage was observed (5/8 clones). A detailed analysis of 34 TCL showed broad cross-reactivity (64.7%) between PPD, p -toluenediamine, Bandrowski's Base, and p -aminoazobenzene. More restricted patterns were found in 8.8%, which responded only to compounds with two or three benzene rings, whereas 26.5% of the clones reacted specifically only to one compound. Conclusion:, More than 60% of the clones showed a broad cross-reactivity pattern. Hence, clinically observed cross-reactivity between different para-amino compounds can be based on a TCR recognizing similar epitopes of these compounds with low specificity. [source] A decreased positivity for CD90 on human mesenchymal stromal cells (MSCs) is associated with a loss of immunosuppressive activity by MSCs,CYTOMETRY, Issue 3 2009Diana Campioni Abstract Biologic and clinical interest in human mesenchymal stromal cells (hMSC) has risen over the last years, mainly due to their immunosuppressive properties. In this study, we investigated the basis of immunomodulant possible variability using hMSC from different sources (amniotic membrane, chorion, and bone marrow from either healthy subjects or patients with hematological malignancies, HM) and having discordant positivity for several immunological markers. The CD90+ hMSC reduced lymphoproliferative response in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) via sHLA-G and IL-10 up-modulation. On the contrary, hMSC showing a significantly lower expression for CD90 antigen, elicited a lymphoproliferative allogeneic response in PHA/PBMCs without any increase in soluble HLA-G and IL-10 levels. These data seems to suggest that CD90 molecule may be considered a novel predictive marker for hMSC inhibitory ability, and might cooperate with HLA-G molecule in regulating suppressive versus stimulatory properties of hMSC. These results may have clinical implication in either transplantation or in regenerative medicine fields. © 2008 Clinical Cytometry Society [source] Validated protocol for FoxP3 reveals increased expression in type 1 diabetes patients,CYTOMETRY, Issue 2 2009Jean Grant Abstract Background FoxP3 has become a key identifier of regulatory T cells. Investigators have used a variety of antibodies and methods for detecting FoxP3 by flow cytometry. To standardize FoxP3 antibody staining for use in clinical trial samples, we tested various antibodies from different vendors, cell preparation protocols and fix/perm reagents, and cell isolation procedures. Using this optimized staining protocol, we evaluated clinical specimens from patients with multiple sclerosis (MS) or type 1 diabetes. Methods FoxP3 antibodies from eBioscience (236A/E7 and PCH101) and BioLegend (206D) were evaluated along with their respective methods and fix/perm reagents for preparation and staining of FoxP3 for flow cytometry. Fresh washed blood and frozen or fresh PBMC were evaluated. Upon optimization of the protocol, clinical samples (frozen PBMC) from patients with MS or type 1 diabetes and healthy control donors were evaluated with the BioLegend antibody. Results Clone 206D from BioLegend yielded optimal staining and the fix/perm reagents from both eBioscience and BioLegend were comparable. Data were also comparable between cells separated by Ficoll (fresh or frozen) and washed blood samples, allowing this protocol to be applicable to different types of samples. We validated this protocol using clinical samples and saw a significant increase in FoxP3 expression in the patients with type 1 diabetes but not in the MS. Conclusions The results from this study will allow the assessment of FoxP3 by flow cytometry on samples from clinical sites that are analyzed in real time on fresh blood or frozen PBMC. © 2008 Clinical Cytometry Society [source] Cell microarray platform for anticancer drug development,DRUG DEVELOPMENT RESEARCH, Issue 5 2007Min-Jung Lee Abstract Pharmacodynamic assessment of whether a drug has interacted with and modified its target is an essential component of molecularly targeted clinical trials. Although many trials are written with the intent to assess tumor biopsies, if available, thus far the great majority of early drug trials have used peripheral blood mononuclear cells (PBMC) as a tumor surrogate. Typically, PBMC are studied by low-throughput techniques such as Western blot. We present the use of a cell-based tissue microarray for assessment of anticancer drug activity in vivo. We demonstrate the utility of this technique for analysis of protein hyperacetylation in response to treatment with the histone deacetylase inhibitor, SNDX-275 in PBMC treated in vitro and in PBMC and bone marrow aspirates from patients in Phase I clinical trials with SNDX-275. We demonstrate that the cell microarray can be used to measure drug response in a high-throughput manner, allowing analysis of an entire trial on one or two glass slides. The cell microarray technique brings the advantages of the tissue microarray platform to the pharmacodynamic assessment of single cells, such as those isolated from bone marrow aspirates, fine needle aspirates, or malignant effusions, and to analysis of PBMC, the most commonly studied surrogate in oncology trials. Drug Dev Res 68:226,234, 2007. Published 2007 Wiley-Liss, Inc. [source] Quantification by real-time PCR of the magnitude and duration of leucocyte-associated viraemia in horses infected with neuropathogenic vs. non-neuropathogenic strains of EHV- 1EQUINE VETERINARY JOURNAL, Issue 3 2006G. P. Allen Summary Reasons for performing study: Neurological disease in horses caused by infection with certain ,paralytic' strains of equine herpesvirus-1 (EHV-1) is a potentially devastating condition the pathogenesis of which is poorly understood. Preliminary observations in both experimentally induced and naturally occurring cases of the central nervous system disease have revealed a more robust cell-associated viraemia in horses infected with paralytic isolates of EHV-1, relative to horses infected with abortigenic isolates. To investigate further this pathogenesis - rdevant question, the present study was performed using a greater number of horses and a more precise method for quantification of EHV-1 DNA present in viraemic leucocytes. Objective: To compare the magnitude and duration of leucocyte-associated viraemia in seronegative, age-matched foals following infection with paralytic vs. abortigenic isolates of EHV-1. Methods: Peripheral blood mononuclear cells (PBMC) were collected from 20 weanling foals at 2, 4, 7, 9, 11, 14 and 21 days after intranasal inoculation with either paralytic or abortigenic isolates of EHV-1. The amount of EHV-1 DNA present in each PBMC sample was measured by real-time quantitative PCR. Results: Foals inoculated with paralytic strains of EHV-1 developed both a greater magnitude and longer duration of PBMC-associated viraemia than foals inoculated with abortigenic strains of the virus. Conclusions: Both the higher magnitude and longer duration of cell-associated viraemia contribute to the risk for development of neurological signs in horses infected with paralytic strains of EHV-1. Potential relevance: Our results provide empirically derived, scientific data that contributes to a better understanding of the pathogenetic basis for the differing abilities of paralytic and abortigenic strains of EHV-1 to cause post infection central nervous system disease in the horse. The findings identify the importance of minimising the quantitative burden of viraemic leucocytes that follows exposure to the virus, by the use of effective therapeutic antiviral drugs and efficacious prophylactic vaccines that stimulate cytotoxic immune responses against EHV-1 infected cells. [source] Irradiated cultured apoptotic peripheral blood mononuclear cells regenerate infarcted myocardiumEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 6 2009H. J. Ankersmit Abstract Background, Acute myocardial infarction (AMI) is followed by post AMI cardiac remodelling, often leading to congestive heart failure. Homing of c-kit+ endothelial progenitor cells (EPC) has been thought to be the optimal source for regenerating infarcted myocardium. Methods, Immune function of viable peripheral blood mononuclear cells (PBMC) was evaluated after co-culture with irradiated apoptotic PBMC (IA-PBMC) in vitro. Viable PBMC, IA-PBMC and culture supernatants (SN) thereof were obtained after 24 h. Reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay were utilized to quantify interleukin-8 (IL-8), vascular endothelial growth factor, matrix metalloproteinase-9 (MMP9) in PBMC, SN and SN exposed fibroblasts. Cell suspensions of viable- and IA-PBMC were infused in an experimental rat AMI model. Immunohistological analysis was performed to detect inflammatory and pro-angiogenic cells within 72 h post-infarction. Functional data and determination of infarction size were quantified by echocardiography and Elastica van Gieson staining. Results, The IA-PBMC attenuated immune reactivity and resulted in secretion of pro-angiogenic IL-8 and MMP9 in vitro. Fibroblasts exposed to viable and IA-PBMC derived SN caused RNA increment of IL-8 and MMP9. AMI rats that were infused with IA-PBMC cell suspension evidenced enhanced homing of endothelial progenitor cells within 72 h as compared to control (medium alone, viable-PBMC). Echocardiography showed a significant reduction in infarction size and improvement in post AMI remodelling as evidenced by an attenuated loss of ejection fraction. Conclusion, These data indicate that infusion of IA-PBMC cell suspension in experimental AMI circumvented inflammation, caused preferential homing of regenerative EPC and replaced infarcted myocardium. [source] Haemodialysis induces mitochondrial dysfunction and apoptosisEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 12 2007D. S. C. Raj Abstract Background Mitochondria play a crucial role in the regulation of the endogenous pathways of apoptosis activated by oxidant stress. Nuclear factor-,B (NF-,B) is a central integration site for pro-inflammatory signals and oxidative stress. Materials and methods Peripheral blood mononuclear cells (PBMC) were isolated from eight end-stage renal disease (ESRD) patients before haemodialysis (Pre-HD) and during the last 10 min of HD (End-HD). A new polysulfone membrane (F70, Fresenius) was used for dialysis. Intracellular generation of reactive oxygen species (ROS), mitochondrial redox potential (,,m) and PBMC apoptosis were determined by flow-cytometry. Results Plasma levels of interleukin-6 (IL-6) (24·9 ± 7·0 vs. 17·4 ± 5·5 pg dL,1, P < 0·05), IL-6 soluble receptor (52·2 ± 4·9 vs. 37·6 ± 3·2 ng dL,1, P < 0·02) and IL-6 gp130 (405·7 ± 41·0 vs. 235·1 ± 38·4 ng dL,1, P < 0·02) were higher end-HD compared to pre-HD. IL-6 secretion by the isolated PBMC (24·0 ± 2·3 vs. 19·3 ± 3·5 pg dL,1, P < 0·02) increased end-HD. Percentage of lymphocytes exhibiting collapse of mitochondrial membrane potential (43·4 ± 4·6% vs. 32·6 ± 2·9%, P < 0·01), apoptosis (33·4 ± 7·1% vs. 23·7 ± 7·7%, P < 0·01), and generation of superoxide (20·7 ± 5·2% vs. 12·5 ± 2·9%, P < 0·02) and hydrogen peroxide (51·1 ± 7·8% vs.38·2 ± 5·9%, P < 0·04) were higher at end-HD than pre-HD. NF-,B activation (3144·1 ± 208·1 vs. 2033·4 ± 454·6 pg well,1, P < 0·02), expression of B-cell lymphoma protein-2 (6494·6 ± 1461 vs. 3501·5 ± 796·5 ng mL,1, P < 0·03) and heat shock protein-70 (9·81 ± 1·47 vs. 6·38 ± 1·0 ng mL,1, P < 0·05) increased during HD. Conclusions Intra-dialytic activation of cytokines, together with impaired mitochondrial function, promotes generation of ROS culminating in augmented PBMC apoptosis. There is concomitant activation of pathways aimed at attenuation of cell stress and apoptosis during HD. [source] Long-term effects of idiotype vaccination on the specific T-cell response in peripheral blood and bone marrow of multiple myeloma patientsEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2007Amir Osman Abdalla Abstract Objectives:, To elucidate long-term effects of idiotype (Id) vaccination on Id-specific T cells of multiple myeloma (MM) patients and compare Id-specific T-cell responses of peripheral blood with those of bone marrow (BM). Materials and methods:, Id-specific T-cell responses of peripheral blood mononuclear cells (PBMC) were compared with those of BM mononuclear cells (BMMC) in 10 MM patients vaccinated with the Id protein at a median time of 41 months since the last immunization. The PBMC responses at late follow-up were also compared with those during active immunization. The responses were assessed by a proliferation assay, enzyme-linked immunospot (ELISPOT) (,-interferon), cytometric bead array (CBA) for secreted cytokines and quantitative real-time polymerase chain reaction (QRT-PCR) for cytokine gene expression. Results:, At the late testing time, an Id-specific response was detected in PBMC of five patients (ELISPOT, CBA, QRT-PCR) and in BMMC of four patients (CBA, QRT-PCR). A response in both compartments was noted only in three patients. The cytokines gene profile was consistent with a predominance of Th2 cells [interleukin (IL)-4, IL-5, IL-10]. Comparison of the Id-specific responses of PBMC during active immunization with those at the late follow-up showed that the frequency and magnitude of the responses had decreased significantly by time (proliferation/ELISPOT) (P < 0.02) and shifted at the gene level from a Th1 to a Th2 profile (P < 0.05). Conclusion:, Id-specific T cells may decline overtime and shift toward a Th2 response and may be found at a similar frequency of patients in blood and BM. [source] Interleukin-10-secreting T cells define a suppressive subset within the HIV-1-specific T-cell populationEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2009Eirik A. Torheim Abstract Recent studies have indicated that Treg contribute to the HIV type 1 (HIV-1)-related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV-1-specific T-cell population. Here, PBMC from HIV-1-infected individuals were stimulated with a 15-mer Gag peptide pool, and HIV-1-specific T cells were enriched by virtue of their secretion of IL-10 or IFN-, using immunomagnetic cell-sorting. Neither the IL-10-secreting cells nor the IFN-,-secreting cells expressed the Treg marker FOXP3, yet the IL-10-secreting cells potently suppressed anti-CD3/CD28-induced CD4+ as well as CD8+ T-cell proliferative responses. As shown by intracellular cytokine staining, IL-10- and IFN-,-producing T cells represent distinct subsets of the HIV-1-specific T cells. Our data collectively suggest that functionally defined HIV-1-specific T-cell subsets harbor potent immunoregulatory properties that may contribute to HIV-1-associated T-cell dysfunction. [source] Activation drives PD-1 expression during vaccine-specific proliferation and following lentiviral infection in macaquesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2008David Abstract Recent data supports that increased expression of PD-1, a negative regulator of immune function, is associated with T cell exhaustion during chronic viral infection. However, PD-1 expression during acute infection and vaccination has not been studied in great detail in primates. Here, we examine PD-1 expression on CD3+ T cells following DNA vaccination or lentiviral infection of macaques. Ex vivo peptide stimulation of PBMC from DNA-vaccinated uninfected macaques revealed a temporal increase in PD-1 expression in proliferating antigen-specific CD8+ T cells. Following the initial increase, PD-1 expression steadily declined as proliferation continued, with a concomitant increase in IFN-, secretion. Subsequent examination of PD-1 expression on T cells from uninfected and lentivirus-infected non-vaccinated macaques revealed a significant increase in PD-1 expression with lentiviral infection, consistent with previous reports. PD-1 expression was highest on cells with activated memory and effector phenotypes. Despite their decreased telomere length, PD-1hi T cell populations do not appear to have statistically significant uncapped telomeres, typically indicative of proliferative exhaustion, suggesting a different mechanistic regulation of proliferation by PD-1. Our data indicate that PD-1 expression is increased as a result of T cell activation during a primary immune response as well as during persistent immune activation in macaques. Supporting Information for this article is available at www.wiley-vch.de/contents/jc_2040/2008/37857_s.pdf [source] Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirusEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2006Andrea Sáez-Borderías Abstract The human NKG2D killer lectin-like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3-kinase (PI3,K) and specifically interacts with different stress-inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus-infected cells. This KLR is commonly expressed by human NK cells as well as TCR,,+ and TCR,,+CD8+ T lymphocytes, but it has been also detected in CD4+ T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)-specific CD4+ T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4+NKG2D+ T lymphocytes that coexpressed perforin. NKG2D was detected in CD28, and CD28dull subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR-dependent activation of CD4+ T cells, triggering proliferation and cytokine production (i.e. IFN-, and TNF-,). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV-specific CD4+ T lymphocytes and thus may have a role in the response against infected HLA class II+ cells displaying NKG2D ligands. [source] CCL17 transgenic mice show an enhanced Th2-type response to both allergic and non-allergic stimuliEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2006Yuichiro Tsunemi Dr. Abstract CC chemokine ligand (CCL)17 is implicated in the pathogenesis of atopic dermatitis (AD). To study the effect of CCL17 produced by keratinocytes (KC) during inflammation, we created transgenic (Tg) mice in which CCL17 is overexpressed in KC. Th2-type contact hypersensitivity (CHS) was enhanced and Th1-type CHS was suppressed in these mice. Increased numbers of CC chemokine receptor (CCR)4+ cells and mast cells infiltrated in Tg mice. Levels of IL-4 mRNA were higher and those of IFN-, mRNA were lower in both acute and chronic CHS. Higher levels of serum IgE were observed after CHS. Numbers of CCR4+ cells among PBMC were increased in Tg mice challenged acutely on the trunk. Chronic irritation with croton oil induced dermatitis and an elevation of serum IgE levels. Tg mice showed enhanced ear swelling after tape stripping. CCL17 was thought to modify the inflammation caused by sensitizing reagents as well as irritant reagents by attracting CCR4+ cells into the lesional skin and creating a Th2-dominant condition. AD-like conditions such as increased number of mast cells and elevated levels of serum IgE were observed. Thus, CCL17 may participate in the pathogenesis of skin diseases such as AD by regulating both allergic and irritant inflammation. [source] IFN-,-producing human T cells directly induce osteoclastogenesis from human monocytes via the expression of RANKLEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2005Shigeru Kotake Abstract The current study explored our hypothesis that IFN-,-producing human T cells inhibit human osteoclast formation. Activated T cells derived from human PBMC were divided into IFN-,-producing T cells (IFN-,+ T cells) and IFN-,-non-producing T cells (IFN-,, T cells). IFN-,+ T cells were cultured with human monocytes in the presence of macrophage-CSF alone. The concentration of soluble receptor activator of NF-,B ligand (RANKL) and IFN-,, and the amount of membrane type RANKL expressed on T cells, were measured by ELISA. In the patients with early rheumatoid arthritis (RA) treated with non-steroidal anti-inflammatory drugs alone, CD4+ T cells expressing both IFN-, and RANKL were detected by flow cytometry. Surprisingly, IFN-,+ T cells, but not IFN-,, T cells, induced osteoclastogenesis from monocytes, which was completely inhibited by adding osteoprotegerin and increased by adding anti-IFN-, antibodies. The levels of both soluble and membrane type RANKL were elevated in IFN-,+ T cells. The ratio of CD4+ T cells expressing both IFN-, and RANKL in total CD4+ T cells from PBMC was elevated in RA patients. Contrary to our hypothesis, IFN-,+ human T cells induced osteoclastogenesis through the expression of RANKL, suggesting that Th1 cells play a direct role in bone resorption in Th1 dominant diseases such as RA. [source] CpG ODN enhance antigen-specific NKT cell activation via plasmacytoid dendritic cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2005Anja Marschner Abstract Human V,24+ V,11+ natural killer T cells (NKT cells) are "natural memory" T cells that detect glycolipid antigens such as ,-galactosylceramide (,-GalCer) presented on CD1d. In the present study we found that highly purified V,24+ NKT cells lack TLR9 mRNA, and thus are not sensitive towards stimulation with CpG oligodeoxynucleotides (ODN). Within PBMC, however, CpG ODN synergistically activated NKT cells stimulated with their cognate antigen ,-GalCer. Depletion of plasmacytoid dendritic cells (PDC) or myeloid dendritic cells (MDC) revealed that both DC subsets were necessary for the synergistic activation of NKT cells by ,-GalCer and CpG ODN. While PDC were responsible for the stimulation of NKT cells with CpG ODN, MDC but not PDC presented ,-GalCer via CD1d. Partial activation of NKT cells was mediated by PDC-derived IFN-,, whereas full activation of NKT cells as indicated by IFN,, production required cell-to-cell contact of PDC and NKT cells in addition to IFN-,; OX40 was involved in this interaction. We conclude that CpG-activated PDC enhance ,-GalCer-specific NKT cell activation, and bias activated NKT cells towards a Th1 phenotype. Our results lead to a novel concept of PDC function: to regulate effector activity of antigen-stimulated T cells in a cell contact-dependent manner without the need of simultaneous presentation of the cognate T cell antigen. [source] Synthetic bacterial lipopeptide analogs facilitate naive CD4+ T,cell differentiation and enhance antigen-specific HLA-II-restricted responsesEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2005Mascia Ghielmetti Abstract Synthetic di- and tri-palmitoylated bacterial lipopeptide analogs (BLpA) can enhance HLA-I-restricted immune responses. Here we show that BLpA indirectly promote antigen-driven differentiation of naive CD4+ T,lymphocytes in vitro, with mechanisms that require DC and are inhibited by CTLA-4/Ig. In mixed cultures of cord blood-derived PBMC and allogeneic DC, P3CSK4 lipopeptide facilitated the transition from CCR7+/CD45RA+/CD62L+ to CCR7,/CD45RA,/CD62Ldim T,cells with kinetics significantly exceeding those obtained with the unlipidated CSK4 analog. Moreover, P3CSK4 and P2CSK4, but neither the mono-palmitoylated PCSK4 analog nor the CSK4 peptide, increased the frequency of IFN-,-producing T,cells expanded under similar conditions. Along with this, P2CSK4 and P3CSK4, but not PCSK4, restored the in vitro antigenicity of MDP-OspA, a non-immunogenic analog of Borrelia burgdorferi major outer surface lipoprotein,A, and enhanced the frequency of in vitro expanded T,cells specific for the tetanus toxoid (TT) and hepatitis,B surface antigen (HBsAg) peptides TT947,967 and HBsAg19,33 and for TT. Altogether, BLpA bearing at least two ester-bonded palmitoyl side chains indirectly enhance antigen-driven CD4+ T,cell differentiation. BLpA adjuvanticity is independent of covalent bonding to Ag and Ag formulation. This information may be helpful to generate more potent recombinant vaccines. [source] Microbial Toll-like receptor ligands differentially regulate CXCL10/IP-10 expression in fibroblasts and mononuclear leukocytes in synergy with IFN-, and provide a mechanism for enhanced synovial chemokine levels in septic arthritisEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 11 2003Paul Proost Abstract The CXC chemokine IFN-,-inducible protein-10 (IP-10/CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T cells and natural killer cells. Peripheral blood mononuclearcells (PBMC) produce low but significant amounts of IP-10/CXCL10 protein upon stimulation with double-stranded (ds) RNA, the Toll-like receptor 3 (TLR3) ligand. IFN-, is a superior IP-10/CXCL10inducer. The bacterial TLR4 and TLR2 ligands, LPS and peptidoglycan (PGN), inhibit IFN-,- or dsRNA-dependent IP-10/CXCL10 production in PBMC, whereas IL-8/CXCL8 production was enhanced. In fibroblasts a different picture emerges with IFN-, inducing moderate and dsRNA provoking strong IP-10/CXCL10 production. Furthermore, treatment of fibroblasts with IFN-, in combination with bacterial LPS or PGN results in a synergistic production of IP-10/CXCL10 and IL-8/CXCL8. The synergistic induction of IP-10/CXCL10 in fibroblasts is reflected by significantly enhanced IP-10/CXCL10 concentrations in synovial fluids of septic compared to osteoarthritis patients to reach on average higher levels than those of IL-8/CXCL8. These high amounts of IP-10/CXCL10 produced by connective tissue fibroblasts not only attract CXCR3 expressing activated Th1 cells and natural killer cells to sites of infection but may also antagonize the CCR3 dependent attraction of Th2 lymphocytes and exert CXCR3-independent, defensin-like antibacterial activity. [source] CD4 T,cell activation by myelin oligodendrocyte glycoprotein is suppressed by adult but not cord blood CD25+ T,cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 3 2003Kajsa Wing Abstract Regulatory T,cells expressing CD25 have been shown to protect rodents from organ-specific autoimmune diseases. Similar CD25+ cells with a memory phenotype exerting suppressive function after polyclonal or allogeneic stimulation are also present in adult human blood. We demonstrate that adult human CD25+ cells regulate the response to myelin oligodendrocyte glycoprotein (MOG), as depletion of CD25+ cells increases responses of PBMC and the addition of purified CD25+ cells suppresses MOG-specific proliferation and IFN-, production of CD4+CD25, T,cells. In contrast, cord blood CD25+ cells do not inhibit responses to self antigens, and only a small subpopulation of cord CD25+ cells expresses the typical phenotype of adult regulatory T,cells (CD45RA, and GITR+) enabling suppression of polyclonal responses. We conclude that activation of self-reactive T,cells in normal healthy individuals is prevented by the presence of self-antigen-specific CD25+ regulatory T,cells and that the majority of these cells mature after birth. [source] Inhibition of T-cell activation in vitro in human peripheral blood mononuclear cells by pimecrolimus and glucocorticosteroids and combinations thereofEXPERIMENTAL DERMATOLOGY, Issue 8 2007Anthony Winiski Abstract:, Pimecrolimus is an ascomycin macrolactam derivative that has been recently approved for the topical treatment of atopic dermatitis. In this study we report for the first time on a direct comparison of the inhibitory activity of pimecrolimus and the glucocorticosteroids betamethasone 17-valerate, dexamethasone and hydrocortisone at the level of T-cell proliferation and cytokine production. Stimulated human peripheral blood mononuclear cell (PBMC) systems were used that are either sensitive or resistant to calcineurin inhibitors or glucocorticosteroids. Pimecrolimus and the glucocorticosteroids inhibited dose-dependently T-cell proliferation and cytokine production in a sensitive system (anti-CD3 mAb-stimulated PBMC) with the following rank order of potency: pimecrolimus , betamethasone 17-valerate , dexamethasone > hydrocortisone. In resistant systems (anti-CD3 plus anti-CD28- or Staphylococcal enterotoxin B-stimulated PBMC), pimecrolimus or the glucocorticosteroids alone exerted either no effect, or only a partial inhibitory effect. However, combinations of pimecrolimus with a glucocorticosteroid synergistically and strongly inhibited T-cell proliferation. Taken together, the data indicate that medium potency glucocorticosteroids, such as betamethasone 17-valerate and dexamethasone, are as potent T-cell inhibitors as pimecrolimus. Furthermore, the experimental evidence suggests that combinations of glucocorticosteroids and pimecrolimus could be used clinically to achieve superior therapeutic efficacy, when monotherapy with the individual agents is unsatisfactory. [source] Immunomodulatory effects of probiotic bacteria DNA: IL-1 and IL-10 response in human peripheral blood mononuclear cellsFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2003Karen Manon Lammers Abstract A new therapeutic approach for inflammatory bowel diseases is based on the administration of probiotic bacteria. Prokaryotic DNA contains unmethylated CpG motifs which can activate immune responses, but it is unknown whether bacterial DNA is involved in the beneficial effects obtained by probiotic treatment. Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated with pure DNA of eight probiotic strains and with total bacterial DNA from human feces collected before and after probiotic ingestion. Cytokine production was analyzed in culture supernatants. Modification of human microflora after probiotic administration was proven by polymerase chain reaction analysis. Here we show that Bifidobacterium genomic DNA induced secretion of the antiinflammatory interleukin-10 by PBMC. Total bacterial DNA from feces collected after probiotic administration modulated the immune response by a decrease of interleukin-1, and an increase of interleukin-10. [source] The study of cell-mediated immune response in recurrent vulvovaginal candidiasisFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2000U. Nawrot Abstract The aim of this work was to examine in vitro the ability of cells from patients with recurrent vulvovaginal candidiasis (RVVC) to cell-mediated immune response. Peripheral blood mononuclear cells (PBMC) and whole blood cells (WBC) of 37 RVVC patients in acute infection and 14 in remission were examined for the ability to proliferation and cytokines production (IFN, TNF, IL-6). As a control, a group of 25 healthy women were examined. The cells were stimulated with Candida antigen (HKCA), LPS and PHA. To indicate the level of cytokines, the following cell-lines were used: A549 for IFN, WEHI 164 for TNF and 7TD1 for IL-6. The proliferation/death of cells was determined by colorimetric test using MTT. Distinct suppression of cell-mediated immune response (CMI) was shown in all patients comparing to the control. Greatest suppression was found in the acute phase of the disease. The ability of cells to proliferate and produce IFN increases only in remission. The data seem to suggest that in this phase of disease, the ability of cell-mediated immune response is restored. It was also indicated that IFN may take part in protection against Candida infection. [source] Evidence of occult HCV genotypes in haemophilic individuals with unapparent HCV mixed infectionsHAEMOPHILIA, Issue 4 2008C. PARODI Summary., Individuals with haemophilia who received non heat-treated factor concentrates were likely to undergo multiple exposures to the hepatitis C virus (HCV). Therefore, HCV mixed-genotype infections might be more frequent in these patients than in the general population. Their prevalence is extremely variable in similar groups of patients tested by different assays due to the fact that currently available genotyping techniques are not suitable to detect multiple HCV genotypes in a viral population. As an HCV viral reservoir, the peripheral blood mononuclear cell (PBMC) might harbor viral variants distinct from the genotypes detected in plasma. We investigated the presence of HCV genotypes in a group of chronically infected haemophilic patients in the PBMC compartment using a non-stimulated cell culture system that allows the detection of the HCV genome in culture supernatants. We compared them to the HCV genotypes found in plasma samples. Cell culture experiments performed with PBMC demonstrated the presence of additional HCV genotypes that were undetected in the corresponding plasma samples with the same genotyping technique. Although mixed infections at HCV genotype level became evident in 5.6% of the patients (16/288), the culture methodology increased the number of HCV infections with multiple genotypes to 62.5% (10/16) (P < 0.0001). Once more, the role of mononuclear cells as HCV viral reservoirs is emphasized. Considering minor strains could influence the outcome of treatment, detection of covert HCV mixed-genotype infections might be essential for choosing the adequate therapeutic regimen. [source] Long-term follow-up after successful interferon therapy of acute hepatitis CHEPATOLOGY, Issue 1 2004Johannes Wiegand Early treatment of acute hepatitis C infection with interferon alfa-2b (IFN-,-2b) prevents chronicity in almost all patients. So far, no data are available on the long-term outcome after interferon (IFN) therapy of acute hepatitis C. The aim of this study was to assess the clinical, virological, and immunological long-term outcome of 31 successfully treated patients with acute hepatitis C infection who were followed for a median of 135 weeks (52-224 weeks) after end of therapy. None of the individuals had clinical evidence of liver disease. Alanine aminotransferase (ALT) levels were normal in all but 1 patient. Serum hepatitis C virus (HCV) RNA was negative throughout follow-up, even when investigated with the highly sensitive transcription-mediated amplification (TMA) assay (cutoff 5-10 IU/mL). In addition, no HCV RNA was detected in peripheral blood mononuclear cells (PBMC) of 15 cases tested. The patients' overall quality-of-life scores as determined by the SF-36 questionnaire did not differ from the German reference control cohort. Ex vivo interferon gamma (IFN-,) ELISPOT analysis detected HCV-specific CD4+ T-helper cell reactivity in only 35% of cases, whereas HCV-specific CD8+ T-cell responses were found in 4 of 5 HLA -A2,positive individuals. Anti-HCV antibody levels decreased significantly during and after therapy in all individuals. In conclusion, early treatment of symptomatic acute hepatitis C with IFN-,-2b leads to a long-term virological, biochemical, and clinical response. Waning of anti-HCV humoral immunity and presence of HCV-specific CD8+ (but not CD4+) T cells highlights the complexity of T-cell and B-cell memory to HCV, which might be significantly altered by IFN treatment. (HEPATOLOGY 2004;40:98,107.) [source] Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responsesIMMUNOLOGY, Issue 1 2010Amit Golding Summary An adequate effector response against pathogens and its subsequent inactivation after pathogen clearance are critical for the maintenance of immune homeostasis. This process involves an initial phase of T-cell effector (Teff) activation followed by the expansion of regulatory T cells (Tregs), a unique cell population that limits Teff functions. However, significant questions remain unanswered about the mechanisms that regulate the balance between these cell populations. Using an in vitro system to mimic T-cell activation in human peripheral blood mononuclear cells (PBMC), we analysed the patterns of Treg and Teff activation, with special attention to the role of type I interferon (IFN-I). Interestingly, we found that IFN-,, either exogenously added or endogenously induced, suppressed the generation of CD4+ FoxP3HI IFN-,Neg activated Tregs (aTregs) while simultaneously promoting propagation of CD4+ FoxP3Low/Neg IFN-,Pos activated Teffs (aTeffs). We also showed that IFN-,-mediated inhibition of interleukin (IL)-2 production may play an essential role in IFN-,-induced suppression of aTregs. In order to test our findings in a disease state with chronically elevated IFN-,, we investigated systemic lupus erythematosus (SLE). Plasma from patients with SLE was found to contain IFN-I activity that suppressed aTreg generation. Furthermore, anti-CD3 activated SLE PBMCs exhibited preferential expansion of aTeffs with a very limited increase in aTreg numbers. Together, these observations support a model whereby a transient production of IFN-, (such as is seen in an early antiviral response) may promote CD4 effector functions by delaying aTreg generation, but a chronic elevation of IFN-, may tip the aTeff:aTreg balance towards aTeffs and autoimmunity. [source] Elevation of interleukin-18 in chronic hepatitis C: implications for hepatitis C virus pathogenesisIMMUNOLOGY, Issue 1pt2 2009Arpita Sharma Summary The outcome of hepatitis C virus (HCV) infection is determined by the interplay between the virus and the host immune response. Interleukin (IL)-18, an interferon-,-inducing factor, plays a critical role in the T helper type 1 (Th1) response required for host defence against viruses, and antibodies to IL-18 have been found to prevent liver damage in a murine model. The present study was conducted to investigate the possible role of IL-18 in the pathogenesis and persistence of HCV. IL-18 levels were measured in sera of 50 patients at various stages of HCV infection (resolved, chronic and cirrhosis) and compared with those of normal controls. IL-18 gene expression was studied in peripheral blood mononuclear cells (PBMC) from each group, and in liver biopsy tissue from patients with chronic hepatitis C. The mean levels of IL-18 in sera were markedly elevated in patients with chronic hepatitis and cirrhosis, and were reduced in patients with resolved HCV infection. The serum IL-18 concentrations were related to the Child,Pugh severity of liver disease in cirrhotic patients. There also existed a strong positive correlation of IL-18 levels with histological activity score and necrosis. IL-18 mRNA expression was significantly up-regulated in the PBMC of cirrhotic patients when compared with other groups, while in the liver, higher levels of IL-18 transcripts were expressed in patients with chronic hepatitis C. The results of our study indicate that IL-18 levels reflect the severity and activity of HCV infection, and may contribute to the pathogenesis and progression of liver disease associated with HCV. [source] Epidermal keratinocytes do not activate peripheral T-cells: interleukin-10 as a possible regulatorIMMUNOLOGY, Issue 3 2008Rocío Isabel Domínguez-Castillo Summary The immunogenicity of allogeneic cultured human epidermal keratinocytes (cHEKs) has been studied in several models with contradictory results. We studied human T-cell activation in an in vitro assay by incubating, for 4 and 24 hr, cHEK confluent sheets with human peripheral blood mononuclear cells (PBMC); parallel HEK cultures were incubated with interferon (IFN)-, to induce the expression of major histocompatibility complex (MHC) molecules before their interaction with PBMC. T-cell activation was evaluated by flow cytometry. T cells neither expressed the early and late activation markers CD69 and CD25, respectively, nor proliferated after incubation with the epidermal sheets, despite the IFN-,-induced expression of MHC and adhesion molecules in cHEKs. Interleukin (IL)-10 was detected in the medium from the co-cultured PBMC and HEK sheets, but not from HEK alone. The results suggest that HEKs are unable to stimulate T lymphocytes through secretion of cytokines that might contribute to the immunosuppressive effect in this in vitro model. [source] Induction of 150-kDa adenosine deaminase that acts on RNA (ADAR)-1 gene expression in normal T lymphocytes by anti-CD3-, and anti-CD28IMMUNOLOGY, Issue 4 2007Dama Laxminarayana Summary We and other investigators have demonstrated up-regulation of the expression of the RNA-editing gene 150-kDa adenosine deaminase that acts on RNA (ADAR1) in systemic lupus erythematosus (SLE) T cells and B cells, peripheral blood mononuclear cells (PBMC), natural killer (NK) cells. The presence of a small proportion of activated T cells is the hallmark of SLE. Therefore, it was hypothesized that 150-kDa ADAR1 gene expression is induced by the physiological activation of T cells. To examine this hypothesis, normal T cells were activated by anti-CD3-, plus anti-CD28 for various time periods from 0 to 48 hr. The expression of 110-kDa and 150-kDa ADAR1, and interleukin (IL)-2 and ,-actin gene transcripts was analysed. An approximately fourfold increase in 150-kDa ADAR1 gene expression was observed in activated T cells. ADAR2 gene transcripts are substrates for ADAR1 and ADAR2 enzymes. Therefore, we assessed the role of the 150-kDa ADAR enzyme in editing of ADAR2 gene transcripts. In activated T cells, site-selective editing of the ,2 site was observed. Previous studies indicate that this site is predominantly edited by ADAR1. In addition to this, novel editing sites at base positions ,56, ,48, ,45, ,28, ,19, ,15, +46 and +69 were identified in activated T cells. On the basis of these results, it is proposed that 150-kDa ADAR1 gene expression is selectively induced in T cells by anti-CD3-, and anti-CD28 stimulation and that it may play a role in site-selective editing of gene transcripts and in altering the functions of several gene products of T cells during activation and proliferation. [source] Impact of class A, B and C CpG-oligodeoxynucleotides on in vitro activation of innate immune cells in human immunodeficiency virus-1 infected individualsIMMUNOLOGY, Issue 4 2007Jeffrey A. Martinson Summary Oligodeoxynucleotides (ODN) with unmethylated deoxycytidyl-deoxyguanosine dinucleotides (CpG-ODNs) stimulate Toll-like receptor 9 (TLR9) in plasmacytoid dendritic cells (pDC) and B cells and activate innate and adaptive immunity. Three classes of synthetic CpG-ODNs, class A, B and C, activate cells through TLR9; our goal was to evaluate their effect on cells from human immunodeficiency virus (HIV)-1+ individuals. We compared the frequencies and the unstimulated activation status of immune effector cells in HIV-1+ and HIV-1, individuals. Fewer pDC, myeloid dendritic cells (mDC), B cells, natural killer (NK) cells and invariant natural killer T cells (iNKT) were present in HIV-1+ peripheral blood mononuclear cells (PBMC) and their baseline activation status was higher than HIV-1, PBMC. Exposure of HIV-1+ PBMC to all classes of CpG-ODNs led to activation and maturation of pDC based on CD86, CD80, and CD83 expression similar to that of cells from HIV-1, individuals. The percentage of CpG-ODN stimulated pDC that express CD40 was dramatically higher when cells were obtained from HIV-1+ than from HIV-1, individuals. B-lymphocytes were activated similarly in HIV-1+ and HIV-1, individuals. mDC, NK and iNKT cell, which lack TLR9, were indirectly activated. Interferon-, (IFN-,) and interferon inducible protein 10 (IP-10) secretion was induced by class A or C but not class B CpG-ODN, but the concentrations were less than those produced by HIV-1, PBMC. HIV-1 infected individuals have fewer innate effector cells that are chronically activated, but these cells can be further activated by CpG-ODN, which suggests that synthetic CpG-ODNs could be used to enhance the immune system in HIV-1 infected individuals. [source] Interleukin-16 inhibits interleukin-13 production by allergen-stimulated blood mononuclear cellsIMMUNOLOGY, Issue 1 2006Souad El Bassam Summary Expression of interleukin (IL)-16 is increased in bronchial mucosal biopsies of atopic asthmatics compared to normal controls. The functional significance of increased expression of IL-16 at sites of allergic inflammation is not yet clear. We have previously shown that IL-16 inhibits IL-5 secretion by allergen-stimulated peripheral blood mononuclear cells (PBMC). We investigated whether IL-16 inhibits the production of other T helper 2 cytokines, namely IL-13 and IL-4, by allergen-specific T cells. PBMC from ragweed-sensitive atopic subjects were stimulated with allergen extract for cytokine production in the presence or absence of rhIL-16. Production of cytokines was assessed by enzyme-linked immunosorbent assay and reverse transcription,polymerase chain reaction. To evaluate whether the modulatory effect of IL-16 on cytokine synthesis was mediated by interferon-, (IFN-,), IL-10, IL-12 or IL-18, allergen-stimulated PBMC were cultured in presence of IL-16 and neutralizing concentrations of relevant antibodies. Allergen-stimulated PBMC produced significantly elevated levels of IL-13 (90,740 pg/ml) as compared to unstimulated PBMC (0,375 pg/ml, P < 0·01). Addition of rhIL-16 resulted in down-regulation of IL-13 mRNA expression as well as significantly reduced amounts of IL-13 released by allergen-stimulated PBMC (0,457 pg/ml, P < 0·001), as observed for IL-5. No effect of IL-16 was observed on IL-4 mRNA expression. Treatment with IL-16 resulted in increased levels of IL-10 and IL-18 in allergen-stimulated cell culture. Neutralization of IFN-,, IL-12, IL-10 or IL-18 did not alter the inhibitory effects of IL-16 on IL-13 and IL-5 secretion by allergen-stimulated PBMC. IL-16 did not modify IL-13 synthesis by anti-CD3-stimulated CD4+ T cells, but it significantly reduced the production of IL-5. These data suggest that IL-16 may play an important immunoregulatory role in allergic states in response to allergen. [source] Increased expression of mRNA encoding interleukin (IL)-4 and its splice variant IL-4,2 in cells from contacts of Mycobacterium tuberculosis, in the absence of in vitro stimulationIMMUNOLOGY, Issue 4 2004Helen A. Fletcher Summary Expression of interleukin (IL)-4 is increased in tuberculosis and thought to be detrimental. We show here that in healthy contacts there is increased expression of its naturally occurring antagonist, IL-4delta2 (IL-4,2). We identified contacts by showing that their peripheral blood mononuclear cells (PBMC) released interferon (IFN)-, in response to the Mycobacterium tuberculosis -specific antigen 6 kDa early secretory antigenic target (ESAT-6). Fresh unstimulated PBMC from these contacts contained higher levels of mRNA encoding IL-4,2 (P=0·002) than did cells from ESAT-6 negative donors (noncontacts). These data indicate that contact with M. tuberculosis induces unusual, previously unrecognized, immunological events. We tentatively hypothesize that progression to active disease might depend upon the underlying ratio of IL-4 to IL-4,2. [source] Dihomo-,-linolenic acid inhibits tumour necrosis factor-, production by human leucocytes independently of cyclooxygenase activityIMMUNOLOGY, Issue 3 2003Maaike M. B. W. Dooper Summary Dietary oils (such as borage oil), which are rich in ,-linolenic acid (GLA), have been shown to be beneficial under inflammatory conditions. Dihomo-GLA (DGLA) is synthesized directly from GLA and forms a substrate for cyclooxygenase (COX) enzymes, resulting in the synthesis of lipid mediators (eicosanoids). In the present study, the immunomodulatory effects of DGLA were investigated and compared with those of other relevant fatty acids. Freshly isolated human peripheral blood mononuclear cells (PBMC) were cultured in fatty acid (100 µm)-enriched medium for 48 hr. Subsequently, cells were stimulated with lipopolysaccharide (LPS) for 20 hr and the cytokine levels were measured, in supernatants, by enzyme-linked immunosorbent assay (ELISA). Phospholipids were analysed by gas chromatography. Fatty acids were readily taken up, metabolized and incorporated into cellular phospholipids. Compared with the other fatty acids tested, DGLA exerted pronounced modulatory effects on cytokine production. Tumour necrosis factor-, (TNF-,) and interleukin (IL)-10 levels were reduced to 60% of control levels, whereas IL-6 levels were not affected by DGLA. Kinetic studies showed that peak levels of TNF-,, occurring early after LPS addition, were inhibited strongly, whereas IL-10 levels were not affected until 15 hr after stimulation. Both the reduction of cytokine levels and the decrease in arachidonic acid levels in these cells, induced by DGLA, were dose dependent, suggesting a shift in eicosanoid-subtype synthesis. However, although some DGLA-derived eicosanoids similarly reduced TNF-, levels, the effects of DGLA were probably not mediated by COX products, as the addition of indomethacin did not alter the effects of DGLA. In conclusion, these results suggest that DGLA affects cytokine production by human PBMC independently of COX activation. [source] | ||||||||||||||||||||||||||||||||||||||||