Development of a polymeric surgical paste formulation for taxol

The purpose of the study was to map the dominant T cell epitope of the CB11 sequence of CII in RTlu haplotype rats and to determine if, when used as a synthetic peptide, it would induce tolerance to protect against CIA. A dominant epitope corresponding to residues 184-198 included in the sequence of the CB11 fragment of bovine CII was identified in proliferation assay using peptides in an epitope scanning system using synthetic peptides of 15 amino acids, overlapping by 12 amino acids. This epitope is bovine-specific, but cross-reacts with the corresponding rat peptide. Minor epitopes in the bovine CB11 sequence was also autoantigenic. Use of independently synthesized and purified 184-198 peptide confirmed its dominance in the T cell responses of arthritic rats. The peptide itself was not arthritogenic. Cells from lymph nodes draining arthritic feet were particularly responsive to the dominant peptide sequence, and showed evidence of epitope spreading to include reactions to at least four subdominant epitopes. Mucosal tolerance was successfully induced by instilling CII into the nose of rats before induction of CIA: this was found to delay the onset of disease, reduce mean disease severity, shift the anti-CII antibody response to favour antibodies of the IgG1, rather than the IgG2b isiotype, and to reduce T cell reactivity to both CII and to the 184-198 peptide. The dominant 184-198 peptide itself had the same tolerogenic effects when given nasally to rats daily, on the 4 days immediately preceding the induction of CIA. Two forms of CIA with acute and delayed disease onset were each modified by pre-treatment with the peptide. This study demonstrates that mucosal tolerance to CII can be induced by delivering it nasally in a way similar to that achieved previously by oral delivery, and that the use of an immunodominant epitope contained in a synthetic peptide will also suppress the immunologic and arthritic responses to collagen.     

Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen

Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 and a minor determinant within CII286-300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.     

Molecular analysis of presentation by HLA-A2.1 of a promiscuously binding V3 loop peptide from the HIV-envelope protein to human cytotoxic T lymphocytes

P18(IIIB) is a highly immunogenic peptide from the V3 loop of the HIV-1 gp160 envelope protein that is presented promiscuously by multiple class I MHC molecules. Understanding the molecular basis for promiscuous presentation may have many practical applications. As the highly prevalent HLA-A2.1 class I molecule is known to present P18(IIIB) for recognition by cytotoxic T lymphocytes (CTL) found in peripheral blood mononuclear cells of HIV+ donors, a P18(IIIB)-specific CTL line was generated from and HLA-A2(+), HIV- donor in order to define the molecular basis for, and ultimately improve upon the binding of, this peptide to HLA-A2.1. The minimal epitope recognized by the line was a decamer, I10, with the sequence RGPGRAFVTI. Interestingly, this decamer is identical to the minimal epitope from P18(IIIB) seen by murine CTL restricted by H-2Dd. A panel of Ala-substituted peptides was employed in MHC-binding and T cell response studies to identify MHC- and TCR-binding residues. Notably, many of the agretopic and epitopic residues identified were identical to those involved in the corresponding interactions of I10 with the H-2Dd MHC molecule and murine I10-specific CTL. The I10 peptide does not contain the described HLA-A2.1 binding motif. Instead a Pro at P3, a Phe at P7 and an Ile at P10 are utilized for MHC binding. Agretopic residue similarities with the hepatitis B nucleocapsid decamer suggest that these residues may comprise an alternative motif of anchors utilized by decamers for binding to HLA-A2.1.     

Characterization of the peptide binding motif of a rhesus MHC class I molecule (Mamu-A*01) that binds an immunodominant CTL epitope from simian immunodeficiency virus

The majority of immunogenic CTL epitopes bind to MHC class I molecules with high affinity. However, peptides longer or shorter than the optimal epitope rarely bind with high affinity. Therefore, identification of optimal CTL epitopes from pathogens may ultimately be critical for inducing strong CTL responses and developing epitope-based vaccines. The SIV-infected rhesus macaque is an excellent animal model for HIV infection of humans. Although a number of CTL epitopes have been mapped in SIV-infected rhesus macaques, the optimal epitopes have not been well defined, and their anchor residues are unknown. We have now defined the optimal SIV gag CTL epitope restricted by the rhesus MHC class I molecule Mamu-A*01 and defined a general peptide binding motif for this molecule that is characterized by a dominant position 3 anchor (proline). We used peptide elution and sequencing, peptide binding assays, and bulk and clonal CTL assays to demonstrate that the optimal Mamu-A*01-restricted SIV gag CTL epitope was CTPYDINQM(181-189). Mamu-A*01 is unique in that it is found at a high frequency in rhesus macaques, and all SIV-infected Mamu-A*01-positive rhesus macaques studied to date develop an immunodominant gag-specific CTL response restricted by this molecule. Identification of the optimal SIV gag CTL epitope will be critical for a variety of studies designed to induce CD8+ CTL responses specific for SIV in the rhesus macaque.     

An immobilised peptide array identifies antibodies to a discontinuous epitope in the extracellular domain of the bovine growth hormone receptor

Using an array of overlapping decapeptides representing the extracellular domain of the bovine (b) growth-hormone receptor (GHR) we have mapped the continuous, dominant epitopes defined by five rabbit and one guinea pig polyclonal antisera to recombinant bovine growth-hormone-binding protein (rbGHBP). We report that six major epitopes are identified by these antisera and that these largely occur in areas of non-ordered secondary structure, although there is some contribution from the extensive beta-sheet structure of GHBP. Similar to our previously described studies for growth hormone (GH), we have again found slight differences between animals in the exact location of these epitopes. Using peptide-affinity chromatography we have isolated a population of antibodies reactive with epitope 1 (the N-terminal epitope:GHBP residues 21-38). Analysis of these antibodies by further peptide affinity chromatography and competitive radioimmunoassay experiments indicated cross-reactivity of epitope-1-specific antibodies with epitope 4 (in the interdomain hinge region of the GHBP:residues 111-126). We suggest that, although separate in the primary structure of the molecule, the tertiary fold exhibited by GHBP may bring into close proximity areas of sequence representing epitope 1 and epitope 4 such that they represent a conformational epitope. Under these conditions our experiments indicate that peptides 1 and 4 may represent partial functional epitopes for this antibody population and consequently demonstrate that this approach may be useful in describing discontinuous epitopes.     

The MHC class I-restricted immune response to HIV-gag in BALB/c mice selects a single epitope that does not have a predictable MHC-binding motif and binds to Kd through interactions between a glutamine at P3 and pocket D

Using a strain of Listeria monocytogenes that stably expresses and secretes HIV gag to deliver this Ag to the MHC class I pathway of Ag processing, we have identified the immunodominant CTL epitope to gag in the BALB/c mouse and shown that it is Kd restricted. The specific motif for the peptides that bind the MHC class I molecule H-2 Kd is believed to be a nonamer with residues tyrosine or phenylalanine in the second amino acid position and leucine or isoleucine in the carboxyl-terminal or ninth amino acid position as dominant anchoring positions. Surprisingly, the identified gag peptide, AMQMLKETI, does not contain an anchoring aromatic residue in position two although competition assays with other Kd-restricted epitopes indicated that it binds to Kd with comparable affinity. Using a theoretical molecular dynamics approach to probe the stability of peptide binding to MHC class I molecules, we show that the absence of an appropriate anchor residue at P2 in AMQMLKETI is compensated by favorable interactions of the glutamine at P3 with pocket D of Kd. These findings were verified experimentally, demonstrating the predictive power of this theoretical approach in analyzing MHC class I/peptide interactions. These studies also indicate that CTL epitope prediction that relies on dominant peptide motifs may not always identify the correct epitope.     

Specificity of the H-2 L(d)-restricted cytotoxic T-lymphocyte response to the mouse hepatitis virus nucleocapsid protein

Cytotoxic T lymphocytes provide protection against persistent infection of the central nervous system by the JHM strain of mouse hepatitis virus. In BALB/c (H-2d) mice, the dominant response is directed against an Ld-restricted peptide in the nucleocapsid protein (APTAGAFFF). Characterization of the fine specificity of this response revealed that the predicted anchor residues at positions 2 and 9 were the most critical for class I binding. Amino acids at positions 7 and 8 were identified as T-cell receptor contact residues. Virus-induced cytotoxic T lymphocytes to other Ld motif-containing nucleocapsid peptides were not detected, despite the identification of two epitopes with reduced Ld affinity. These data suggest that mutations within four residues of the dominant epitope could contribute to the persistence of the JHM strain of mouse hepatitis virus.     

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256-270 glycopeptide to H-2Aq and H-2Ap molecules

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.     

Analysis of peptides associated with class II MHC molecules, HLA-DR3: implication for the prediction of peptides useful for vaccines

In order to characterize peptide binding motifs of MHC class II molecules HLA-DR3, we have sequenced pool or single peptides eluted from the binding groove. Anchor residues identified are in agreement with peptide binding and sequencing studies reported by different groups. Four positions seem to be dominant (i, i + 3, i + 5, i + 8) while 2 secondary positions (i + 1, i + 2) could cooperate to facilitate binding. According to all the criteria define here and the literature, we propose an anchor motif specific for DR3, which has been tested on the sequence of an antigen from Schistosoma mansoni. Three out of 6 putative epitopes identified share common sequences with immunodominant regions determined in humans and by experimental immunizations in animal models. Extended to other alleles, this approach could be suitable to define potentially immunodominant peptides useful for vaccines.     

Epitope mapping of C1 inhibitor autoantibodies from patients with acquired C1 inhibitor deficiency

We report six patients with acquired C1 inhibitor (C1-inh) deficiency associated with serum C1-inh autoantibodies and circulating cleaved (96 kDa), functionally inactive C1-inh. In three patients, all of whom had IgG-kappa paraproteins in their sera, the Abs were IgG-kappa. In the remaining three patients, the Abs were IgM (2 kappa, 1 lambda). These data suggest that all the Abs were monoclonal. The autoantibodies recognized two synthetic peptides (peptides 2 and 3), which spanned the reactive center of C1-inh. Binding to peptide 3 (residues 448-459) was greater than to peptide 2 (residues 438-449), suggesting that the epitope recognized by the autoantibodies was expressed principally by peptide 3. Both peptides inhibited the binding of the autoantibodies to C1-inh. None of the autoantibodies recognized peptide 1 (residues 428-440), and this peptide did not inhibit the binding of the autoantibodies to C1-inh. The use of substituted peptides suggested that residues Q452 and Q453 made significant contributions to the epitope, and computer modeling studies showed their side chains to be surface exposed in the intact molecule. However, computer modeling also showed that none of the side chains of the polar residues in peptide 2 were sufficiently close to Q452 and Q453 to be able to contribute to a shared epitope. As peptide 2 could inhibit the binding of C1-inh autoantibodies to peptide 3 and vice versa, we conclude that an autoepitope also exists in peptide 2. Computer modeling and the use of substituted peptides suggested that the sequence LLVF (residues 446-449) in peptide 2 is structurally similar to the sequence QQPF (residues 452-455) in peptide 3. We therefore conclude that there are two potential epitopes in the intact C1-inh molecule that are capable of binding to C1-inh autoantibodies.     

Mapping a conserved conformational epitope from the M protein of group A streptococci

The carboxyl terminus of the M protein of group A streptococci (GAS) is highly conserved and contains epitopes that have been shown to induce opsonic antibodies and protection against GAS infection. This region of the protein can also stimulate T cells, which can react in vitro with heart antigens. Since different segments of the carboxyl terminus may be involved in immunity to GAS and in the pathogenesis of autoimmune disease (rheumatic heart disease), it is important to precisely define critical epitopes. However, the M protein is known to be a coiled coil, and a critical immunodominant antibody-binding epitope within this region (peptide 145, a 20-mer with the sequence LRRDLDASREAKK-QVEKALE) is shown here to be conformational. Thus, small synthetic overlapping peptides of 8-12 amino acids in length that span peptide 145 (p145) were unable to capture antibodies present in p145-immune mouse sera or in endemic human sera, even though antibodies raised to these small peptides coupled to diphtheria toxoid could bind the smaller peptides and, in some cases, p145. A series of mutated peptides in which every residue of p145 was sequentially altered also failed to identify critical residues for antibody binding. We thus devised a strategy to produce chimeric peptides in which small peptides copying the M protein sequence were displayed within a larger 28-mer peptide derived from the sequence of the GCN4 leucine zipper DNA binding protein of yeast. A 12-amino-acid window of the p145 sequence was inserted into the GCN4 peptide in such a way as to preserve any potential helical structure. The window was moved along one residue at a time to give a series of peptides representing p145. Circular dichroism demonstrated that these larger chimeric peptides and p145, but not a shorter 12-mer peptide, displayed alpha-helical potential in 50% trifluoroethanol. Certain chimeric peptides efficiently captured antibodies specific for p145 and thus enabled us to map the minimal antibody-binding sequence. RRDLDASREAKK, referred to as J(1)2. The chimeric peptide containing this sequence, referred to as J2, was able to inhibit opsonization of GAS by human antisera containing anti-peptide 145 antibodies. The T-cell response from p145-immunized responder B10.BR mice to J2 and J(I)2 was much lower than the response to p145 and mapped to a different peptide.     

Generation of neutralizing antipeptide antibodies to the enzymatic domain of Pseudomonas aeruginosa exotoxin A

Burn patients suffer a break in the physical barrier (skin), which, when combined with their generalized state of immunodeficiency, creates an open window for opportunistic infections, mainly with Pseudomonas aeruginosa. Infection of the burn wound has always been a major factor in retardation of wound healing, and sepsis remains the leading cause of death in burn patients. Because studies have shown that topical treatment with antiexotoxin A (ETA) antibodies significantly increases survival in rats infected with toxin-producing strains of P. aeruginosa, we examined 11 synthetic peptides encompassing 12 to 45 amino acid (aa) residues, representing what were predicted by computer analysis to be the most hydrophilic and antigenic regions of ETA. These synthetic peptides were injected into rabbits for antibody production. Different groups of rabbits were immunized with a combination of peptides, with each combination representing one of the three distinct domains of ETA. Animals immunized with various peptide combinations produced peptide-specific antibodies that exhibited cross-reactivity to ETA. Two major epitopes were identified on the ETA molecule by experiments with peptide-specific antibodies in enzyme-linked immunosorbent assay and immunoprecipitation. One of these epitopes was located in the translocation domain (II) (aa 297 to 310), while the other was mapped to the last 13 aa residues at the carboxy-terminal end of the enzymatic domain (III) (aa 626 to 638). Of these two regions, the epitope in the enzymatic domain induced a much higher level of neutralizing antibodies that abrogated the cytotoxic activity of ETA in vitro. Antibodies to this epitope blocked the ADP-ribosyltransferase activity of ETA and appeared to interfere with binding of the substrate elongation factor 2 to the enzymatic active site of the ETA molecule. We conclude that polyclonal, as well as monoclonal, antibodies to short peptides, representing small regions of ETA, may have therapeutic potential in passive immunization or topical treatment of burn patients infected with toxin-producing strains of P. aeruginosa.     

Capitated risk-bearing managed care systems could improve end-of-life care

A surface protein antigen (PAc) of Streptococcus mutans, in particular the A-region of this PAc molecule, has been noted as a possible target in research for an effective dental caries vaccine. To identify the antigenic peptide binding to major histocompatibility complex (MHC) class II (HLA-DR) molecules in the A-region, we prepared a panel of overlapping synthetic peptides in the second unit of the A-region, and established that a simple enzyme-linked immunosorbent assay (ELISA) binding assay could be achieved by incubating the DR-crude. Binding to DR molecules of these peptides from nine donors was investigated by using the ELISA binding assay. It was revealed that the PAc(316-334) peptide bound more strongly to the HLA-DR molecule in seven out of nine subjects. In particular, DR8 (DRB1*0802), DR5 (DRB1*1101) and DR6 (DRB1*1402 and *1405), which bound strongly to PAc(316-334) peptide, were identified. Moreover, we synthesized glycine-substituted peptide analogues of the peptide and examined the binding motif of the binding region. As a result, the multiple binding motif in DR8, DR5 and DR6 was found in L-RV-K-A. It is suggested that a peptide vaccine for dental caries that is more effective for humans, with fewer adverse side-effects, could be designed by combining the multiple binding motif with the B-cell epitope to produce only the inhibiting antibody against dental caries. The peptide could therefore be useful for peptide vaccine development in the general human population.     

Human melanoma patients recognize an HLA-A1-restricted CTL epitope from tyrosinase containing two cysteine residues: implications for tumor vaccine development

To identify shared epitopes for melanoma-reactive CTL restricted by MHC molecules other than HLA-A*0201, six human melanoma patient CTL lines expressing HLA-A1 were screened for reactivity against the melanocyte differentiation proteins Pmel-17/gp100, MART-1/Melan-A, and tyrosinase, expressed via recombinant vaccinia virus vectors. CTL from five of the six patients recognized epitopes from tyrosinase, and recognition of HLA-A1+ target cells was strongly correlated with tyrosinase expression. Restriction by HLA-A1 was further demonstrated for two of those tyrosinase-reactive CTL lines. Screening of 119 synthetic tyrosinase peptides with the HLA-A1 binding motif demonstrated that nonamer, decamer, and dodecamer peptides containing the sequence KCDICTDEY (residues 243-251) all reconstituted the CTL epitope in vitro. Epitope reconstitution in vitro required high concentrations of these peptides, which was hypothesized to be a result of spontaneous modification of cysteine residues, interfering with MHC binding. Substitution of serine or alanine for the more N-terminal cysteine prevented modification at that residue and permitted target cell sensitization at peptide concentrations 2 to 3 orders of magnitude lower than that required for the wild-type peptide. Because spontaneous modification of sulfhydryl groups may also occur in vivo, tumor vaccines using this or other cysteine-containing peptides may be improved by amino acid substitutions at cysteine residues.     

B-cell epitopes recognized by IgE from patients sensitive to Amb a 5

BACKGROUND: The response to allergens characterized by IgE-mediated hypersensitivity is selective. The search for the inherited contribution to atopy has among other things, focused on the linkage of sensitivity to the presence of specific alleles in the DR and DQ locus. More than 90% of the responders to Amb a 5, an allergen from ambrosia artemisifolia, are DR-2 positive. This relationship is logically linked to the T-cell epitope presentation by the HLA complex. OBJECTIVES: This study aims to investigate a possible relationship between T-cell epitopes, B-cell epitopes and the alleles of the DR and DQ loci in Amb a 5 sensitive DR-2+ and DR-2- individuals. METHODS: Inhibition of solid state Elisa assays by IgE-enriched and IgG-depleted, heated sera. The inhibition was carried out in checkerboard pattern, bidirectionally; A inhibits B and B inhibits A. RESULTS: The B-cell epitopes defined by the inhibition pattern were all found to be conformational. Three different epitope patterns (A, B, C) were recognized. The IgE and IgG complexes were found in only one responder. The DR and DQ locus alleles were all sequenced. Although all the individuals studied responding to Amb a 5 show presence of alleles such as 1501, associated with DR-2, our data indicates no correlation between the B-cell epitopes recognized and the DR and DQ locus alleles. A well known, general T-cell motif was recognized in the known sequence of Amb a 5. CONCLUSIONS: Our investigation suggests that the choice of B-cell recognition is regulated independently of a putative link between T-cell epitope recognition and the D locus.     

T cell recognition of hypervariable region-1 from hepatitis C virus envelope protein with multiple class II MHC molecules in mice and humans: preferential help for induction of antibodies to the hypervariable region

Hypervariable region-1 (HVR1) from the hepatitis C virus (HCV) envelope protein is thought to be a target for neutralizing Abs. To explore HVR1 recognition by helper T cells, and their role in Ab responses, we attempted to generate helper T cells specific for HVR1 in mice of three MHC types, and with PBMC from HCV-infected HLA-diverse humans. In both species, HVR1 was presented by >1 class II MHC molecule to CD4+ helper T cells and showed surprising interisolate cross-reactivity. The epitope for two DR4+ patients was mapped to a more conserved C-terminal sequence containing a DR4 binding motif, possibly accounting for cross-reactivity. Strikingly, Abs to patients' own HVR1 sequences were found only in patients with T cell responses to HVR1, even though all had Abs to envelope protein, suggesting that induction of Abs to HVR1 depends on helper T cells specific for a sequence proximal to the Ab epitope. Thus, helper T cells specific for HVR1 may be functionally important in inducing neutralizing Abs to HCV. These results may be the first example of "T-B reciprocity," in which proximity of a helper T cell epitope determines Ab epitope specificity, in a human disease setting.     

The effect of epitope variation on the profile of cytotoxic T lymphocyte responses to the HIV envelope glycoprotein

To address the relationship between viral and host factors during HIV infection, we analyzed the effect of viral mutations on T cell responses in seropositive, asymptomatic HLA-A2+ individuals using four envelope (env)-specific peptides with the HLA-A*0201 binding motif. We showed that the natural sequence variation was frequent within epitopes located in the C-terminal region of the env glycoprotein and was largely responsible for a lower env-specific cytotoxic T lymphocyte (CTL) activity in the peptide-stimulated cultures. The highest CTL responses in vitro were induced with conserved epitopes D1 and 4.3 that mapped to the N-terminal region of the env glycoprotein. These peptides exhibited high binding affinity for HLA-A*0201 molecules and stimulated CD8+ T cells of relatively limited TCR Vbeta chain repertoire. Decreased CTL activities to the D1 epitope were observed in the absence of any detectable viral mutation, and were associated with lower proliferative responses and expression of the CD28 antigen. Results of this study demonstrate that the degree of sequence variation within a stimulatory epitope of the viral quasispecies, as well as proliferative potential of the effector cells, are among the factors underlying decreased CTL activity in HIV-infected patients. These experiments also provide evidence that the D1 peptide might be useful for the development of vaccines and immune-based therapy.     

Specificity of the T-cell responses in covalently linked peptides each comprising of a T helper epitope

The selection of T-cell specificities in chimeric peptides comprising of two T helper epitopes (288-302 and 240-252) from the fusion protein of measles virus was investigated. The resulting chimeric peptides (288-P-240 and 240-P-288) were shown to be immunogenic by inducing proliferative responses in both B10.s and C57BL/6 strains of mice. In B10.s mice immunization with the chimeric peptides resulted in proliferative T-cell responses only to the constituent 288-302 peptide, whereas in C57BL/6 mice no proliferative responses to the constituent 288-302 or 240-252 peptides were detected. In vivo competition studies between the 288-302 and 240-252 peptides for binding to the I-As molecule have shown that the peptide 288-302 was dominant in B10.s mice and competed with the non-dominant 240-252 peptide for the induction of an in vivo response. The absence of any proliferative T-cell response to the constituent 288-302 and 240-252 peptides after immunization of C57BL/6 mice with the 288-P-240 or 240-P-288 chimeric peptides, suggests that the dominant T-cell responses might have shifted to newly formed T cell epitope(s) as a result of the covalent linkage of the two peptides. In conclusion, these results indicate that the selection of Th epitopes within chimeric peptides is dependent not only on the amino acid composition of the epitope but also on the context of the epitope within the chimera and the haplotype of the mouse strain used.     

Use of T cell receptor/HLA-DRB1*04 molecular modeling to predict site-specific interactions for the DR shared epitope associated with rheumatoid arthritis

OBJECTIVE: To use molecular modeling tools to analyze the potential structural basis for the genetic association of rheumatoid arthritis (RA) with the major histocompatibility complex (MHC) "shared epitope," a set of conserved amino acid residues in the third hypervariable region of the DRbeta chain. METHODS: Homology model building techniques were used to construct molecular models of the arthritis-associated DRB1*0404 molecule and a T cell receptor (TCR) from T cell clone EM025, which is specific for DR4 molecules containing the shared epitope sequence. Interactive graphics techniques were used to orient the TCR on the DR molecule, guided by surface complementarity analysis. RESULTS: The predicted TCR-MHC-peptide complex involved multiple interactions and specificity for the shared epitope. TCR residues CDR1beta D30, CDR2beta N51, and CDR3beta Q97 were positioned to potentially participate in hydrogen bond interactions with the shared epitope DRbeta residues Q70 and R71. CONCLUSION: These results suggest a structural mechanism in which specific TCR recognition and possibly Vbeta selection are directly influenced by the disease-associated MHC polymorphisms.     

Involvement of testosterone in the induction of hepatic microsomal cytochrome P-450 2B1/2 (P-450 2B1/2) by 1-benzylimidazole in male and female rats: sex-differentiated induction of P-450 2B1/2 species

Collagen-induced arthritis (CIA) is an animal model for the human autoimmune disease rheumatoid arthritis (RA). CIA can be induced in several species including primates by immunization with heterologous type-II collagen (CII). Polyclonal antibodies are formed upon immunization with CII that exhibit a broad range of epitope specificities (some that cross-react with hose CII); however, only antibodies directed against certain specific epitopes on CII are arthritogenic. Recently, the importance of cognate interactions between T-cells and B-cells to the induction of CIA was demonstrated by administration of monoclonal antibodies against a T-cell surface protein, gp39. Blocking the interaction of T-cell gp39, with its receptor/ligand on the surface of B-cells (CD40), completely blocked induction of CIA in mice. A concomitant reduction in the level of anti-CII IgG produced in anti-gp39-treated animals was observed, demonstrating the crucial importance of T-cell:B-cell interactions via gp39:CD-40 binding to the primary immune response to CII in vivo and therefore to the induction of CIA. Other features of CIA are important in elucidating the condition and this article will deal with some important issues.