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Healthy Donors (healthy + donor)
Kinds of Healthy Donors Selected AbstractsA novel gene, MDS2, is fused to ETV6/TEL in a t(1;12)(p36.1;p13) in a patient with myelodysplastic syndromeGENES, CHROMOSOMES AND CANCER, Issue 1 2002María D. Odero ETV6/TEL is the first transcription factor identified that is specifically required for hematopoiesis within the bone marrow. This gene has been found to have multiple fusion partners of which 16 have been cloned. Fluorescence in situ hybridization (FISH) analysis in a patient with myelodysplastic syndrome (MDS) revealed a t(1;12)(p36;p13) involving ETV6, with the breakpoint in this gene between exon 2 and exon 3. We report here the cloning of a novel ETV6 partner located on 1p36.1, involved in the t(1;12). 3, RACE-PCR from RNA identified a novel sequence fused to exon 2 of ETV6. Database searches localized this sequence in a bacterial artificial chromosome (BAC) mapped to 1p36 by fingerprint analysis. This result was confirmed by FISH using this BAC as probe. 5, and 3, RACE experiments with primers from this novel sequence were carried out on RNA from a healthy donor and identified a novel full-length mRNA, which we named MDS2 (myelodysplastic syndrome 2). RT-PCR experiments were performed on a panel of human cDNAs to analyze the expression pattern of this gene and they revealed four splicing variants. RT-PCR analysis showed that ETV6-MDS2, but not the reciprocal MDS2-ETV6 fusion transcript, was expressed in the bone marrow of the patient. The product of the ETV6-MDS2 fusion transcript predicts a short ETV6 protein containing the first 54 amino acids of ETV6 plus four novel amino acids, lacking both the PTN and the DNA-binding domains. Possible mechanisms to account for the development of MDS in this patient are discussed. © 2002 Wiley-Liss, Inc. [source] High sensitivity of chemiluminescent methodology for detection of clonal CDR3 sequences in patients with acute lymphoblastic leukemiaHEMATOLOGICAL ONCOLOGY, Issue 2 2004E. Leal Abstract Detection of minimal residual disease (MRD) in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been achieved using several radioactive labelling methodologies; however, limited information exists about the use of chemiluminescent labelling. Although many malignant disorders are related to cytogenetic alterations, there is not a consistent chromosomal translocation that could serve as a tumour marker for the monitoring of MRD. ALL are derived from B-lymphocytes in 80% of cases. In the early stages of their maturation, the immunoglobulin heavy chain genes (IgH) undergo rearrangements among their V, D, and J segments, giving rise to the Complementary Determining Regions (CDR). Among these, CDR3 is considered unique for each lymphocyte and used as a tumour-specific marker in B-ALL patients. In this study, the CDR3 was labelled with digoxigenin and used as a patient-specific probe to test its sensitivity for further detection of MRD. Fourteen pretreatment samples of bone marrow (BM) or peripheral blood (PB) from B-ALL patients were included. Tumour-specific probes were designed from each clonal product by elimination of the consensus sequences. Ten digoxigenin-labelled probes were hybridized with a mixture of their respective clonal DNA and the polyclonal product from a normal healthy donor, in serial dilutions from 1:1 up to 1:107. A sensitivity range of 1:103,1:106 was obtained, with an average of 1:105. Crossed tests performed in four patients, showed right probe specificity in all cases. We propose that the design of allele-specific probes with chemiluminescent labelling, represents a reliable, sure and sensitive alternative methodology for MRD detection in patients with B-cell lymphoproliferative disorders. Copyright © 2004 John Wiley & Sons, Ltd. [source] Phage display of human antibodies from a patient suffering from coeliac disease and selection of isotype-specific scFv against gliadinIMMUNOLOGY, Issue 2 2003Claudio Rhyner Summary Coeliac disease (CD), a gastrointestinal illness characterized by intestinal malabsorption, results from gluten intolerance accompanied with immunological responses towards gliadin, an ethanol-soluble protein fraction of wheat and other cereals. The role of gliadin in eliciting immune responses in CD is still partly unclear; however, the occurrence of anti-gliadin in the sera of patients suffering from CD correlates well with clinical symptoms. In this work we report the construction of isotype-specific, phage-displayed scFv libraries from peripheral blood lymphocytes of a patient with CD and from a healthy control individual. VH and VL chains were amplified by reverse transcription,polymerase chain reaction (RT,PCR) using a set of oligonucleotides recognizing all human variable gene families. The three scFv libraries (IgA, IgG and IgM) were selectively enriched for gliadin-binding phage. After four rounds of affinity selection, polyclonal enrichment of gliadin-binding phage was observed in all libraries from the CD patient but in none from the healthy donor. Phagemid particles generated from single clones were demonstrated to be gliadin-specific, as shown by strongly positive enzyme-linked immunosorbent assay (ELISA) and BiaCore signals. The VH and VL chains from samples of these monoclonal isotype-specific phage were sequenced to identify the most common variable regions used by the immune system to elicit antibody responses against gliadin. [source] Simultaneous pancreas and kidney transplantation from organ donation after cardiac deathANZ JOURNAL OF SURGERY, Issue 4 2009Nancy Suh Abstract Background:, The concept of organ donation after cardiac death (DCD) historically precedes the current practice of organ procurement from heartbeating donors meeting the brainstem death criteria. DCD has not gained widespread interest, however, due partly to initial fears that transplantation of such organs leads to suboptimal outcome. Methods:, Available data on long-term outcomes following simultaneous pancreas and kidney transplant (SPK) from DCD donors were reviewed, and it was found that the long-term outcome is comparable to SPK from heartbeating donors. Australia's first SPK from a DCD donor was performed. Results:, The patient received a kidney and a pancreas from a young healthy donor after cardiac death, and at the time of writing was well with functioning grafts. Conclusion:, SPK from donation after cardiac death is safe and should continue to be available for patients in need. [source] Granulocyte colony-stimulating factor-induced Sweet syndrome in a healthy donorBRITISH JOURNAL OF HAEMATOLOGY, Issue 2 2006Naoki Oiso No abstract is available for this article. [source] Flow cytometric differential of leukocyte populations in normal bone marrow: Influence of peripheral blood contamination1,CYTOMETRY, Issue 1 2009R. A. Brooimans Abstract Background: Availability of immunophenotypic reference values for the various leukocyte populations distributed in bone marrow may be helpful to recognize abnormal bone marrow development and, therefore, useful as first screening of individuals with suspected hematological malignancies or other hematopoietic disorders. Methods: A single tube four-color staining panel (CD66abce/CD14/CD45/CD34) together with a predefined gating strategy was utilized to immunologically differentiate the distribution of the major leukocyte populations in bone marrow aspirates of healthy donors. The sample-blood erythrocyte ratio was applied to assess the amount of blood contamination of marrow and account for this in the marrow value estimates. Results: The frequency of the major leukocyte populations in bone marrow of 134 normal donors were for granulocytes: mean, 69.4%; SD, 10.3%; monocytes: mean, 4.7%; SD, 2.3%; lymphocytes: mean, 18.3%; SD, 8.7%. The frequency of the immature cell population that included precursor cells of each of the cell lineages among other cell types were mean 5.0%; SD 2.2%. The mean percentage of CD34 positive cells was 1.5%; SD 0.7%. Our results showed further that the frequency of cell populations, of which the presence is restricted to the bone marrow (e.g., CD34+ progenitor cells), is influenced by the degree of peripheral blood admixture. Between the total immature cells and purity of the bone marrow, there was a significant positive correlation demonstrated, whereas a negative correlation was found between the percentages of both lymphocytes as monocytes and the purity of the bone marrow. Conclusions: With a single tube-staining panel, we obtained reference values for flow cytometric assessment of all relevant leukocyte populations present in bone marrow that can be used as a frame of reference for better recognition of individuals with abnormal hematopoiesis. In addition, we have demonstrated the influence of the degree of peripheral blood admixture in the bone marrow aspirates on those reference values. © 2008 Clinical Cytometry Society [source] Performance of calibration standards for antigen quantitation with flow cytometry in chronic lymphocytic leukemiaCYTOMETRY, Issue 6 2007Eva D. Rossmann Abstract Background: The fluorescence intensities of CD3, CD4 on T cells and CD20, CD22 molecules on B cells were quantitatively measured on lymphocytes from chronic lymphocytic leukemia (CLL) patients and healthy donors. Methods: The performance of three different types of microbeads was compared, i.e. Quantum molecules of equivalent soluble fluorochrome (Q-MESF), Quantum simply cellular (QSC), and QuantiBRITEÔ (QB). As all PE-conjugates had a F/P ratio of 1:1, the MESF units represented also the antibody binding capacity (ABC). Results: The ABCs of CD4 and CD20 antigens estimated with QSC (ABCQSC) were higher than those assigned with QB (ABCQB) with an average difference 49%. Higher numbers of antigenic sites were obtained with Q-MESF than with QSC for CD20 antigen. On the contrary, CD4 antigenic sites numbers estimated with QSC were higher than those estimated with Q-MESF. ABC values estimated with Quantum MESF PE (ABCQ-MESF) were ,15% higher than ABCQSC, whereas ABCQ-MESF was ,49% higher than ABCQB. Statistically significant correlations were found between the values obtained using various standards. The present study is the first to report down-regulation of CD3 antigen on T cells from patients with CLL. Conclusions: This study emphasizes the relevance of quantitative measurement of fluorescence intensity by flow cytometry as a standardized approach to measure and interpret the expression of some CLL markers and reduce variability of results obtained at different sites in multi-center clinical studies. © 2007 Clinical Cytometry Society [source] Cells meeting our immunophenotypic criteria of endothelial cells are large plateletsCYTOMETRY, Issue 2 2007Michiel H. Strijbos Abstract Background Circulating endothelial cells (CEC) are shed from damaged vasculature, making them a rational choice to serve as surrogate marker for vascular damage. Currently, various techniques and CEC definitions are in use, and their standardization and validation is needed. A flow cytometric single platform assay defining CEC as forward light scatter (FSC)low-to-intermedate, sideward light scatter (SSC)low, CD45,, CD31++ and CD146+ is a promising approach to enumerate CEC because of its simplicity (Mancuso et al., Blood 2001;97:3658,3661). Here, we set out to confirm the endothelial nature of these cells. Methods We isolated cells with a FSClow-to-intermediate, SSClow, CD31++, CD45dim immunophenotype (termed "cells meeting our immunophenotypic criteria for endothelial cells" [CMOIC]) from healthy donors to study the expression of endothelium-associated markers using several techniques. Special attention was paid to reagents identifying the endothelial cell-specific marker CD146. We compared antigen expression patterns of CMOIC with those of the HUVEC endothelial cell line and lymphocytes. Electron microscopy was used to detect the presence of endothelial cell-specific Weibel,Palade bodies in the sorted cells. Results CD146 expression was negative on CMOIC for all tested CD146 mAbs, but positive on HUVEC cells and a minor subset of T lymphocytes. Using flow cytometry, we found no expression of any endothelium-associated marker except for CD31 and CD34. HUVEC cells were positive for all endothelial markers except for CD34. Evaluation of CMOIC morphology showed a homogenous population of cells with a highly irregular nucleus-like structure and positive endothelial immunohistochemistry. CMOIC contained neither nuclei nor DNA. Electron microscopy revealed the absence of a nucleus, the absence of endothelial specific Weibel,Palade bodies, and revealed CMOIC to be large platelets. Conclusion The vast majority of cells with the immunophenotype FSClow-to-intermediate, SSClow, CD45,, CD31++ do not express CD146 and are large platelets rather than endothelial cells. © 2007 Clinical Cytometry Society. [source] Increased immature hematopoietic progenitor cells CD34+/CD38dim in myelodysplasiaCYTOMETRY, Issue 2 2006Mariela B. Monreal Abstract Background Myelodysplastic syndromes (MDS) are clonal disorders affecting hematopoietic progenitor cells (HPC). Despite the relevance of clonal CD34+ cells in developing MDS, only few studies analyze the phenotype of this cell population. The aim of this study was to evaluate phenotypic changes on HPC in MDS that could reflect abnormalities in the differentiation process of stem cells. Methods We analyzed the expression of CD38 and HLA-DR on CD34+ cells by flow cytometry in 36 patients with MDS, as well as in healthy donors (n = 12) and patients with other hematological disorders: non-Hodgkin lymphomas and multiple myeloma, both in complete remission (CR) (n = 32); acute lymphoblastic leukemia in CR (n = 17); de novo acute myeloblastic leukemia (AML) at diagnosis (n = 22) and in CR (n = 37); and AML secondary to MDS at diagnosis (n = 19). Cases with available karyotype were grouped according to the International Prognostic Scoring System (IPSS). Results Compared to normal BM, the fraction of immature HPC, characterized as CD34+bright, intermediate FSC/SSC, and CD38dim, was significantly increased in high risk MDS and secondary AML, but not in low risk MDS, (P , 0.001, P = 0.03, and P = 0.7). De novo AML showed decreased immature HPC. High numbers of immature HPC correlated with higher IPSS risk groups (P = 0.05) and showed significant impact on disease progression (P = 0.03). Conclusion Our study confirms that evaluation of CD38 expression pattern on HPC is an easy and reproducible test that allows evaluating the immature subset of progenitor cells. Increased immature HPC in high risk MDS and secondary AML may reflect blocked differentiation of CD34+ cells in these diseases. © 2006 International Society for Analytical Cytology [source] 22-ene-25-oxa-vitamin D: a new vitamin D analogue with profound immunosuppressive capacitiesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 5 2005C. Daniel Abstract Background, The biologic role of 1,25-dihydroxyvitamin D3, such as anti-inflammatory functions, reduction of cytokine production by T cells and immunoglobulin production by B cells, is well established. However, its clinical use as an immunosuppressive agent is limited because of the hypercalcemic toxicity occurring after systemic application. The purpose of this study was to investigate the immunmodulatory effects of 22-ene-25-oxa-vitamin D (ZK156979), a novel low calcemic vitamin D analogue. Materials and methods, Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated using the Ficoll Hypaque technique, cultured for 24 h and treated with different concentrations of ZK156979 ranging from 10,5 to 10,10 mol L,1 compared with 1,25-dihydroxyvitamin D3[10,5,10,10 mol L,1] following phytohaemagglutinin (PHA) stimulation. Interferon gamma (IFN,), tumour necrosis factor alpha (TNF,), interleukin 1 beta (IL-1,), interleukin 10 (IL-10) and interleukin 4 (IL-4) secretion in supernatants were measured by ELISA. Results, ZK156979 inhibited the PHA-induced Th1-response (IFN, and TNF, levels) and the macrophage-product IL-1, in a concentration-dependent manner (10,10,10,5 mol L,1) with the efficiency on cytokine expression compared with 1,25-dihydroxyvitamin D3 being slightly reduced. In contrast, ZK156979 and 1,25-dihydroxyvitamin D3 both affected the Th2 response, leading to significantly increased IL-10- and IL-4 secretion. Conclusions, ZK156979 is a member of novel vitamin D analogues revealing prominent immunomodulatory and suppressive characteristics with distinctive inhibition of Th1-cytokines whereas the Th2 compartment is augmented, thus providing a considerable therapeutic potential in T-cell -mediated diseases. [source] Retinol binding protein isolated from acute renal failure patients inhibits polymorphonuclear leucocyte functionsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 11 2004G. Cohen Abstract Background, Protein factors accumulating in sera of patients with end-stage renal disease (ESRD) that interfere with the nonspecific immune response by inhibiting essential functions of polymorphonuclear leucocytes (PMNLs) have previously been described. No such factor has been isolated from acute renal failure (ARF) patients to date. Materials and methods, Using a three-step chromatographic procedure involving ion exchange, size exclusion and hydrophobic interaction chromatography we purified the apo- and holo-form of retinol binding protein (RBP) from high-flux dialyser (polyacrylonitrile; AN69) ultrafiltrates of patients with ARF. Their effect on the chemotaxis of PMNLs isolated from healthy donors was determined by the under-agarose method. Whole-blood assays applying flow cytometry were used to assess phagocytosis and the oxidative metabolism of PMNLs. Apoptosis was assessed by determining the DNA content using propidium iodide. Results, Isolated apo- and holo-forms of RBP were truncated on their C-terminus as determined by mass spectrometry. All isolates significantly inhibited the chemotactic movement of PMNLs obtained from healthy donors and the PMNL oxidative metabolism stimulated by E. coli. These effects were concentration dependent. Retinol binding protein had no influence on the PMNL oxidative metabolism stimulated by PMA and on PMNL phagocytosis. Commercially available RBP isolated from urine influenced PMNL functions in the same way. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 significantly attenuated the phagocytosis-induced respiratory burst and RBP did not lead to a further decrease. Polymorphonuclear leucocyte apoptosis was significantly inhibited by RBP. Conclusions, The apo- and holo-forms of RBP isolated from the ultrafiltrate of ARF patients inhibit PMNL chemotaxis, oxidative metabolism and apoptosis. Therefore, RBP may be considered a uraemic toxin contributing to a disturbed immune defence. [source] Resveratrol modulates apoptosis and oxidation in human blood mononuclear cellsEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 9 2003G. A. Losa Abstract Background, We examined the effect of resveratrol (RS), a nonflavonoid polyphenolic phytoalexin found in grapes and red wine, and RS coincubated with the oxidant 2-deoxy-D-ribose (dR), on apoptosis and on the oxidative metabolic status of normal human peripheral blood mononuclear cells (PBMNCs) isolated ex vivo from healthy donors. Material and methods, Apoptosis was measured by changes of membrane permeability to propidium iodide (PI), plasma membrane exposure of phosphatidylserine (PS) and intracellular caspase activity. Oxidative status was assessed by recording the intracellular glutathione concentration (GSH), the activities of the enzymes y -glutamyltransferase (y- GT) and glutathione-S-transferase (GST), and intracellular lipid peroxidation (MDA). Results, Neither apoptotic nor oxidative parameters were affected by culturing PBMNCs in medium containing RS up to 20 µM for 5 days, while the frequency of cells with intermediate permeability to PI (17% ± 5) increased at 50 µM of RS. Thus resveratrol was slightly toxic, but there was little apoptosis in these cells. Peripheral blood mononuclear cells were also grown first in medium plus RS for 24 h and then for 96 h in medium containing RS plus 10 mM of dR, an oxidant sugar that is apoptogenic for human lymphocytes. The apoptotic changes triggered by dR were counteracted by the phytoalexin in a dose-dependent manner, but RS activity was absent at the lowest concentration (5 µM) and significantly reduced at the highest concentration used (50 µM). In PBMNCs coincubated with 20 µM of RS and 10 mM of dR the antioxidant effect of RS manifested with a significant reduction of caspases-3, -8, y- GT, GST activities and MDA content. Conclusions, Peripheral blood mononuclear cells acquire antioxidant capacity when treated with RS. Grape resveratrol may make a useful dietary supplement for minimizing oxidative injury in immune-perturbed states and human chronic degenerative diseases. [source] Quantitative, phenotypic, and functional evaluation of basophils in myelodysplastic syndromesEUROPEAN JOURNAL OF CLINICAL INVESTIGATION, Issue 10 2001W. Füreder Background The myelodysplastic syndromes (MDS) are a group of clonal haematological disorders characterized by cytopenia(s), reduced differentiation-capacity of myeloid cells, and impaired leukocyte function. However, little is known so far about basophil granulocytes in MDS. Design We have compared the numbers, phenotype and function of basophils in MDS patients with those in healthy subjects. A total numer of 23 patients with MDS (refractory anaemia, n = 8; refractory anaemia with ringsideroblasts, n = 7; refractory anaemia with excess of blasts/refractory anaemia with excess of blasts in transformation, n = 8) and 20 healthy donors were included. Results The numbers of blood basophils in MDS patients (34·6 ± 62·9 ,L,1) was lower compared to healthy controls (58·6 ± 64·9 ,L,1). Correspondingly, whole blood histamine levels were lower in MDS patients (MDS 34·1 ± 29·1 ng mL,1 vs. normal donors 72·0 ± 36·9 ng mL,1). Like ,normal' basophils, basophils in MDS expressed interleukin-3 receptor , (CD123), E-NPP3 (CD203c), CR1 (CD35), CR3 (CD11b), CR4 (CD11c), membrane co-factor protein (CD46), decay-accelerating factor (CD55) and membrane attack complex inhibitory factor (CD59), as well as receptors for C3a, C5a (CD88), and IgE. Recombinant human (rh) C5a and anti-IgE induced significant release of histamine from basophils in both groups of donors without significant differences between MDS and healthy controls. Conclusions The absolute numbers of basophils in MDS patients are lower than in normal donors. However, basophils in MDS do not differ from their ,normal counterparts' in terms of complement receptor expression, IgE-receptor expression, or functional responses to respective ligands. [source] Memory B cells from a subset of treatment-naïve relapsing-remitting multiple sclerosis patients elicit CD4+ T-cell proliferation and IFN-, production in response to myelin basic protein and myelin oligodendrocyte glycoproteinEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2010Christopher T. Harp Abstract Recent evidence suggests that B- and T-cell interactions may be paramount in relapsing-remitting MS (RRMS) disease pathogenesis. We hypothesized that memory B-cell pools from RRMS patients may specifically harbor a subset of potent neuro-APC that support neuro-Ag reactive T-cell proliferation and cytokine secretion. To test this hypothesis, we compared CD80 and HLA-DR expression, IL-10 and lymphotoxin-, secretion, neuro-Ag binding capacity, and neuro-Ag presentation by memory B cells from RRMS patients to naïve B cells from RRMS patients and to memory and naïve B cells from healthy donors (HD). We identified memory B cells from some RRMS patients that elicited CD4+ T-cell proliferation and IFN-, secretion in response to myelin basic protein and myelin oligodendrocyte glycoprotein. Notwithstanding the fact that the phenotypic parameters that promote efficient Ag presentation were observed to be similar between RRMS and HD memory B cells, a corresponding capability to elicit CD4+ T-cell proliferation in response to myelin basic protein and myelin oligodendrocyte glycoprotein was not observed in HD memory B cells. Our results demonstrate for the first time that the memory B-cell pool in RRMS harbors neuro-Ag specific B cells that can activate T cells. [source] Frequent occurrence of high affinity T cells against MELOE-1 makes this antigen an attractive target for melanoma immunotherapyEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2010Yann Godet Abstract We recently showed that the infusion of tumor infiltrating lymphocytes specific for the MELOE-1 antigen was associated with a prolonged relapse-free survival for HLA-A2+ melanoma patients who received tumor infiltrating lymphocytes therapy. Here, we characterized the MELOE-1/A2-specific T-cell repertoire in healthy donors and melanoma patients to further support an immunotherapy targeting this epitope. Using tetramer enrichment followed by multicolor staining, we found that MELOE-1-specific T cells were present in the blood of healthy donors and patients at similar frequencies (around 1 in 1×105 CD8+ cells). These cells mainly displayed a naïve phenotype in 4/6 healthy donors and 3/6 patients, whereas high proportions of memory cells were observed in the remaining individuals of both groups. There was a recurrent usage of the V,12.1 chain for 17/18 MELOE-1-specific T-cell clones derived from healthy donors or patients, associated with diverse V, chains and V(D)J junctional sequences. All clones derived from melanoma patients (9/9) were reactive against the MELOE-136,44 peptide and against HLA-A2+ melanoma cell lines. This study documents the existence of a large TCR repertoire specific for the MELOE-1/A2 epitope and its capacity to give rise to antitumor CTL that supports the development of immunotherapies targeting this epitope. [source] Differential capacity of T cell priming in naive donors of promiscuous CD4+ T cell epitopes of HCV NS3 and Core proteinsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 6 2007Florence Abstract To understand the inter-individual and virus-independent variability of CD4+ T cell responses to HCV components, we evaluated the effect on these responses of HLA II molecules in uninfected healthy donors. Using HLA II-specific binding assays, we identified, in the Core and NS3 proteins, 21 long fragments and 24 15-mer peptides that bound to four to eight of the most preponderant HLA II molecules. We then evaluated the priming capacity of eight long promiscuous peptides in 12 HLA-unrelated healthy donors. The NS3 1250,1264 peptide primed T cells in all the naive donors, while five others were stimulating in at least half of the individuals. We also report sequences that bind to multiple HLA II molecules but are weakly immunogenic. We therefore conclude that (i) broad HLA II specificity is only a prerequisite for a peptide to be stimulating in multiple individuals, and (ii) promiscuous peptides widely differ in their capacity to prime CD4+ T cells from uninfected healthy donors. We suggest that these priming differences result from inter-individual variations in the peptide-specific T cell repertoire. Interestingly, five of the most immunogenic peptides we identified correspond to frequently targeted T cell epitopes in infected patients. [source] Comparative analysis of NK cell subset distribution in normal and lymphoproliferative disease of granular lymphocyte conditionsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 10 2004Véronique Pascal Abstract We have characterized the heterogeneity of human blood NK cell subsets defined by expression of KIR, lectin like receptors and NK cell differentiation markers within a cohort of 51 healthy Caucasian individuals. High inter-individual variability in cell surface expression of most NK cell markers is observed. Range values defining NK cell subsets in healthy donors were further used as references to characterize 14 patients with NK-type lymphoproliferative disease of granular lymphocytes (NK-LDGL). Alterations of the KIR repertoire were noted in all NK-LDGL patients. NK cell expansions were classified as oligoclonal KIR+ or as non-detectable KIR (ndKIR) using anti-KIR2DL1/2DS1, anti-KIR2DL2/2DL3/2DS2, anti-KIR3DL1 and anti-KIR2DS4 monoclonal antibodies. A major reduction in the size of the CD56bright NK cell subset was a constant feature of NK-LDGL. Altered distribution of CD94+, CD161+, and CD162R+ NK cell subsets was also observed in NK-LDGL patients. Considering the potential role of NK cells in eliminating tumors or virus-infected cells, the reference values defined in this study should be valuable to characterize both quantitative and qualitative alterations of the NK cell repertoire in pathological conditions and to monitor NK cell reconstitution following hematopoietic transplantation. [source] Alpha B-crystallin is not a dominant peripheral T-cell autoantigen in multiple sclerosis amongst SardiniansEUROPEAN JOURNAL OF NEUROLOGY, Issue 5 2003S. Sotgiu The heat shock protein alpha B-crystallin appears to be the dominantly recognized autoantigen in the early demyelinative process of multiple sclerosis (MS) in brain of patients. In Sardinia, MS is linked to human leucocyte antigen (HLA)-DR alleles that might influence the production of cytokines from peripheral lymphocytes. We tested the nature of peripheral anti-alpha B-crystallin-specific T-cell response in the context of predisposing HLA haplotypes both in MS patients and healthy controls. The alpha B-crystallin specific T-cell lines were generated by using the ,split-well' technique. The results indicate that the presence of short-term T-cell lines towards alpha B-crystallin is numerically comparable between the two groups and not restricted to MS-predisposing HLA-DR alleles. As for the T-cell characterization, CD4+ anti-alpha B-crystallin T cells secreting high levels of interferon- , are similarly identified in MS and healthy donors. In conclusion, the peripheral response towards the myelin antigen alpha B-crystallin is neither quantitatively nor qualitatively peculiar to MS, in contrast to the theoretical paradigm suggesting peripheral activation of myelin-reactive T cells to be the prerequisite for MS induction. [source] Human melanocytes can be isolated, propagated and expanded from plucked anagen hair folliclesEXPERIMENTAL DERMATOLOGY, Issue 6 2010Christina Dieckmann Please cite this paper as: Human melanocytes can be isolated, propagated and expanded from plucked anagen hair follicles. Experimental Dermatology 2010; 19: 543,545. Abstract:, Herein, we report a technically simple method for isolation and culture of human follicular melanocytes based on explant cultures of epilated hair follicles. This technique does not require any surgical intervention and allows the isolation and cultivation of follicular melanocytes from a comparatively small amount of raw material. Generally, 30,60 human anagen hair follicles have been plucked from the scalp of healthy donors and cultivated under low oxygen pressure (5%). After a short period of time cells of various types were growing out from the outer root sheath (ORS) of the hair follicles. Under the selected culture conditions, most of the cells other than melanocytes have been eliminated and a nearly 100% pure population of melanocytes has been achieved, as confirmed by immunohistochemical analyses for melanocyte-specific markers, for example, Tyrosinase-1, S-100 and premelanosomal antigens. These melanocytes derived from the ORS were proliferating for up to 2 months. [source] Deficient translocation of c-Rel is associated with impaired Th1 cytokine production in T cells from atopic dermatitis patientsEXPERIMENTAL DERMATOLOGY, Issue 1 2005Karsten Dieckhoff Abstract:, Decreased production of T helper type 1 (Th1) cytokines, such as interferon-, (IFN-,) or interleukin-2 (IL-2), is a hallmark of atopic diseases. While accessory signals from antigen-presenting cells may be missing, T cells themselves may be suppressed in their ability to produce substantial amounts of Th1 cytokines. We show, in this study, that T cell receptor (TCR)-activated T cells from atopic dermatitis (AD) patients proliferate less than control T cells and produce lower amounts of IFN-, and IL-2, but comparable amounts of IL-4. Because mice lacking the nuclear factor kappa B (NF-,B) transcription factors , p65 or c-Rel , show reduced Th1, but undisturbed Th2 responses, we investigated the role of c-Rel and p65 for Th1 cytokine production in T cells from healthy and severe AD patients. TCR-activated primary T cells from healthy donors treated with c-Rel antisense oligonucleotides produced lower levels of IL-2 and IFN-, and proliferated less efficiently than the corresponding control T cells. Moreover, transfection of primary T cells with c-Rel or p65 enhanced proliferation and production of IL-2 and IFN-,. Nuclear extracts of activated primary T cells from AD donors bound weakly to NF-,B-specific oligonucleotides, compared to extracts from healthy control T cells. Western blotting studies revealed that nuclear, but not cytosolic, extracts from T cells of AD patients lacked significant amounts of c-Rel and p65. T cell clones derived from AD patients failed to sufficiently translocate c-Rel and p65 into the nucleus following activation. Thus, impaired nuclear translocation of c-Rel and p65 may determine an impaired Th1 cytokine response in AD. [source] Infiltrating cells and related cytokines in lesional skin of patients with chronic idiopathic urticaria and positive autologous serum skin testEXPERIMENTAL DERMATOLOGY, Issue 5 2003M. Caproni Abstract:, In approximately one-third of patients with chronic idiopathic urticaria (CIU), autoantibodies against the high-affinity IgE receptor and/or against IgE can be detected and a wheal-and-flare response can be provoked by the intradermal injection of autologous serum (ASST). In this study we aimed to further characterize the inflammatory response observed in the subgroup of CIU patients with positive ASST and serum-evoked histamine-release in vitro from basophils in comparison with unaffected skin and healthy donors. An immunohistochemical analysis of infiltrating cells (CD4, MPO, EG1, EG2, tryptase), cytokines (IL-4, IL-5, IFN-,), chemokines and chemokine receptors (IL-8, CCR3, CXCR3), and adhesion molecules (ICAM-1, VCAM-1, ELAM-1) was performed on seven selected patients (four males and three females; median age: 45 years; range: 22,57) and five healthy donors. Cytokine evaluation was also performed in five psoriatic patients to obtain an additional control. In spontaneous wheals we observed an increased number of CD4+ T lymphocytes when compared with the controls, and an increased number of neutrophils and eosinophils, whereas mast cells did not show a significant variation. A significant expression for IL-4 and IL-5 could only be observed in lesional skin, while IFN-, showed a slight expression in the same site. Chemokine receptors CCR3 and CXCR3 did not show a defined polarized response in either lesional or unaffected skin. An increased expression of all cellular adhesion molecules (CAMs) studied was detected in spontaneous wheals. The lack of a significant difference in the expression of tryptase + mast cells, T lymphocytes, IL-8, CXCR3 and CCR3, a few CAMs between the lesional and unaffected skin of CIU patients suggests a wide immunological activation that involves not only lesional tissues, but possibly extends to the whole of the skin's immune system. [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] Identification of genes with abnormal expression changes in acute myeloid leukemiaGENES, CHROMOSOMES AND CANCER, Issue 1 2008Derek L. Stirewalt Acute myeloid leukemia (AML) is one of the most common and deadly forms of hematopoietic malignancies. We hypothesized that microarray studies could identify previously unrecognized expression changes that occur only in AML blasts. We were particularly interested in those genes with increased expression in AML, believing that these genes may be potential therapeutic targets. To test this hypothesis, we compared gene expression profiles between normal hematopoietic cells from 38 healthy donors and leukemic blasts from 26 AML patients. Normal hematopoietic samples included CD34+ selected cells (N = 18), unselected bone marrows (N = 10), and unselected peripheral bloods (N = 10). Twenty genes displayed AML-specific expression changes that were not found in the normal hematopoietic cells. Subsequent analyses using microarray data from 285 additional AML patients confirmed expression changes for 13 of the 20 genes. Seven genes (BIK, CCNA1, FUT4, IL3RA, HOMER3, JAG1, WT1) displayed increased expression in AML, while 6 genes (ALDHA1A, PELO, PLXNC1, PRUNE, SERPINB9, TRIB2) displayed decreased expression. Quantitative RT/PCR studies for the 7 over-expressed genes were performed in an independent set of 9 normal and 21 pediatric AML samples. All 7 over-expressed genes displayed an increased expression in the AML samples compared to normals. Three of the 7 over-expressed genes (WT1, CCNA1, and IL3RA) have already been linked to leukemogenesis and/or AML prognosis, while little is known about the role of the other 4 over-expressed genes in AML. Future studies will determine their potential role in leukemogenesis and their clinical significance. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat. © 2007 Wiley-Liss, Inc. [source] D324N single-nucleotide polymorphism in the FLT3 gene is associated with higher risk of myeloid leukemiasGENES, CHROMOSOMES AND CANCER, Issue 4 2006Susanne Schnittger Mutations within the FLT3 gene are of growing importance for classification, risk assessment, and therapeutic targeting of acute myeloid leukemia (AML). We analyzed 656 AML patients for a recently described single-nucleotide polymorphism (SNP) in the third immunoglobulin-like domain of the extracellular region of FLT3. The FLT3 D324N variant was present in 42 cases (6.4%), but it was not associated with a specific AML subtype and did not show an elevated leukocyte count, as do other FLT3 mutations. In remission samples, a 50% ratio of the normal to the D324N variant was detectable. Stably expressed in IL-3 dependent Ba/F3 cells, the D324N variant did not confer receptor autophosphorylation, factor independent growth, or increased resistance to apoptotic cell death in response to varying doses of FLT3 ligand. In 400 healthy donors, the FLT3 D324N variant was detected in 6 cases (1.5%) and segregated in a family. Thus, it was shown to be a polymorphism with a lower frequency in healthy controls than in patients with AML (P < 0.001). In addition, 21 of 234 CML (9.0%) and 7 of 155 ALL (4.5%) cases carried the FLT3 D324N. Our data suggest that the FLT3 D324N variant might be associated with a predisposition to different subtypes of leukemia. © 2005 Wiley-Liss, Inc. [source] Cell culture,produced hepatitis C virus does not infect peripheral blood mononuclear cells,HEPATOLOGY, Issue 6 2008Svetlana Marukian Hepatitis C virus (HCV) replicates primarily in the liver, but HCV RNA has been observed in association with other tissues and cells including B and T lymphocytes, monocytes, and dendritic cells. We have taken advantage of a recently described, robust system that fully recapitulates HCV entry, replication and virus production in vitro to re-examine the issue of HCV infection of blood cell subsets. The HCV replicase inhibitor 2,C-methyl adenosine was used to distinguish HCV RNA replication from RNA persistence. Whereas cell culture,grown HCV replicated in Huh-7.5 hepatoma cells, no HCV replication was detected in B or T lymphocytes, monocytes, macrophages, or dendritic cells from healthy donors. No blood cell subset tested expressed significant levels of Claudin-1, a tight junction protein needed for HCV infection of Huh-7.5 cells. A B cell line expressing high levels of Claudin-1, CD81, and scavenger receptor BI remained resistant to HCV pseudoparticle infection. We bypassed the block in HCV entry by transfecting HCV RNA into blood cell subsets. Transfected RNA was not detectably translated and induced high levels of interferon-,. Supernatants from HCV RNA,transfected macrophages inhibited HCV replication in Huh-7.5 cells. Conclusion: We conclude that multiple blocks prevent blood cells from supporting HCV infection. (HEPATOLOGY 2008;48:1843-1850.) [source] Quantitative analysis of human mitochondrial DNA using a real-time PCR assayHIV MEDICINE, Issue 3 2003K Gourlain Objectives Known for their ability to inhibit the human DNA polymerase-,, nucleoside analogues induce toxic effects on mitochondria ranging from increased serum lactate levels to fatal lactic acidosis. DNA polymerase-, ensures the mitochondrial DNA (mtDNA) replication and, thus, its inhibition leads to the decrease of the mtDNA. We describe a real-time PCR assay for mtDNA quantification associating DNA extraction procedures applied on peripheral blood mononuclear cells (PBMCs) and subcutaneous adipose tissues and to study the antiretroviral effect on mitochondria. Methods Total DNA was extracted from PBMCs and subcutaneous adipose tissues. Nuclear and mitochondrial genes were amplified to determine the number of copies of mtDNA per cell using a cyt-b recombinant plasmid as standard control. We analysed eight HIV-infected asymptomatic patients never treated, four patients who had been treated for 6 months with highly active antiretroviral therapy (HAART) and six non-infected donors. Results The mtDNA quantification gave rise to reproducible results as the mean coefficients of variation were 1.09% for replicates of samples undertaken 10 times within the same run, and 5.78% and 3.7% for replicates tested in five different runs at 1:100 and 1:1000 dilutions, respectively. Median levels of mtDNA in PBMCs of healthy donors, naive and treated HIV-infected patients were 2.94, 2.78 and 1.93 log HIV-1 RNA copies/mL, respectively. Whereas DNA from PBMCs was shown to be devoid of inhibitors, subcutaneous adipose tissues needed an extra treatment as they were found to be highly inhibited. Conclusions The method generated consistent and reproducible results and was successfully applied to DNAs extracted from PBMCs and subcutaneous adipose tissues with adapted extraction. The mtDNA changes in PBMCs were found to be fast as they fall off after 6 months' therapy, decreasing from 2.78 to 1.93 log copies/mL. [source] Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling,HUMAN MUTATION, Issue 6 2009Steven F. Dobrowolski Abstract Identifying mitochondrial DNA (mtDNA) sequence variants in human diseases is complicated. Many pathological mutations are heteroplasmic, with the mutant allele represented at highly variable percentages. High-resolution melt (HRM or HRMA) profiling was applied to comprehensive assessment of the mitochondrial genome and targeted assessment of recognized pathological mutations. The assay panel providing comprehensive coverage of the mitochondrial genome utilizes 36 overlapping fragments (301,658,bp) that employ a common PCR protocol. The comprehensive assay identified heteroplasmic mutation in 33 out of 33 patient specimens tested. Allele fraction among the specimens ranged from 1 to 100%. The comprehensive assay panel was also used to assess 125 mtDNA specimens from healthy donors, which identified 431 unique sequence variants. Utilizing the comprehensive mtDNA panel, the mitochondrial genome of a patient specimen may be assessed in less than 1 day using a single 384-well plate or two 96-well plates. Specific assays were used to identify the myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) mutation m.3243A>G, myoclonus epilepsy, ragged red fibers (MERRF) mutation m.8344A>G, and m.1555A>G associated with aminoglycoside hearing loss. These assays employ a calibrated, amplicon-based strategy that is exceedingly simple in design, utilization, and interpretation, yet provides sensitivity to detect variants at and below 10% heteroplasmy. Turnaround time for the genotyping tests is about 1,hr. Hum Mutat 30,1,8, 2009. © 2009 Wiley-Liss, Inc. [source] Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-GuérinIMMUNOLOGY, Issue 1 2004Semih Esin Summary The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3, cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-, (IFN-,)-producing cells were NK cells, with a peak IFN-, production at 24,30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16,20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-, production and cytotoxic activity, on negatively selected CD56+ CD3, cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections. [source] Identification of SPARC as a candidate target antigen for immunotherapy of various cancersINTERNATIONAL JOURNAL OF CANCER, Issue 6 2010Mitsuhiro Inoue Abstract To establish efficient anticancer immunotherary, it is important to identify tumor-associated antigens (TAAs) directing the immune system to attack cancer. A genome-wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC-derived CTL epitopes. We previously identified H-2Kd -restricted and SPARC-derived CTL epitope peptides in BALB/c mice, of which H-2Kd -binding peptide motif is comparable with that of HLA-A24 binding peptides. By using these peptides, we tried to induce HLA-A24 (A*2402)-restricted and SPARC-reactive human CTLs and demonstrated an antitumor immune response. The SPARC-A24-1143,151 (DYIGPCKYI) and SPARC-A24-4225,234 (MYIFPVHWQF) peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA-A24 (A*2402). Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy. [source] Reduced numbers of IL-7 receptor (CD127) expressing immune cells and IL-7-signaling defects in peripheral blood from patients with breast cancerINTERNATIONAL JOURNAL OF CANCER, Issue 7 2007Nalini Kumar Vudattu Abstract Interleukin-7-receptor-signaling plays a pivotal role in T-cell development and maintenance of T-cell memory. We studied IL-7R, (CD127) expression in PBMCs obtained from patients with breast cancer and examined IL-7 receptor-mediated downstream effects defined by STAT5 phosphorylation (p-STAT5). Reduced numbers of IL-7R,-positive cells were identified in CD4+ T-cells as well as in a CD8+ T-cell subset defined by CD8,/, homodimer expression in patients with breast cancer. PBMCs obtained from healthy donors (n = 19) and from patients with breast cancer (n = 19) exhibited constitutive p-STAT5 expression in the range of 0,6.4% in CD4+ T-cells and 0,4% in CD8+ T-cells. Stimulation with recombinant human IL-7 for 15 min increased p-STAT5 expression up to 36,97% in CD4+T-cells and to 26,90% in CD8+T-cells obtained from healthy control donors (n = 19). In contrast, PBMCs obtained from 13/19 patients with breast cancer did not respond to IL-7 as defined by STAT5 phosphorylation, despite expression of IL-7R, on T-lymphocytes. T-cells were further characterized for IL- 2 and IFN-, production induced by PMA/Ionomycin. PBMCs from 9/19 patients with breast cancer showed decreased IL-2 and IFN-, production combined with IL-7-signaling defects; PBMCs from 4 patients with breast cancer exhibited deficient IL-7-signaling, yet intact cytokine production. Reduced numbers of IL-7R,-positive cells and nonresponsiveness to IL-7, defined by lack of STAT5 phosphorylation, characterizes the immunological profile in T-cells from patients with breast cancer. © 2007 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||||||||