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G Duinkerken

Publications and source records attributed to G Duinkerken.

14 recordsLinked to original sources
PubMed2002

Modulation of autoimmunity to beta-cell antigens by proteases.

AIMS/HYPOTHESIS: Proteases are used in therapy for autoimmune diseases yet the mechanism of their action remains to be determined. We studied the immunological basis of protease therapy in the context of Type I (insulin-dependent) diabetes mellitus. METHODS: We studied the effects of proteases (trypsin, papain, chymotrypsin, bromelain) on immune reactivity of a series of autoreactive T-cell clones from prediabetic subjects and patients with a recent onset of Type I diabetes and specific to the autoantigens GAD65, IA-2 and insulin-secretory granule protein. RESULTS: Cell surface expression of adhesion, co-stimulatory and homing molecules on both antigen-presenting cells and T cells was changed after protease treatment. Cytokine analyses showed a selective inhibition of proinflammatory (Th-1) but not Th-2 cytokine production. Autoreactive T-cell proliferation was inhibited at pharmacological serum concentrations, whereas non-specific proliferation to phytohaemagglutinin was not affected at these concentrations. Preincubation experiments on T cells and antigen-presenting cells separately showed that this effect was mediated by APCs, but not T-cells. CONCLUSION/INTERPRETATION: Proteases have pleiotropic immunological effects supporting an immunomodulatory potential for the intervention of chronic inflammatory diseases and Th-1 mediated oedema formation.

Amino Acid Sequence↗
PubMed2001

Molecular mimicry in type 1 diabetes mellitus revisited: T-cell clones to GAD65 peptides with sequence homology to Coxsackie or proinsulin peptides do not crossreact with homologous counterpart.

Type 1 diabetes mellitus is a T-cell mediated autoimmune disease in which the insulin-producing pancreatic beta cells are selectively destroyed. Molecular mimicry and T-cell crossreactivity to beta-cell autoantigens and environmental agents with sequence similarities have been a proposed mechanism underlying the pathogenesis of type 1 diabetes, but actual crossreactivity has not yet been demonstrated. We isolated and investigated T cells reactive to GAD65 peptides and homologous peptides of the Coxsackie virus protein P2C and proinsulin from recent onset type 1 diabetes patients, and tested their fine specificity and cytokine production profile. Six T-cell lines specific for GAD65 peptides (amino acids 491-530) with homology to proinsulin (B20-C14) were isolated from six newly diagnosed patients with type 1 diabetes, but none of the stable T-cell lines crossreacted to the homologous proinsulin peptides. Similarly, none of four T-cell lines reactive to GAD65 peptides (amino acids 247-280) with sequence homology to Coxsackie P2C (amino acids 30-50) crossreacted to the homologous viral peptide. Two T-cell lines corecognized a GAD65 peptide and a Coxsackie P2C peptide. However, the antigen-specific T-cell clones from these T-cell lines were reacting either with the GAD65 peptide or the Coxsackie P2C peptide using different restriction elements without crossreacting to the homologous peptide. Our data demonstrate that homologous peptides previously proposed to serve as targets for crossreactivity indeed are immunogenic. Yet, T-cell clones did not crossreact with linear sequence homologies, despite strong T-cell responses to individual peptides.

Amino Acid Sequence↗
PubMed1999

GAD65-Reactive T cells in a non-diabetic stiff-man syndrome patient.

GAD65 (glutamic acid decarboxylase) is an important autoantigen in both type 1 (insulin-dependent) diabetes mellitus (IDDM) and the neurological autoimmune disease stiff-man syndrome (SMS), and is expressed in pancreatic islets as well as the nervous system. Still, only 30% of SMS patients also have type 1 diabetes. To study regulation of T cell responsiveness to GAD65, we investigated a non-diabetic SMS patient with HLA-DR3/7 (predisposing to type 1 diabetes) and high levels of type 1 diabetes-associated autoantibodies against GAD65 and islet cells, and compared the results with those of her diabetic son and two other SMS patients. T cell responses to GAD65 were repeatedly absent in primary stimulation, whereas IA-2, islet antigen and tetanus toxoid induced significant T cell proliferation. However, after in vitro restimulation, GAD65 reactive T cell lines and clones were obtained that were HLA-DR3 restricted, and cross-reactive with a homogenate of purified human pancreatic islets. These T cells produced the immunoregulatory cytokine IL-10 in combination with IFN-gamma and IL-4 (Th0). The dominant T cell epitope was mapped to the central region of GAD65. Although no primary response to whole GAD65 was detectable, the naturally processed GAD65 peptide epitope was recognized vigorously in the primary stimulation assay. The lack of detectable primary T cell responses to GAD65, together with the GAD65-specific cytokine production of restimulated T cells, suggest that GAD65-specific cellular autoimmunity in this patient is suppressed and may be related to the absence of diabetes despite humoral autoreactivity and genetic predisposition.

Adult↗
PubMed1999

Auto- and alloimmune reactivity to human islet allografts transplanted into type 1 diabetic patients.

Allogeneic islet transplantation can restore an insulin-independent state in C-peptide-negative type 1 diabetic patients. We recently reported three cases of surviving islet allografts that were implanted in type 1 diabetic patients under maintenance immune suppression for a previous kidney graft. The present study compares islet graft-specific cellular auto- and alloreactivity in peripheral blood from those three recipients and from four patients with failing islet allografts measured over a period of 6 months after portal islet implantation. The three cases that remained C-peptide-positive for >1 year exhibited no signs of alloreactivity, and their autoreactivity to islet autoantigens was only marginally increased. In contrast, rapid failure (<3 weeks) in three other cases was accompanied by increases in precursor frequencies of graft-specific alloreactive T-cells; in one of them, the alloreactivity was preceded by a sharply increased autoreactivity to several islet autoantigens. One recipient had a delayed loss of islet graft function (33 weeks); he did not exhibit signs of graft-specific alloimmunity, but developed a delayed increase in autoreactivity. The parallel between metabolic outcome of human beta-cell allografts and cellular auto- and alloreactivity in peripheral blood suggests a causal relationship. The present study therefore demonstrates that T-cell reactivities in peripheral blood can be used to monitor immune mechanisms, which influence survival of beta-cell allografts in diabetic patients.

Adult↗
PubMed1998

Cloned T cells from a recent onset IDDM patient reactive with insulin B-chain.

Insulin-dependent diabetes mellitus (IDDM) results from selective autoimmune destruction of insulin producing beta-cells. T-cell reactivity and autoantibodies to several islet proteins such as insulin, GAD and IA-2 are associated with IDDM in mice and men. In NOD mice, the majority of T cells from insulitis specifically recognize the insulin B-chain peptide amino acid 9-22, in contrast to the periphery where the precursor frequency is much lower. It is important to note that these cells are diabetogenic. Surprisingly, the same insulin B-chain region contains epitopes recognized by protective T cells. In fact, autoimmune diabetes in NOD mice could be prevented by prophylactic treatment with this immunodominant T-cell epitope. In humans, however, no immunodominant regions of insulin have yet been defined. We have isolated and characterized a human insulin-specific T-cell clone that was derived from peripheral blood of a newly diagnosed IDDM patient. This patient displayed weakly positive primary T-cell responses to insulin. The peptide recognized by the clone was mapped to the insulin B chain (B:11-27). Functionally, the human insulin-specific CD4+ T cells displayed a Th1/0 like cytokine profile and were restricted by HLA-DR. The previously proposed alternative superantigen-like binding of insulin-B chain peptide outside of the peptide binding groove of HLA-DR could not be confirmed, since T-cell recognition was inhibited in competition experiments of insulin-B chain peptide with HLA-DR16 binding influenza peptide HA307-319. Our results indicate that human clonal T cells isolated from a recent onset IDDM patient recognize an epitope overlapping with the insulin B-chain region that is immunodominant and potentially therapeutic in NOD mice. This observation may be useful in studying the role of insulin-specific T cells in IDDM, and may eventually help to establish peptide-based immunotherapies in IDDM.

Adolescent↗
PubMed1997

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries.

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the beta cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.

Amino Acid Sequence↗
PubMed1997

Th1-like cytokine production profile and individual specific alterations in TCRBV-gene usage of T cells from newly diagnosed type 1 diabetes patients after stimulation with beta-cell antigens.

In order to study cytokine production profile (IFN-gamma, IL-4 and TNF-alpha) and TCRBV-gene usage of peripheral autoreactive T cells from IDDM patients, we have generated antigen-specific T cell lines with either tetanus toxoid, insulinoma membranes or a single beta-cell protein, recombinant ICA69, which has been shown to be a target of both autoantibodies and T cells in IDDM. By semi-quantitative polymerase chain reaction (PCR) analysis, we have determined the composition of the T cell receptor repertoire of these T cell lines and compared this with the general peripheral repertoire. T cell responses against beta-cell antigens and tetanus toxoid (TT) were shown to be associated with IFN-gamma and TNF-alpha production, suggestive of a Th1-like phenotype of the T-cell lines. The production of IFN-gamma was significantly higher in T-cell lines generated with ISG compared to those generated with TT. The cytokine production profiles of the T-cell lines generated with ICA69 did not provide an obvious explanation for the inverse relation between cellular and humoral responses to this protein observed earlier. Upon stimulation with beta-cell antigens, outgrowth of T cells using a restricted set of TCRBV elements was observed in newly diagnosed IDDM patients. However, this skewing in TCRBV-gene expression was patient-specific rather than antigen-associated, since the T-cell repertoire that is used for the recognition of these antigens was, overall, heterogeneous.

Adolescent↗
PubMed1996

T-cell epitope analysis using subtracted expression libraries (TEASEL): application to a 38-kDA autoantigen recognized by T cells from an insulin-dependent diabetic patient.

Studies on circulating T cells and antibodies in newly diagnosed type 1 diabetic patients and rodent models of autoimmune diabetes suggest that beta-cell membrane proteins of 38 kDa may be important molecular targets of autoimmune attack. Biochemical approaches to the isolation and identification of the 38-kDa autoantigen have been hampered by the restricted availability of islet tissue and the low abundance of the protein. A procedure of epitope analysis for CD4+ T cells using subtracted expression libraries (TEASEL) was developed and used to clone a 70-amino acid pancreatic beta-cell peptide incorporating an epitope recognized by a 38-kDa-reactive CD4+ T-cell clone (1C6) isolated from a human diabetic patient. The minimal epitope was mapped to a 10-amino acid synthetic peptide containing a DR1 consensus binding motif. Data base searches did not reveal the identity of the protein, though a weak homology to the bacterial superantigens SEA (Streptococcus pyogenes exotoxin A) and SEB (Staphylococcus aureus enterotoxin B) (23% identity) was evident. The TEASEL procedure might be used to identify epitopes of other autoantigens recognized by CD4+ T cells in diabetes as well as be more generally applicable to the study low-abundance autoantigens in other tissue-specific autoimmune diseases.

Amino Acid Sequence↗
PubMed1996

Imogen 38: a novel 38-kD islet mitochondrial autoantigen recognized by T cells from a newly diagnosed type 1 diabetic patient.

Cell-mediated autoimmune attack directed against islet proteins of approximately 38 kD in size has been associated with type 1 diabetes. A novel murine cDNA encoding an antigen of this size was cloned using a screening procedure based on the proliferative response of a human diabetic T cell clone (1C6) to a recombinant antigen epitope library. Membrane preparations from COS 7 cells transfected with the full-length 1,267-bp cDNA elicited a proliferative response from the reporter T cells comparable to that of the defined peptide epitope and native insulinoma antigen. In vitro translation and transfection experiments suggested that the protein is initially synthesized as a 44-kD protein and then processed to the native 38-kD form through the proteolytic removal of a 54-aa NH2-terminal mitochondrial targeting sequence. Differential centrifugation, Percoll density gradient centrifugation, and immunofluorescence studies confirmed localization of the antigen to mitochondria. Northern blot, Western blot, and 1C6 T cell proliferation assays showed that, although imogen 38 was more highly expressed in beta cell than alpha cell lines, it was also present in other tissues. It is concluded that imogen 38 may be a target for bystander autoimmune attack in diabetes rather than a primary autoantigen.

Amino Acid Sequence↗
PubMed1996

HLA-associated inverse correlation between T cell and antibody responsiveness to islet autoantigen in recent-onset insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is a T cell-dependent immune-mediated disease. Recently, a novel islet cell antigen (ICA69) recognized by autoantibodies was described. We tested T cell responsiveness to ICA69 in peripheral blood of patients with recent onset IDDM (n = 46), patients with long-standing IDDM (n = 44), non-diabetic age-matched, islet cell autoantibody- and glutamic acid decarboxylase (GAD)65 antibody-negative first-degree relatives of IDDM patients (n = 15) and rheumatoid arthritis patients (n = 22). T cell responsiveness was significantly higher in recent onset IDDM patients, compared to IDDM patients post-disease onset, non-diabetic first degree relatives and rheumatoid arthritis patients (p < 0.001). In responding IDDM patients a significant inverse correlation between T cell and autoantibody responsiveness to ICA69 was observed (p < 0.0005). Immunogenetic evaluation revealed an association of HLA-DR3 with T cell responsiveness to ICA69 (p < 0.02) and absence of ICA69-reactive autoantibodies (p < 0.04). The increased T cell reactivity to ICA69 in the absence of antibody reactivity at onset of IDMM is associated with an HLA class II immune response gene, and therefore suggestive of a genetically controlled selective activation of T helper subsets to a specific autoantigen in humans.

Antibody Formation↗
PubMed1996

Increased numbers of in vivo activated T cells in patients with recent onset insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus is a T-cell dependent immune mediated disease. Conflicting results regarding the distribution of various T lymphocyte phenotypes in recently diagnosed diabetic patients and in patients with established IDDM compared to controls have been reported. In the present study we evaluated phenotypic characteristics of lymphocytes in IDDM patients. Lymphocytes from 77 newly diagnosed IDDM patients, 58 IDDM patients with disease duration > 6 months and 30 non-diabetic controls (including patients with several inflammatory conditions) were analyzed for membrane expression of CD4, CD8, CD45RA, CD45RO and CD27 molecules by FACS analysis. No differences in the percentage of CD8+ T cells were found between any of the groups. However, the percentage of CD4+ T cells, and consequently the CD4/CD8 ratio were significantly increased in PBL of recently diagnosed diabetic patients compared to the non-diabetic control group (P < 0.005). Interestingly, the fraction of lymphocytes coexpressing CD45RO and CD45RA molecules was significantly increased in recent onset IDDM patients, as well as IDDM patients with disease for a longer duration, compared to controls (P < 0.0009 and P < 0.007, respectively). IDDM patients had a lower percentage of resting memory T cells (CD45RO+ CD27+) than the non-diabetic controls. The proportion of CD45RO+ lymphocytes lacking the CD27 molecule ((re)-activated memory cells) was similar in IDDM patients and non-diabetic controls. Our findings confirm and extend previous observations that a disturbance in lymphocyte subset distribution is present in patients with IDDM showing an increase in the percentages of circulating CD4 lymphocytes.

Adolescent↗
PubMed1995

T-cell reactivity to beta-cell membrane antigens associated with beta-cell destruction in IDDM.

Insulin-dependent diabetes mellitus (IDDM) results from a T-cell-mediated destruction of the insulin-producing beta-cells. In this study, we designed a sensitive assay to detect and identify islet cell-reactive T-cells in patients with newly diagnosed IDDM. The relation between T-cell recognition of beta-cell antigens with IDDM and the pathogenesis of the disease (the beta-cell destruction process) was tested in a large group of IDDM patients and compared with T-cell responses in nondiabetic children with other chronic inflammations and in immunologically normal, age-matched control subjects. The results demonstrate that peripheral blood T-cells reacting with a beta-cell membrane preparation enriched for insulin-secretory granule antigen were detectable in the majority of newly diagnosed IDDM patients (27 of 40 [67%]; mean stimulation index [SI] 37.0). Such reactivity was reduced postonset in IDDM patients proportionally to the duration of the disease (11 of 30 [37%]; mean SI 8.7). Nondiabetic age-matched control subjects showed no responses or moderate responses to the granule preparation (4 of 48 [8%]; mean SI 3.4). The magnitude of the T-cell response was significantly greater in newly diagnosed IDDM patients than in IDDM patients tested at least 2 years postonset (P < 0.001). Two children in remission for insulin dependency (so-called honeymoon period) displayed exceptionally high proliferative responses to insulin-secretory granules (mean SI 86.7). These results imply that T-cell recognition of insulin-secretory granule antigens is associated with IDDM and in particular with the immune-mediated process of beta-cell destruction.

Adolescent↗