Biomedical subjects

J W Drijfhout

Publications and source records attributed to J W Drijfhout.

At least 19 recordsLinked to original sources

PubMed2002

Human T cell responses to peptides of the Mycobacterium leprae 45-kD serine-rich antigen.

In order to identify T cell epitopes within the Mycobacterium leprae 45-kD serine-rich antigen, we analysed responses to overlapping 17-mer peptides encompassing the whole antigen in non-exposed UK controls, Pakistani leprosy patients and tuberculosis patients in both the United Kingdom and Pakistan. This antigen has been described as M. leprae-specific, although it has a hypothetical homologue in M. tuberculosis. Human peripheral blood mononuclear cells were stimulated with peptide for 5 days and IFN-gamma measured in supernatants by ELISA. Some peptides were recognized more frequently by T cells from tuberculoid leprosy patients than those from UK controls, suggesting that such T cell epitopes might have diagnostic potential, while other peptides induced greater responses among UK control subjects. Short-term cell lines confirmed that these assays detected specific T cell recognition of these peptides. However, many tuberculosis patients also recognized these potentially specific peptides suggesting that there could be a true homologue present in M. tuberculosis.

Amino Acid Sequence↗

PubMed2002

The use of DODT as a non-malodorous scavenger in Fmoc-based peptide synthesis.

We have synthesized a random group of peptides and performed cleavages using various cleavage cocktails including 3,6-dioxa-1,8-octanedithiol (DODT). Purity of the peptides was compared to that obtained with standard protocols for cleavage using RP-HPLC and Maldi-Tof mass spectrometry. We show that stinking thiols can be replaced by the almost odourless (DODT) without negatively affecting the purity of the end product.

Amino Acid Sequence↗

PubMed2002

Molecular mimicry in type 1 diabetes: immune cross-reactivity between islet autoantigen and human cytomegalovirus but not Coxsackie virus.

Type 1 diabetes is caused by a T cell-mediated autoimmune destruction of the pancreatic beta cells. Molecular mimicry between viral pathogens and beta cell protein has been a popular theory to explain loss of tolerance in type 1 diabetes. However, functional data in support of this hypothesis have been lacking, presumably because the homologies were defined on the basis of linear similarities in peptide sequences, which ignores the criteria of HLA versus T cell receptor contact residues in peptide epitopes required for T cell recognition. We applied a HLA-binding dedicated peptide microarray analysis using autoreactive T cell clones specific for the autoantigen GAD65 to determine the algorithm of T cell recognition by this given T cell clone. The subsequent database search identified a 100% fit with cytomegalovirus peptide, which was subsequently shown to be recognized by these clonal T cells. However, T cell clones reactive with linear homologies previously described as putative candidates for T cell cross-reactivity between GAD65 and Coxsackie virus peptide were unable to recognize the homologous counterparts.

Amino Acid Sequence↗

PubMed2001

Specific inhibition of the classical complement pathway by C1q-binding peptides.

Undesired activation of the complement system is a major pathogenic factor contributing to various immune complex diseases and conditions such as hyperacute xenograft rejection. We aim for prevention of complement-mediated damage by specific inhibition of the classical complement pathway, thus not affecting the antimicrobial functions of the complement system via the alternative pathway and the lectin pathway. Therefore, 42 peptides previously selected from phage-displayed peptide libraries on basis of C1q binding were synthesized and examined for their ability to inhibit the function of C1q. From seven peptides that showed inhibition of C1q hemolytic activity but no inhibition of the alternative complement pathway, one peptide (2J) was selected and further studied. Peptide 2J inhibited the hemolytic activity of C1q from human, chimpanzee, rhesus monkey, rat, and mouse origin, all with a similar dose-response relationship (IC(50) 2-6 microM). Binding of C1q to peptide 2J involved the globular head domain of C1q. In line with this interaction, peptide 2J dose-dependently inhibited the binding of C1q to IgG and blocked activation of C4 and C3 and formation of C5b-9 induced via classical pathway activation, as assessed by ELISA. Furthermore, the peptide strongly inhibited the deposition of C4 and C3 on pig cells following their exposure to human xenoreactive Abs and complement. We conclude that peptide 2J is a promising reagent for the development of a therapeutic inhibitor of the earliest step of the classical complement pathway, i.e., the binding of C1q to its target.

Amino Acid Sequence↗

PubMed2001

Cytomegalovirus in autoimmunity: T cell crossreactivity to viral antigen and autoantigen glutamic acid decarboxylase.

Antigens of pathogenic microbes that mimic autoantigens are thought to be responsible for the activation of autoreactive T cells. Viral infections have been associated with the development of the neuroendocrine autoimmune diseases type 1 diabetes and stiff-man syndrome, but the mechanism is unknown. These diseases share glutamic acid decarboxylase (GAD65) as a major autoantigen. We screened synthetic peptide libraries dedicated to bind to HLA-DR3, which predisposes to both diseases, using clonal CD4(+) T cells reactive to GAD65 isolated from a prediabetic stiff-man syndrome patient. Here we show that these GAD65-specific T cells crossreact with a peptide of the human cytomegalovirus (hCMV) major DNA-binding protein. This peptide was identified after database searching with a recognition pattern that had been deduced from the library studies. Furthermore, we showed that hCMV-derived epitope can be naturally processed by dendritic cells and recognized by GAD65 reactive T cells. Thus, hCMV may be involved in the loss of T cell tolerance to autoantigen GAD65 by a mechanism of molecular mimicry leading to autoimmunity.

Antigens, Viral↗

PubMed2001

Identification of three non-VNTR MUC1-derived HLA-A*0201-restricted T-cell epitopes that induce protective anti-tumor immunity in HLA-A2/K(b)-transgenic mice.

The human epithelial mucin MUC1 is over-expressed in more than 90% of carcinomas of the breast, ovary, and pancreas as well as in some other tumours, making it a potential target for tumour immunotherapy. We have identified several MUC1-derived peptides mapping outside the variable number tandem repeat region that comply with the peptide-binding motif for HLA-A*0201 and that become processed into stable major histocompatibility complex-peptide complexes as assessed by in vitro assays. In A2/K(b) transgenic mice, 3 peptides, namely MUC(79-87) (TLAPATEPA), MUC(167-175) (ALGSTAPPV) and MUC(264-272) (FLSFHISNL) elicit peptide-specific cytotoxic T lymphocyte (CTL) immunity, which protects these mice against a challenge with MUC1, A2/K(b)-expressing tumour cells. These peptides therefore represent naturally processed MUC1-derived CTL epitopes that could be used as components in peptide-based vaccines and for the analysis of anti-MUC1 CTL responses in A*0201-positive patients with MUC1-expressing tumours.

Animals↗

PubMed2001

The identification of a common pathogen-specific HLA class I A*0201-restricted cytotoxic T cell epitope encoded within the heat shock protein 65.

Bacterial antigens recognized by CD8(+) T cells in the context of MHC class I are thought to play a crucial role in protection against pathogenic intracellular bacteria. Here, we demonstrate the induction of HLA-A*0201-restricted CD8(+) T cell responses against six new high-affinity HLA-A*0201-binding CTL epitopes, encoded within an immunodominant and highly conserved antigen of Mycobacteria, the heat shock protein 65 (hsp65). One of these epitopes, Mhsp65(9(369)), is identical in a large number of pathogenic bacteria, and is recognized in a CD8-independent fashion. Mhsp65(9(369)) could be presented by either mycobacterial hsp65-pulsed target cells or BCG-infected macrophages. Interestingly, T cells specific for this epitope did not recognize the corresponding human hsp65 homologue, probably due to structural differences as revealed by modeling studies. Furthermore, in vitro proteasome digestion analyses show that, whereas the mycobacterial hsp65 epitope is efficiently generated, the human hsp65 homologue is not, thus avoiding the induction of autoreactivity. Collectively, these findings describe high-affinity HLA class I-binding epitopes that are naturally processed and are recognized efficiently by MHC class I-restricted CD8(+) T cells, providing a rational basis for the development of subunit vaccine strategies against tuberculosis and other intracellular infectious diseases.

Amino Acid Sequence↗

PubMed2001

Urokinase-receptor/integrin complexes are functionally involved in adhesion and progression of human breast cancer in vivo.

Interactions between specific cell-surface molecules, which include the urokinase receptor (uPAR) and integrins, are crucial to processes of tumor invasion and metastasis. Here we demonstrate that uPAR and beta1-integrins may cluster at distinct sites at the cell surface of metastatic MDA-MB-231 breast cancer cells and form functional complexes. Attachment assays performed in the presence of a synthetic peptide (p25), which interferes with the formation of uPAR-integrin complexes, reveal that uPAR is able to regulate the adhesive function of integrins in breast cancer cells. On dissociation of the uPAR-integrin complexes by p25, tumor cell attachment to the extracellular matrix was either decreased (vitronectin) or increased (fibronectin). Moreover, the tumor cells display remarkable morphological changes when cultured on fibronectin in the continuous presence of p25, leading to increased cell spreading and attachment. In marked contrast to control conditions, increased cellular adhesion to fibronectin after p25 treatment was entirely beta1-integrin-mediated. The role of uPAR-integrin complexes in tumor progression was studied in an in vivo bone xenograft model. Stably transfected MDA-MB-231 cells that overexpress p25 showed a significant reduction in tumor progression in bone (P < or = 0.0001 versus mock-control). In line with these observations, continuous administration of p25 (25 microg/mouse/day, osmotic minipumps) for 28 days resulted in significantly reduced tumor progression of MDA-MB-231 cells in bone (P < or = 0.005) when compared to scrambled control peptide. In conclusion, our data demonstrate that uPAR can act as an adhesion receptor in breast cancer and is capable of regulating integrin function. Our findings strongly suggest that adhesive and proteolytic events are tightly associated in metastatic breast cancer cells and that functional integrin-uPAR complexes are involved in tumor progression in vivo.

Bone Neoplasms↗

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↗

PubMed2000

Identification of major epitopes of Mycobacterium tuberculosis AG85B that are recognized by HLA-A*0201-restricted CD8+ T cells in HLA-transgenic mice and humans.

CD8(+) T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8(+) CTLs, epitopes for classical MHC class I-restricted CD8(+) T cells, which most likely represent a majority among CD8(+) T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/K(b) transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4(+) T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8(+) T cells against Ag85B of M. tuberculosis in HLA-A2/K(b) transgenic mice and HLA-A*0201(+) humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8(+) CTLs were identified. These CD8(+) T cells produced IFN-gamma and TNF-alpha and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8(+) T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201(+) individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8(+) CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8(+) T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.

Acyltransferases↗

PubMed2000

Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent.

Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components. We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E. coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products. In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins. Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed. Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents.

2-Propanol↗

PubMed2000

Antigen arrays in T cell immunology.

The screening of compound arrays in in vitro bioassays has developed into a powerful tool for the identification of biologically active substances. In the past decade, this technology has increasingly been applied to immunology. As the specificity of the immune system is determined by antigen detection via receptors on B and T cells, targeting the specificity of these immune receptors with random arrays is unique in its ability to generate general and quantitative information on cellular (cross-)reactivity. Synthetic array studies have been useful for identification of epitopes and antigens from databases by defining recognition patterns, isolation of synthetic peptides capable of modulating T cell responsiveness, characterisation of TCR promiscuity, and identification of functionally cross-reacting peptides that are potentially involved in molecular mimicry.

Combinatorial Chemistry Techniques↗

PubMed2000

Structure of celiac disease-associated HLA-DQ8 and non-associated HLA-DQ9 alleles in complex with two disease-specific epitopes.

The association of celiac disease (CD) with HLA-DQ2 and HLA-DQ8 is indicative of preferential mucosal T cell recognition of gluten fragments bound to either DQ allele. We have recently identified two gluten-derived, HLA-DQ8-restricted T cell stimulatory peptides, one each from gliadin and glutenin, recognized by specific T cell clones derived from the small intestine of CD patients. We have now performed molecular modeling and examined the fine specificity of these peptides in complex with HLA-DQ8. There is only one binding register for both peptides, with glutamine residues at the p1 and p9 anchor positions. Both T cell clones recognize substituted peptides at p1 and p9, but poorly so at p2-p8, especially the gliadin-specific clone. Contrasting patterns of recognition of p9Gln --> Glu peptide variants (both predicted as better DQ8 binders by modeling) were observed: enhancement of recognition for the gliadin peptide, yet complete absence thereof for the glutenin peptide. The double-substituted gliadin peptide variant p1/9Gln --> Glu, which can also arise by pepsin/acid/transglutaminase treatment, shows a considerable increase in sensitivity of recognition, consistent with better binding of this peptide to DQ8, as predicted by energy minimization. Surprisingly, the two native peptides are also recognized by their respective T cell clones in the context of the related molecule HLA-DQ9 (beta57Asp(+)). The p1/9Gln --> Glu gliadin peptide variant is likewise recognized, albeit with a 10-fold lower sensitivity, the first reported p9Glu binding in a beta57Asp(+) MHC II allele. Our results have important implications for the pathogenesis of autoimmune disease and the possible manipulation of aberrant responses thereof.

Alleles↗

PubMed2000

T-cell lines reactive to an immunodominant epitope of the tyrosine phosphatase-like autoantigen IA-2 in type 1 diabetes.

Type 1 diabetes is the result of destruction of the insulin-secreting beta-cells of the pancreas by a process in which T-cells play a central role. A tyrosine phosphatase-like protein, IA-2, is a major target for autoantibodies and T-cells in the disease. In this study, we have further characterized the T-cell response to IA-2 by the generation and characterization of T-cell lines. T-cell lines responsive to IA-2 antigen were generated from 17 of 32 patients and 3 of 10 control subjects. Antigen specificity was confirmed in lines from six diabetic patients and one control individual by demonstration of responses to synthetic IA-2 peptides and epitope mapping. Five lines from diabetic patients responded to one of two peptides representing amino acids 831-850 and 841-860 of IA-2. The overlapping portion may therefore represent an immunodominant region of the molecule. The sixth patient-derived line responded to a peptide representing amino acids 751-770 of IA-2 presented by the DR 4 (DRB1*0401) allele that confers susceptibility to type 1 diabetes. Primary T-cell responses to peptides of the immunodominant region were detected in 9 of 19 (47%) type 1 diabetic patients and 16 of 22 (73%) nondiabetic siblings, consistent with this region having immunostimulatory properties. The study reports for the first time T-cell lines reactive to IA-2 from diabetic patients and defines an immunodominant region of the molecule.

Adolescent↗

PubMed1999

Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin.

Elevated MMP activities are implicated in tissue degradation in, e.g., arthritis and cancer. The present study was designed to measure MMP enzyme activity in plasma. Free active MMP is unlikely to be present in plasma: upon entering the circulation, active MMP is expected to be captured by the proteinase inhibitor alpha 2-macroglobulin (alpha 2M). Reconstituted MMP-13/alpha 2M complex was unable to degrade collagen (MW 300,000) in contrast to the low-molecular-weight fluorogenic substrate (MW < 1500). Limited access of high-MW substrates to the active site of MMPs captured by alpha 2M presents the most likely explanation. Consistently, the high-MW inhibitor TIMP (MW approximately 28,000) was unable to inhibit MMP/alpha 2M enzyme activity, whereas the low-MW inhibitor BB94 (MW approximately 500) effectively suppressed enzyme activity. By using fluorogenic substrates with Dabcyl/Fluorescein as quencher/fluorophore combin-ation, sensitive MMP-activity assays in plasma were achieved. Spiking of active MMP-13 and MMP-13/alpha 2M complex, and inhibitor studies with TIMP-1 and BB94, indicated that active MMPs are efficiently captured by alpha 2M in plasma. MMP activity was even detected in control plasma, and was significantly increased in plasma from rheumatoid arthritis patients.

Collagen↗

PubMed1999

Identification of a conserved universal Th epitope in HIV-1 reverse transcriptase that is processed and presented to HIV-specific CD4+ T cells by at least four unrelated HLA-DR molecules.

CD4+ Th cells play an important role in the induction and maintenance of specific T cell immunity. Indications for a protective role of CD4+ T cells against HIV-1 infection were found in subjects who were able to control HIV-1 viremia as well as in highly HIV-1-exposed, yet seronegative, individuals. This study describes the identification of an HIV-1-specific Th epitope that exhibits high affinity binding as well as high immunogenicity in the context of at least four different HLA-DR molecules that together cover 50-60% of the Caucasian, Oriental, and Negroid populations. This HIV-1 reverse transcriptase-derived peptide (RT171-190) is highly conserved among different HIV-1 isolates. Importantly, stimulation of PBL cultures from HIV-1 seronegative donors with this peptide resulted in Thl-type lymphocytes capable of efficient recognition of HIV-1-pulsed APCs. Taken together, these data indicate that peptide RT171-190 constitutes an attractive component of vaccines aiming at induction or enhancement of HIV-1-specific T cell immunity.

Amino Acid Sequence↗

PubMed1999

Differential binding of viral peptides to HLA-A2 alleles. Implications for human papillomavirus type 16 E7 peptide-based vaccination against cervical carcinoma.

Several cancer immune intervention protocols aim at inducing T cell immunity against antigens presented by HLA-A2, the most common human MHC class I molecule. In the context of HLA-A*0201, we previously identified two cytotoxic T lymphocyte epitopes (E7(11-20) and E7(86-93)) encoded by the human papillomavirus type 16 E7 (HPV16 E7) oncoprotein, which is a tumor-specific antigen for cervical carcinoma. This study reports that the two HPV16 epitopes and a control hepatitis B virus epitope bind equally well to five HLA-A2 alleles (A*0201, A*0202, A*0203, A*0204, and A*0209). These HLA-A2 variants display comparable binding characteristics in accordance with the A2 supertype (M. F. Del Guercio et al., J. Immunol. 1995. 154: 685-693). Cervical carcinoma patients expressing these alleles may benefit from vaccination with the two HPV16 E7 peptides. In contrast, none of the peptides tested bound to A*0207 or A*0208, whereas heterogeneous binding was observed for A*0205 and A*0206. Therefore, the amino acid substitutions that discriminate these HLA-A2 variants from A*0201 affect antigen presentation. Taken together, our findings have implications for application of the A2 supertype concept and for vaccination with A*0201-binding peptides, in particular HPV16 E7 peptides.

Alleles↗

PubMed1999

Quantitative determination of TCR cross-reactivity using peptide libraries and protein databases.

A single T cell clone can be activated by many different peptides in the context of a particular HLA molecule. To quantify the number of peptides that can be recognized by a CD4(+) T cell clone, we screened a one-bead-one-peptide synthetic peptide library and a protein database for peptides that stimulate an HLA-DR3-restricted, human glutamic acid decarboxylase (GAD65)-reactive CD4(+) T cell clone. Both the library screening and the database analysis indicated that this T cell clone is able to recognize approximately 10(6) 11-mer peptides at low nanomolar concentration. Furthermore, we determined that the frequency of cross-reactivity increased only 1.5-3 times when the peptide concentration increased 10 times, in the range of 0.01 - 1 microM. These data imply that there is a considerable potential for T cell cross-reactivity and are useful for studies on the role of molecular mimicry in the etiology of T cell-mediated disease.

Amino Acid Sequence↗