Biomedical subjects

M Schlesier

Publications and source records attributed to M Schlesier.

At least 19 recordsLinked to original sources

PubMed2005

Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.

The functional interaction of BAFF and APRIL with TNF receptor superfamily members BAFFR, TACI and BCMA is crucial for development and maintenance of humoral immunity in mice and humans. Using a candidate gene approach, we identified homozygous and heterozygous mutations in TNFRSF13B, encoding TACI, in 13 individuals with common variable immunodeficiency. Homozygosity with respect to mutations causing the amino acid substitutions S144X and C104R abrogated APRIL binding and resulted in loss of TACI function, as evidenced by impaired proliferative response to IgM-APRIL costimulation and defective class switch recombination induced by IL-10 and APRIL or BAFF. Family members heterozygous with respect to the C104R mutation and individuals with sporadic common variable immunodeficiency who were heterozygous with respect to the amino acid substitutions A181E, S194X and R202H had humoral immunodeficiency. Although signs of autoimmunity and lymphoproliferation are evident, the human phenotype differs from that of the Tnfrsf13b-/- mouse model.

Amino Acid Sequence↗

PubMed2005

Effects of of didanosine-related depletion of mtDNA in human T lymphocytes.

The normal metabolism of mitochondria in T lymphocytes is unknown, as are the effects from nucleoside-analogue reverse-transcriptase inhibitors that impair mitochondrial polymerase- gamma . We isolated peripheral-blood CD4 and CD8 T lymphocytes from 6 healthy men and stimulated them with anti-CD3 and anti-CD28 antibodies, in the presence and in the absence of didanosine (ddI). In the absence of ddI, mitosis of T lymphocytes was paralleled by a transient up-regulation of both mtDNA and production of lactate. In CD4 lymphocytes, 10-day incubation with ddI at concentrations of 11.8 mu mol/L, 35.4 mu mol/L, 59.0 mu mol/L, and 118.0 mu mol/L induced (1) a concentration-dependent reduction of both mtDNA (to 73%, 29%, 24%, and 23%, respectively, of the levels in control samples) and subunit II of mtDNA-encoded cytochrome c oxidase (to 86%, 81%, 55%, and 31%, respectively, of the levels in control samples) and (2) a concentration-dependent increase in production of lactate (to 139%, 222%, 276%, and 312%, respectively, of the levels in control samples). Activation of lymphocytes (which was measured in terms of expression of CD25) was unaffected. Mitochondrial depolarization (assessed by staining with JC-1) was observed as early as day 7 of incubation. All changes were time dependent and also were observed in isolated CD8 lymphocytes. Electron microscopy revealed enlarged mitochondria with vacuoles, inclusions, and reduced electron density. ddI at a concentration of 11.8 mu mol/L induced changes that bordered statistical significance. After stimulation, there was a wide range in the change of mtDNA content in lymphocytes. Therefore, mtDNA measurements in blood are not necessarily a marker for the mitochondrial toxicity of ddI. Nevertheless, ddI does lead to depletion of mtDNA in lymphocytes and to functional impairment.

Adult↗

PubMed2005

Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method.

Recent reports have described reduced populations of CD27+ memory B cells and increased percentages of undifferentiated B cells in peripheral blood of patients with common variable immunodeficiency (CVID). This work has prompted two attempts to classify CVID based on rapid flow cytometric quantification of peripheral blood memory B cells and immature B cells. Evidence to support the hypothesis that such in vitro B cell classification systems correlate with clinical subtypes of CVID is being sought. For the classification to be useful in routine diagnosis, it is important that the flow cytometric method can be used without prior separation of peripheral blood mononuclear cells (PBMC). We have examined 23 CVID patients and 24 controls, using both PBMC and whole blood, and find an excellent correlation between these methods. The reproducibility of the method was excellent. We classified the CVID patients by all three of the existing classifications, including secretion of immunoglobulin by B cells in vitro as described by Bryant, as well as the more recent flow cytometric classification methods. Only one patient changed classification as a result of using whole blood.

Adolescent↗

PubMed2004

Reduction of soluble complement receptor 2/CD21 in systemic lupus erythomatosus and Sjogren's syndrome but not juvenile arthritis.

A soluble form of the complement receptor CD21 (sCD21) is shed from the lymphocyte surface. The amount of sCD21 in serum may modulate immunity as sCD21 levels are correlated with several clinical conditions. We report here the serum levels of sCD21 in juvenile arthritis (JA), systemic lupus erythematosus (SLE) and Sjogren's syndrome (SS). Using enzyme-linked immunosorbent assay, we determined sCD21 levels in SLE, SS and JA patients. Mann-Whitney test for nonparametric two-tail P value was performed to obtain statistical significance. Cytometrical analysis of synovial fluid leucocytes of JA patients was done on a FACSsort. While sCD21 levels in SLE and SS are reduced to levels previously found in rheumatoid arthritis (RA), JA sCD21 levels were normal. sCD21 levels did not correlate with clinical parameters and immunophenotype of synovial cells. CD4 T cells in the synovium were almost all of the CD45RO memory type and 13 of 40 patients displayed synovial expansion of gammadeltaT cells. CD21 shedding in JA differs from RA/SS/SLE. JA sCD21 levels in synovial fluid are always lower compared to blood levels of the same patients. Analysis of JA synovial T cells indicates a T-cell driven response.

Arthritis, Juvenile↗

PubMed2002

Impaired up-regulation of CD70 and CD86 in naive (CD27-) B cells from patients with common variable immunodeficiency (CVID).

CVID is characterized by reduced serum levels of all switched immunoglobulin isotypes (IgG, IgA, IgE) predisposing patients to recurrent infections of their respiratory and gastrointestinal tract. Correspondingly, most CVID patients exhibit a severely decreased proportion of class switched memory B cells (CD19+CD27+IgD-IgM-IgG+ or IgA+) in their peripheral blood (CVID type I). We previously identified a subgroup of CVID patients showing a significantly reduced expression of CD86 and CD137 following activation in vitro of PBMC or purified B cells (CD19+) with anti-IgM plus IL-2. Here we extend our previous studies by asking whether highly purified, cell-sorted naive B cells show already an expression defect of B cell surface molecules relevant in activation (CD39, CD69), differentiation (CD24, CD27, CD38) or T-B interaction (CD25, CD70, CD86). We stimulated cell-sorted, naive B cells (CD19+CD27-IgM+IgDhighIgG-IgA-) from 10 CVID patients and 10 healthy controls for 4 days with anti-IgM plus IL-2 in the absence or presence of autologous CD4+ T cells and measured the expression of the referred surface molecules. Based on reduced or normal numbers of switched memory B cells the CVID patients had previously been classified into eight type I patients and two type II patients, respectively. Interestingly, only the molecules CD25, CD70 and CD86, all relevant in cognate T-B interaction, showed a significantly lower expression in naive B cells from CVID patients compared to controls. While coculture with autologous CD4+ T cells normalized the CD25 expression, CD70 and CD86 expression remained subnormal, notably in the eight CVID patients of type I. These findings strongly suggest an intrinsic signalling or expression defect for CD70/CD86 at the level of naive B cells in type I CVID patients.

Adult↗

PubMed2000

Impaired up-regulation of CD86 in B cells of "type A" common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is characterized by defective B cell maturation and antibody formation resulting in low serum antibody levels of most or all Ig isotypes. A specific subgroup of patients ("type A") has normal numbers of mature surface (s)IgM / sIgD- positive circulating B cells. However, since these lymphocytes do not respond to in vitro stimulation by differentiation and Ig synthesis, they seem to suffer from so far unknown intrinsic defects. Analyzing the expression pattern of a large set of B cell activation-specific surface markers, we found that type A CVID patients show a highly reduced expression of the CD28 / CTLA-4 ligand CD86 (B7-2) and of the lymphocyte activation marker CDw137 when compared to B cells of healthy donors and non-type-A CVID patients. The lowered CD86 expression levels were found to correlate with reduced levels of CD86 mRNA. Since combined stimulation via B cell antigen receptor and CD40 cross-linking did not rescue the defects in CD86 and CDw137 expression, B cells of CVID type A patients resemble functionally unresponsive lymphocytes incapable of cooperating with T cells. The fact that these cells accumulate in type A CVID patients suggests a causal relationship with the pathogenesis of this disease.

Adult↗

PubMed2000

Presence of IgE antibodies to bovine serum albumin in a patient developing anaphylaxis after vaccination with human peptide-pulsed dendritic cells.

Dendritic cells are professional antigen-presenting cells that can be generated in vitro either from monocytes or from CD34+ peripheral blood progenitor cells by using recombinant cytokines. These cells have potential implications for immunotherapeutic approaches in the treatment of cancer and other diseases. We have conducted a phase I study in melanoma patients using peptide-pulsed dendritic cells cultured in medium supplemented with 10% fetal calf serum (FCS) and a cocktail of cytokines. Peptide-pulsed dendritic cells were injected intravenously at 2-week intervals. Here we report on a case of type I hypersensitivity anaphylactic reaction after repetitive vaccination with autologous peptide-pulsed cells. Pre-vaccination and post-vaccination serum samples were evaluated for the presence of antibodies to FCS and bovine serum albumin (BSA). A retrospective study in 7 patients vaccinated with FCS-cultured dendritic cells demonstrated the presence of IgG and IgM antibodies to FCS and BSA after vaccination in 6 out of 7 patients. However, IgE antibodies were absent in all patients with the exception of the patient developing anaphylaxis. The patient's serum was demonstrated to contain a strong IgE response directed against BSA. In contrast, 2 patients vaccinated with dendritic cells cultured under serum-free conditions developed no antibodies to FCS and BSA after repetitive vaccination. We suggest that patients can be sensitized with an IgE response against BSA leading to anaphylactic reactions. On the basis of these data, dendritic cells cultured in autologous serum or under serum-free conditions are recommended for therapeutic applications in vivo.

Anaphylaxis↗

PubMed2000

Successful IL-2 therapy for relapsing herpes zoster infection in a patient with idiopathic CD4+ T lymphocytopenia.

Idiopathic CD4+ T lymphocytopenia (ICL) has been defined by the center of disease control as a rare cause of immunodeficiency with a variable clinical course and an unknown aetiology. Here we describe a 65-year old patient with relapsing generalized herpes zoster infection due to ICL and a severe panlymphocytopenia. In vitro assays revealed an enhanced activation of CD8+ T cells and an increased sensitivity of activated CD4+ T cells for cell death. The clinical outcome was substantially improved after starting the patient on a subcutaneous therapy with IL-2.

Adult↗

PubMed1999

Autoreactive human T cell lines recognizing ribosomal protein L7.

Sera of patients suffering from systemic lupus erythematosus (SLE) frequently contain oligoclonal IgG autoantibodies with high affinity for the ribosomal protein L7 (rpL7). The humoral autoimmune response to rpL7 apparently is driven by antigen and T cell dependent. In order to analyze the T cell response to rpL7 we cultured peripheral blood lymphocytes of healthy individuals and SLE patients in the presence of recombinant rpL7. After 10 days, the cytokine response to re-stimulation with rpL7 was examined using a spot-ELISA. Measuring IFN-gamma secretion, the T cells of two patients and four healthy donors showed a significant increase in the number of spots as compared to control cells. Secretion of IL-4 or IL-10 was not detected. From the antigen-stimulated primary cultures we established by limiting dilution cloning six rpL7-reactive, IFN-gamma-secreting T cell lines which show a CD3+CD4+CD8- phenotype. One line additionally was shown to be positive for HLA-DR and CD45R0, but negative for CD27 and CD31. The cell lines carry alphabeta TCR chains which differ from each other in sequence and specificity. rpL7 fragments rich in basic amino acids could be identified as epitopes recognized by the TCR of three cell lines. Recognition of rpL7 is HLA-DR6 restricted or respectively HLA-DP restricted in the two cell lines analyzed.

Amino Acid Sequence↗

PubMed1997

A double-blind, placebo-controlled, crossover therapy study with natural human IL-2 (nhuIL-2) in combination with regular intravenous gammaglobulin (IVIG) infusions in 10 patients with common variable immunodeficiency (CVID).

Ten CVID patients with defective IL-2 synthesis in vitro were treated with nhuIL-2 in a placebo-controlled, double blind, crossover therapy study during a period of 12 months. No severe side-effects of nhuIL-2 were recorded. Marginal serum nhuIL-2 levels were measurable in individual patients only during the therapy phase. Serum levels of soluble IL-2 receptors were unaffected by the therapy. nhuIL-2 and placebo groups did not differ significantly with respect to requirement of IVIG substitutions which were performed whenever serum IgG levels dropped below 5 g/l: a total of 53 IVIG infusions (corresponding to 17.6 g IgG/month per patient) was necessary during the placebo phase, and 48 infusions (16.4 g IgG/month per patient) during the nhuIL-2 treatment phase. Thus, nhuIL-2 therapy was ineffective in improving spontaneous IgG synthesis in vivo. Nevertheless, the group of patients receiving nhuIL-2 during the first 6 months of the study exhibited a significant reduction of severe infections (n = 25) during the following 6 months of placebo treatment (n = 7) (P<0.045). The infection score dropped in this group from 181 to 23 (P<0.015). Patients of the second group receiving first placebo and then nhuIL-2 did not experience a significant difference in number and score of infectious episodes: 25 infections were recorded during the first 6 months and 24 during the following 6 months. We suppose that nhuIL-2 therapy of CVID patients reduces susceptibility to severe infections, possibly via the induction of a specific antibody response, which is effective at the earliest 6 months after initiating nhuIL-2 therapy.

Adult↗

PubMed1997

Transient CD80 expression defect in a patient with variable immunodeficiency and cyclic neutropenia.

CD80 expression on stimulated B cells of 9 common variable immunodeficiency (CVID) patients and 10 healthy control individuals was examined. Here we report on an exceptional CVID patient with recurrent episodes of cyclic neutropenia, skin vasculitis and recurrent infections associated with a transient, but reproducible CD80 expression defect on stimulated B cells. Concomitantly serum Ig levels were markedly reduced and B cell counts were low. Interestingly, after recovery from an episode of neutropenia and vasculitis we observed an improvement of the CD80 expression as well as an increase in the number of B cells and a recovery of IgM and IgG production in vivo and in vitro.

B-Lymphocytes↗

PubMed1996

Severe combined immunodeficiency due to defective binding of the nuclear factor of activated T cells in T lymphocytes of two male siblings.

Peripheral blood lymphocytes (PBL) and alloreactive T cell lines of two male infants born to consanguinous parents and presenting with severe combined immunodeficiency (SCID) showed a pronounced deficiency in T cell activation. Although phenotypically normal, the proliferative response of the childrens' T cells was strongly reduced but could be improved by the addition of interleukin-2 (IL-2). Furthermore both childrens' T cells were unable to produce the cytokines IL-2, interferon-gamma (IFN-gamma), IL-4 and tumor necrosis factor-alpha (TNF-alpha). This multiple cytokine production deficiency could not be restored by IL-2 or co-stimulatory signals provided by antigen-presenting cells (APC). Moreover, mRNA for IL-2 and IFN-gamma could not be detected. In contrast, expression of the activation-dependent cell surface markers CD25 and CD69 was within normal limits. To determine whether the functional defect of the patients' T cells was due to the absence or abnormal binding of transcription factors involved in cytokine gene expression, electrophoretic mobility shift assays were used to examine the DNA binding of AP-1, Oct, CREB, SP1, NF-kappa B and the nuclear factor of activated T cells (NF-AT) to their respective response elements in the promoter of the IL-2 gene. Whereas AP-1, NF-kappa B, Oct, CREB and SP1 displayed normal binding activities in nuclear extracts, the binding of NF-AT to its IL-2 promoter response element was barely detectable both before and after T cell stimulation. Our results strongly suggest that this NF-AT/DNA binding defect is responsible for the multiple cytokine deficiency and the SCID phenotype observed in the two infant brothers.

Antigen-Presenting Cells↗

PubMed1995

Activated CD4+ and CD8+ T-cell subsets in Wegener's granulomatosis.

Several lines of evidence argue in favour of an involvement of T cells in the pathogenesis of Wegener's granulomatosis (WG). These include the presence of highly specific IgG autoantibodies to proteinase 3, perivascular T-cell infiltrates and elevated amounts of soluble interleukin-2 (IL-2) receptors in patient's serum. In order to further address this question we evaluated by double immunofluorescence and flow cytometry the expression of several cell surface molecules associated with T-cell activation. As compared to healthy controls (n = 15), the CD4+ subset was significantly diminished, while the percentage of CD8+ T cells was elevated in WG patients (n = 24). Within the CD4+ T-cell subset we found a highly significant increase in activation/memory markers (CD25, CD29, HLA-DR). Within the CD8+ T-cell subset the expression of CD11b, CD29 and CD57 was significantly elevated, while the expression of VD28 was reduced. The use of 10 V beta-, 1 V alpha- and 1 V gamma-specific monoclonal reagents failed to reveal any significant bias in the peripheral T-cell receptor V-gene repertoire of WG patients. There was also no correlation between T-cell activation markers and laboratory parameters [C-reactive protein (CRP), ESR], disease duration or therapy. A significant correlation was found only for the degree of organ involvement and the increase in CD4+ T cells coexpressing HLA-DR, as well as the increase in CD57 expression on CD8+ T cells. In conclusion, both CD4+ and CD8+ T-cell subsets were activated in WG. Cytotoxic CD8+CD57+CD11b+CD28- T cells may directly contribute to damage of vascular endothelium.

Adult↗

PubMed1995

Common variable immunodeficiency (CVID) and MxA-protein expression in blood leucocytes.

The underlying immunopathogenic mechanism of CVID has been suspected to involve a chronic viral infection or an autoimmune condition. However, formal proof of viral infection is lacking. Measurement of MxA-protein in leucocyte lysates is a sensitive test for evaluating the activation of the host's interferon system. Both viral infections and autoimmune diseases such as systemic lupus erythematosus (SLE) strongly induce MxA-protein in peripheral leucocytes. We therefore examined 15 patients with longlasting hypogammaglobulinaemia for MxA-protein induction in vivo: 13 patients suffered from CVID, one from hyper-IgM syndrome, and one patient had chronic B lymphocytic leukaemia associated with immunoglobulin deficiency and chronic papilloma virus infection (condylomata accuminata). Only the latter patient exhibited a strong MxA-protein expression; two CVID patients were borderline positive, and the remaining 12 patients including the hyper-IgM syndrome were MxA-protein-negative. There was no relationship between MxA expression and low CD4/CD8 ratios or increased CD8/CD57+ T cell counts, although both conditions are often observed in CVID as well as in chronic viral infections. When exposed in vitro to interferon-alpha (IFN-alpha), peripheral blood leucocytes of four MxA-negative patients were capable of producing normal amounts of MxA-protein. Taken together, these results argue against a viral or autoimmune pathogenesis of CVID.

Adult↗

PubMed1995

T cell lines recognizing the 70-kD protein of U1 small nuclear ribonucleoprotein (U1snRNP).

In sera of patients with mixed connective tissue disease (MCTD) high titres of IgG autoantibodies to U1snRNP-specific proteins (70 kD, A, C) are found, suggesting an antigen-driven and T-cell-dependent process. In order to establish U1snRNP-specific T cell lines we cultured under various culture conditions mononuclear cells from MCTD patients and healthy donors with a highly purified UsnRNP preparation from HeLa cells. Nine T cell lines were established by limiting dilution cloning from two MCTD patients and five T cell lines from a healthy individual. All T cell lines expressed the TCR alpha beta/CD3 complex. Surprisingly, most of the T cells lines exhibited the CD8 phenotype. Irrespective of this phenotype, all T cell lines showed a proliferative response to an N-terminal part (aa 51-195) of recombinant U1-specific 70-kD protein. One CD8+ T cell clone exhibited cytotoxic activity against an autologous B cell line pulsed with snRNP or recombinant fragments (aa 51-95 and aa 51-88). Interestingly, two T cell lines proliferated in response to four recombinant polypeptides representing different parts of the U1snRNP 70-kD protein. Since regions of sequence homology are distributed over the 70-kD molecule, it is suggested that conserved motifs may be recognized by the T cell lines.

Adult↗

PubMed1995

Two novel cationic staphylococcal proteins induce IL-2 secretion, proliferation and immunoglobulin synthesis in peripheral blood mononuclear cells (PBMC) of both healthy controls and patients with common variable immunodeficiency (CVID).

Two cationic proteins, a neutral phosphatase (NP-tase) and a 70-kD protein (p70) were isolated from Staphylococcus aureus by ion exchange chromatography. We compared their properties to those of the well established B cell mitogen of whole, fixed Staph. aureus strain Cowan I cells (SAC). Both purified proteins were able to induce immunoglobulin synthesis in PBMC cultures of healthy donors. NP-tase and p70 also induced immunoglobulin synthesis of PBMC from those patients with CVID who were also responsive to SAC plus IL-2 stimulation. Immunoglobulin synthesis in response to NP-tase and to p70 was time- and dose-dependent and could be inhibited by addition of specific antibodies against the proteins. In contrast to SAC, no addition of exogenous IL-2 was necessary to obtain maximal immunoglobulin synthesis induced by NP-tase or p70. However, neither protein was able to induce immunoglobulin synthesis in B cell-enriched cultures. High amounts of IL-2 were found in supernatants of PBMC from healthy donors following stimulation with low concentrations of NP-tase or p70, and this was associated with vigorous lymphocyte proliferation. Both proteins behave like typical antigens, and not like lectins or superantigens, since an NP-tase-stimulated T cell line showed an antigen-specific, MHC-restricted secondary response. In addition, no preferential T cell receptor V beta chain usage was found with eight V beta-specific MoAb. It is likely that the two proteins induce antigen-specific T cell activation, which is then followed by polyclonal activation of B cells via CD40 receptors and cytokine release.

Adult↗

PubMed1994

Monocyte differentiation and accessory function: different effects on the proliferative responses of an autoreactive T cell clone as compared to alloreactive or antigen-specific T cell lines and primary mixed lymphocyte cultures.

An autoreactive T cell clone derived from a patient with reactive arthritis, two alloreactive T cell lines, two antigen-specific T cell lines and allogeneic resting T cells were analyzed for their responses to monocytes and macrophages derived from monocytes by in vitro differentiation. The autoreactive T cell clone strongly proliferated in response to fresh monocytes and to macrophages derived from a 7 day culture, but only poorly to monocytes cultured for 2 days. In contrast, alloreactive and antigen-specific T cell lines proliferated to all stimulatory cells equally well. Finally, primary mixed lymphocyte reactions could be stimulated by both fresh and 2-day cultured monocytes, but not by in vitro derived macrophages. The impaired response of the autoreactive T cell clone to 2-day cultured monocytes could not be attributed to reduced expression of several well-defined surface molecules nor to induction of nonresponsiveness. Neither allogeneic monocytes nor cytokines (IL-1, IL-2, IL-4, IL-6) could correct the defective response of the autoreactive T cell clone. However, preculture of monocytes in the presence of interferon-gamma, IL-1, IL-4 or IL-6 retained their stimulatory capacity. Our interpretation of the selectively impaired response of the autoreactive T cell clone is that it most likely recognizes a differentiation-dependent monocyte/macrophage-specific peptide.

Antigen-Presenting Cells↗

PubMed1994

Impaired granulocyte oxidative burst and decreased expression of leucocyte adhesion molecule-1 (LAM-1) in patients with Wegener's granulomatosis.

Neutrophils are the target of autoantibodies in Wegener's granulomatosis (WG). In this study, granulocyte function and surface marker expression were investigated in patients with WG. The oxidative burst in response to phorbol myristate acetate (PMA) was tested with granulocytes of 25 patients with histologically proven WG. A significantly diminished percentage of oxygen radical-producing cells was found in patients with active disease. Surface antigen expression of CD11b and LAM-1 was analysed on granulocytes of 20 patients with WG. Whereas the expression of CD11b was normal, surface expression of LAM-1 was decreased in nine cases with WG. The decrease of LAM-1 correlated with disease activity. Phagocytosis of Escherichia coli was tested in 10 patients with WG, and normal values were found in all cases. We conclude that down-regulation of LAM-1 may be a marker of disease activity in WG. The altered response to PMA may indicate functional changes in granulocyte reactivity due to autoantibody-induced damage of the granulocyte membrane.

Cell Adhesion Molecules↗