Chimeric T helper-B cell peptides induce protective response against Japanese encephalitis virus in mice

Virus neutralizing MAb binding and T helper cell stimulating peptide epitopes from structural and non-structural proteins of Japanese encephalitis virus were delineated. It was observed that priming by T helper peptides potentiated neutralizing antibody response against JE virus. Immunization with chimeric T helper - B cell peptides could thus protect mice from lethal challenge with JE virus.     

13C n.m.r. study of L-alanine peptides

The di-, tri-, and tetrapeptides of L-alanine have been studied in aqueous solution by 13C n.m.r. spectroscopy at 25 and 50 MHz. By using selectively 13C enriched analogs containing either 90% 13C methyl or carbonyl carbons and measurements as a function of pH, assignment of the chemical shifts of the peptides has been made. T1 and NOE measurements of the peptides in their cationic, anionic, and zwitterionic states have been recorded as a function of concentration. The results show considerable segmental motion along the backbone carbons of the peptides, with only small changes occurring in the dynamic motions of the peptides as their charge states are altered. The lack of concentration dependence of the chemical shift and T1 values, as well as the similarity of T1 values for individual peptides in the three charge states, indicate that the peptides do not self-associate in aqueous solution.     

Comprehensive analysis of HLA-DR- and HLA-DP4-restricted CD4+ T cell response specific for the tumor-shared antigen survivin in healthy donors and cancer patients

Because of the wide distribution of the survivin Ag in a variety of tumors, we have investigated the survivin-specific CD4+ T cell response in healthy donors and cancer patients. Screening of the entire sequence of survivin for HLA class II binding led to the identification of seven HLA-DR promiscuous peptides, including four HLA-DP4 peptides. All of the peptides were able to prime in vitro CD4+ T cells of eight different healthy donors. The peptide-specific T cell lines were stimulated by dendritic cells loaded with the recombinant protein or with the lysates of tumor cells. The high frequency of responders (i.e., immunoprevalence) was provided by a wide reactivity of multiple peptides. Six peptides were T cell stimulating in at least half of the donors and were close to CD8+ T cell epitopes. HLA-DR molecules were more frequently involved in T cell stimulation than were HLA-DP4 molecules, and hence immunoprevalence relies mainly on HLA-DR promiscuity in the survivin Ag. In two cancer patients a spontaneous CD4+ T cell response specific for one of these peptides was also observed. Based on these observations, the tumor-shared survivin does not appear to be the target of immune tolerance in healthy donors and cancer patients and is a relevant candidate for cancer vaccine.     

Identification of a Human Cyclin D1-Derived Peptide that Induces Human Cytotoxic CD4 T Cells

Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and ⁵¹Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.     

Characterization of desmoglein‐3 epitope region peptides as synthetic antigens: analysis of their in vitro T cell stimulating efficacy,cytotoxicity, stability,and their conformational features

Desmoglein‐3 (Dsg3) adhesion protein is the main target of autoantibodies and autoreactive T cells in Pemphigus vulgaris (PV) autoimmune skin disorder. Several mapping studies of Dsg3 T cell epitope regions were performed, and based on those data, we designed and synthesized four peptide series corresponding to Dsg3 T cell epitope regions. Each peptide series consists of a 17mer full‐length peptide (Dsg3/189–205, Dsg3/206–222, Dsg3/342–358, and Dsg3/761–777) and its N‐terminally truncated derivatives, resulting in 15 peptides altogether. The peptides were prepared on solid phase and were chemically characterized. In order to establish a structure–activity relationship, the solution conformation of the synthetic peptides has been investigated using electronic circular dichroism spectroscopy. The in vitro T cell stimulating efficacy of the peptides has been determined on peripheral blood mononuclear cells isolated from whole blood of PV patients and also from healthy donors. After 20 h of stimulation, the interferon (IFN)‐γ content of the supernatants was measured by enzyme‐linked immunosorbent assay. In the in vitro conditions, peptides were stable and non‐cytotoxic. The in vitro IFN‐γ production profile of healthy donors and PV patients, induced by peptides as synthetic antigens, was markedly different. The most unambiguous differences were observed after stimulation with 17mer peptide Dsg3/342–358, and three truncated derivatives from two other peptide series, namely, peptides Dsg3/192–205, Dsg3/763–777, and Dsg3/764–777. Comparative analysis of in vitro activity and the capability of oligopeptides to form ordered or unordered secondary structure showed that peptides bearing high solvent sensibility and backbone flexibility were the most capable to distinguish between healthy and PV donors. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Functional HLA-DR T cell epitopes of CEA identified in patients with colorectal carcinoma immunized with the recombinant protein CEA

A baculovirus-produced recombinant CEA (rCEA) protein comprising the extracellular region was used for vaccination of CRC patients with or without GM-CSF as an adjuvant cytokine. Ten patients with a significant proliferative T cell response against rCEA were selected for T cell epitope mapping. Fifteen-aa-long overlapping peptides covering the entire aa sequence of the external domain of CEA were used in a proliferation assay. In six of the patients a repeatable T cell response against at least one peptide was demonstrated. For the first time, nine functional HLA-DR epitopes of CEA were defined. Two of the peptides were recognized by more than one patient, i.e., two and three patients, respectively. Those 15-mer peptides that induced a proliferative T cell response fitted to the actual HLA-DR type (SYFPEITHI). The affinity of the native peptides for the T cell receptor was in the low to intermediate range (scores 6–19). The 15-mer peptides also contained 9-mer peptide sequences that could be predicted to bind to the actual HLA-ABC genotypes (SYFPEITHI/BIMAS). Blocking experiments using monoclonal antibodies indicated that the proliferative T cell response was both MHC class I and II restricted. The defined HLA-DR T cell epitopes were spread over the entire CEA molecule, but a higher frequency was noted towards the C-terminal. Peptides with a dual specificity may form a basis for production of subunit cancer vaccines, but modifications should be done to increase the T cell affinity, thereby optimizing the antitumoral effects of the vaccine.Abbreviations aa amino acid - CRC colorectal carcinoma - GM-CSF granulocyte/monocyte colony stimulating factor - CEA carcino-embryonic antigen - BCP baculovirus control protein - MHC major histocompatibility complex - pp peptide - TAA tumor associated antigen     

Functional analysis of birch pollen allergen Bet v 1-specific regulatory T cells

Allergen-specific immunotherapy using peptides is an efficient treatment for allergic diseases. Recent studies suggest that the induction of CD4+ regulatory T (Treg) cells might be associated with the suppression of allergic responses in patients after allergen-specific immunotherapy. Our aim was to identify MHC class II promiscuous T cell epitopes for the birch pollen allergen Bet v 1 capable of stimulating Treg cells with the purpose of inhibiting allergic responses. Ag-reactive CD4+ T cell clones were generated from patients with birch pollen allergy and healthy volunteers by in vitro vaccination of PBMC using Bet v 1 synthetic peptides. Several CD4+ T cell clones were induced by using 2 synthetic peptides (Bet v 1(141-156) and Bet v 1(51-68)). Peptide-reactive CD4+ T cells recognized recombinant Bet v 1 protein, indicating that these peptides are produced by the MHC class II Ag processing pathway. Peptide Bet v 1(141-156) appears to be a highly MHC promiscuous epitope since T cell responses restricted by numerous MHC class II molecules (DR4, DR9, DR11, DR15, and DR53) were observed. Two of these clones functioned as typical Treg cells (expressed CD25, GITR, and Foxp3 and suppressed the proliferation and IL-2 secretion of other CD4+ T cells). Notably, the suppressive activity of these Treg cells required cell-cell contact and was not mediated through soluble IL-10 or TGF-beta. The identified promiscuous MHC class II epitope capable of inducing suppressive Treg responses may have important implication for the development of peptide-based Ag-specific immunotherapy to birch pollen allergy.     

NMR studies of peptide T, an inhibitor of HIV infectivity, in an aqueous environment.

The synthetic octapeptide peptide T (ASTTTNYT) has been shown to interfere with binding of the HIV-1 envelope glycoprotein gp120 to the chemokine receptor R5, thus preventing viral infection. This study investigated the degree of conformational order of two analogs of peptide T, one biologically active (D-Ala peptide T amide) and one inactive (D-Ala, D-Tyr peptide T amide) using nuclear magnetic resonance (NMR) spectroscopy in an aqueous environment, both in solution and in the frozen solid state. Standard solution NMR techniques such as DQFCOSY, HMQC, ROESY and inversion recovery measurements have been utilized to characterize these peptides. Solid state NMR experiments were likewise employed to study the peptides in a frozen glycerol:water mixture. The NMR results indicate that the monomeric form of both peptide T analogs have considerable conformational heterogeneity. Solid state NMR studies indicate aggregation of D-Ala peptide T, possibly into a beta-sheet structure, at concentrations higher than 10 mM.     

Random screening of proteins for HLA-A*0201-binding nine-amino acid peptides is not sufficient for identifying CD8 T cell epitopes recognized in the context of HLA-A*0201

HLA-A2 is the most frequent HLA molecule in Caucasians with HLA-A*0201 representing the most frequent allele; it was also the first human HLA allele for which peptide binding prediction was developed. The Bioinformatics and Molecular Analysis Section of the National Institutes of Health (BIMAS) and the University of Tübingen (Syfpeithi) provide the most popular prediction algorithms of peptide/MHC interaction on the World Wide Web. To test these predictions, HLA-A*0201-binding nine-amino acid peptides were searched by both algorithms in 19 structural CMV proteins. According to Syfpeithi, the top 2% of predicted peptides should contain the naturally presented epitopes in 80% of predictions (www.syfpeithi.de). Because of the high number of predicted peptides, the analysis was limited to 10 randomly chosen proteins. The top 2% of peptides predicted by both algorithms were synthesized corresponding to 261 peptides in total. PBMC from 10 HLA-A*0201-positive and CMV-seropositive healthy blood donors were tested by ex vivo stimulation with all 261 peptides using crossover peptide pools. IFN-gamma production in T cells measured by CFC was used as readout. However, only one peptide was found to be stimulating in one single donor. As a result of this work, we report a potential new T cell target protein, one previously unknown CD8-T cell-stimulating peptide, and an extensive list of CMV-derived potentially strong HLA-A*0201-binding peptides that are not recognized by T cells of HLA-A*0201-positive CMV-seropositive donors. We conclude that MHC/peptide binding predictions are helpful for locating epitopes in known target proteins but not necessarily for screening epitopes in proteins not known to be T cell targets.     

Alpha-fetoprotein (AFP)-derived peptides as epitopes for hepatoma immunotherapy: a commentary

The various immunological roles of human alpha-fetoprotein (HAFP), and its correlation with hepatomas, that is, hepatocellular carcinomas (HCCs), are not often addressed together in biomedical reports considering that HAFP is an established biomarker for hepatomas. Studies reporting measurement of HAFP serum levels in hepatoma patients in basic/clinical research settings has greatly increased over the years. Recent reports have now expanded our base knowledge in the mounting of an immune response against AFP, a self antigen, during hepatoma tumorigenesis. Advances in the detection and identification of AFP-derived peptide epitopes are opening new vistas of knowledge regarding the immunological role of AFP-peptides as T cell stimulating antigens in the course of hepatoma growth and progression. The present commentary addresses HAFP-derived peptides as immunologic responders in HCC and their use in the study and generation of AFP-peptide sensitized T cells directed against hepatoma cells. Attempts were further made to relate the AFP-derived peptide epitopes to T cell activities during the course of hepatoma immunotherapies and to profile the traits and properties of the peptides themselves. Hence, the present commentary was divided into two sections; (1) the characterization, properties, and traits of AFP peptide epitopes, and (2) the use of AFP-derived peptides in the therapeutic induction of T cells primed against hepatoma cells using both in vivo and in vitro models.     

Characterization of human CD4 helper T cell responses against Aurora kinase A

Aurora kinase A (Aurora-A) is a cell cycle-associated serine–threonine kinase that is overexpressed by various types of cancer and is highly associated with poor prognosis. Since the expression of Aurora-A in normal tissues has been shown to be significantly lower as compared to tumor cells, this protein is being considered as a potential tumor-associated antigen for developing immunotherapies. The goal in the present study was to identify CD4 helper T lymphocyte (HTL) epitopes for Aurora-A for the design of T cell-based immunotherapies against Aurora-A-expressing tumors. Synthetic peptides corresponding to potential HTL epitopes were identified from Aurora-A and used to stimulate CD4 T lymphocytes in vitro to generate antigen-specific HTL clones that were evaluated for antigen specificity, MHC restriction and for their ability to interact with Aurora-A-expressing tumor cells. The results show that two peptides (Aurora-A_161–175 and Aurora-A_233–247) were effective in generating HTL responses that were restricted by more than one MHC class II allele (i.e., promiscuous responses). The CD4 HTL clones were able to directly recognize Aurora-A-expressing tumor cells in an antigen-specific and MHC class II-restricted manner and some of the clones displayed cytolytic activity toward Aurora-A + tumor cells. Both of these peptides were capable of stimulating in vitro T cell responses in patients with bladder cancer.     

Determination of T cell epitopes on the S1 subunit of pertussis toxin.

To understand the immunologic characteristics of pertussis toxin molecule and to explore the possibility of developing a synthetic vaccine, T cell epitopes on the enzymatic S1 subunit of pertussis toxin were studied by measuring the proliferative response of immune murine lymph node cells and T cell lines to Ag and to synthetic peptides. The maximum in vitro T cell proliferative response was obtained by stimulating immune lymphoid cells with 20 nM of the enzymatic S1 subunit. When the T cell proliferative response of murine lymphoid cells with different MHC backgrounds was tested, only mice bearing the H-2d haplotype were high responder to the S1 subunit. To determine T cell epitopes on the S1 subunit, the proliferative response of BALB/c immune lymphoid cells to several synthetic S1 peptides was measured. Only the peptide containing amino acid residues, 65-79, was recognized by BALB/c lymphoid cells and was confirmed to contain a T cell epitope by generating S1 specific BALB/c T cell line. By using this T cell line, the response of BALB/c mice to the S1 subunit as well as to peptide 65-79 was shown to be restricted to the I-Ad sublocus of class II Ag. Finally, we showed that lymph node cells of mice immunized with peptide 65-79 respond to the native S1 subunit.     

Mechanical interactions between dendritic cells and T cells correlate with T cell responsiveness

Ag recognition is achieved through the communication across intercellular contacts between T cells and APCs such as dendritic cells (DC). Despite remarkable progress in delineating detailed molecular components at the intercellular contacts, little is known about the functional roles of physical cross-junctional adhesion between T and DC in shaping T cell responses. In addition, the mechanisms underlying sensitivity and specificity of Ag discrimination by T cells at intercellular contacts remain to be elucidated. In this study, we use single-cell force spectroscopy to probe the mechanical interactions between DC and T cells in response to stimulation with a panel of altered peptide ligands. The results show that intercellular interactions of DC-T cell conjugates exhibited different ranges of interaction forces in peptide-dependent manners that match the ability of the peptides to activate T cells. Elevated calcium mobilization and IL-2 secretion by T cells were only promoted in response to antigenic peptides that induce strong interaction forces, suggesting that mechanically stable DC-T cell contacts are crucial for driving T cell activation. Strong interactions were not solely dependent on cell-surface molecules such as TCRs and the adhesion molecule LFA-1, but were also controlled by cytoskeletal dynamics and the integrity of membrane lipid rafts. These data provide novel mechanical insights into the effect of Ag affinity on intercellular contacts that align with T cell responsiveness.     

Role of endogenous peptides in murine allogenic cytotoxic T cell responses assessed using transfectants of the antigen-processing mutant 174xCEM.T2.

One model to explain the high frequency of alloreactive T cells proposes that allogeneic MHC molecules are recognized together with host cell-derived peptides. A model system was developed to investigate the relevance of this mechanism by expression of H-2Dd or H-2Ld in 174xCEM.T2 (T2) cells. This human cell line contains a mutation in its Ag-processing pathway that should restrict the association of endogenous peptides with cell surface class I molecules. CTL generated by stimulating C57BL/6 (H-2b) responder cells with H-2Dd or H-2Ld transfectants of the human B cell line C1R or the murine T cell lymphoma EL4 were assayed for their ability to recognize alloantigenic determinants on these transfectants. The major fraction of the H-2Dd-specific allogeneic CTL response, generated in a MLC or under clonal limiting dilution conditions, was composed of T cells that recognized H-2Dd expressed on C1R or EL4 cells, but failed to recognize this molecule on T2 cells. Clonal analysis indicated that approximately one-third of these CTL recognized determinants that were unique to H-2Dd expressed on C1R stimulator cells whereas the remainder recognized determinants that were also found on EL4 transfectants. Less than 10% of H-2Dd-reactive CTL recognized the T2 transfectant, and these clones also killed C1R-Dd and EL4-Dd. This result suggests that the great majority of H-2Dd-specific alloreactive CTL recognize determinants that are formed by a complex of H-2Dd with endogenous peptides that are absent or significantly reduced in T2 cells. Based on recognition of human or murine transfectants, these CTL exhibit some level of specificity for the structure or composition of the bound peptides. Examination of allogeneic CTL specific for H-2Ld revealed populations similar to those described for H-2Dd. In addition, a major new population was present that recognized determinants shared between C1R-Ld and T2-Ld but not present on EL4-Ld. These results are consistent with the idea that the alloreactive response to H-2Ld is also largely dependent on the presence of bound peptide. However, they also may indicate that the H-2Ld molecule expressed on T2 cells is occupied by one or more peptides that are shared with other human, but not murine, cells. The significance of these results to current models of alloreactivity is discussed.     

Molecular mapping of a histocompatibility-restricted immunodominant T cell epitope with synthetic and natural peptides: implications for T cell antigenic structure

We have prepared synthetic and natural peptides that have allowed delineation of a major antigenic site of myoglobin recognized by histocompatibility (I-Ed)-restricted T cell clones. The smallest peptide capable of stimulating T cell proliferation consisted of residues 136-146. Residues Glu 136, Lys 140, and Lys 145 were essential for antigenicity, whereas Lys 133 and Tyr 146 added potency but were not required for antigenicity. The periodicity of these residues suggests that folding the peptide into its native alpha-helical structure may be essential for antigenicity either by forming a hydrophilic binding site for the T cell receptor or by participating in antigen presentation. The same folding could also produce a hydrophobic site on the opposite side of the alpha-helix that could participate in hydrophobic interactions. The extrapolation of these findings to other known peptide antigens suggests that this tendency to form an amphipathic alpha-helix may be a general property of antigenic sites recognized by T cells, perhaps due to a different functional role of each type of site in eliciting T cell responses.     

An antigen-specific helper T cell hybridoma produces an antigen-specific suppressor inducer molecule with identical antigenic fine specificity. Implications for the antigen recognition and function of helper and suppressor inducer T cells

We have previously described a T cell hybridoma, A.1.1, that responds to specific Ag (P18, a synthetic polypeptide of defined sequence) in the context of I-Ad by producing lymphokines. Herein we report that this cell also releases, into culture supernatants and ascites fluid, an Ag-specific activity that functions in the induction of suppression of anti-SRBC PFC responses. This suppressive activity requires a) Ag-non-specific accessory molecules from a T suppressor inducer factor, b) Ly-2+ T cells in the assay cultures, and c) the specific Ag (P18) conjugated to the SRBC in the assay cultures. The specificity of the A.1.1-derived activity was demonstrated by the absence of suppression in cultures containing SRBC, BSA-SRBC, or conalbumin-SRBC rather than P18-SRBC. Further, the A.1.1-derived activity bound to, and could be eluted from, P18 but not conalbumin. Using a panel of synthetic variant peptides, we have mapped the critical residues in P18 required for Ag/I-Ad induced activation of A.1.1. These peptides were tested for their ability to act as targets for the A.1.1-derived suppressive activity when conjugated to SRBC and added to assay cultures. All peptides capable of stimulating the A.1.1 T cells to release lymphokines were similarly effective in the suppressor assay. Thus, the recognition of Ag by the T cells and by the T cell-derived activity appeared to be identical. The A.1.1-derived molecule was found to be capable of inducing L3T4- T cells to act as suppressor T cells following culture. These suppressor cells were active in inhibiting anti-SRBC responses in the absence of P18 and bore the Ly-2 surface marker. Thus, it is likely that the function of this Ag-specific molecule is to induce Ly-2+ suppressor T cells and thereby cause the inhibition of the response. This function is distinct from that normally associated with helper T cells and may shed new light on the possible relationship between the cell surface T cell receptor for Ag and Ag-specific T suppressor inducer molecules.     

Mapping the fine specificity of a cytolytic T cell response to HIV-1 nef protein.

Epitope mapping of a MHC class I-restricted cytotoxic T cell response to nef, a regulatory protein of HIV, was performed with fresh PBMC from HIV-seropositive donors and target cells pulsed with a panel of overlapping peptides of the nef protein. These nef-specific CTL recognized a synthetic peptide of 10 residues derived from a nonamphipathic, highly conserved region of the nef protein in association with the HLA A3.1 molecule. Using human cell transfectants expressing mutations of the A3 molecule, we demonstrated that the amino acid at position 152 of the A3.1 molecule appears to be critical for detection of this response. Thus, rapid analysis of the epitopes of HIV proteins stimulating CTL responses can be achieved using a combination of fresh donor PBMC and target cells pulsed with synthesized peptides.     

Immunization of mice with lipopeptides bypasses the prerequisite for adjuvant. Immune response of BALB/c mice to human immunodeficiency virus envelope glycoprotein.

In vivo priming of CTL requires the association with MHC class I molecules of peptides derived from the processing of endogenously produced proteins. Immunization with exogenous proteins or peptides rarely induces MHC class I-restricted CTL unless they are associated with lipidic compounds. The capacity to induce CTL was compared in synthetic peptides and simple lipopeptides containing the Immunodominant MHC class I H-2Dd-restricted T-cell epitope of HIV-1 gp160. In contrast with free peptides in saline, lipopeptides induced strong primary CTL responses in vivo. These CTL were able to lyse cells infected with a recombinant vaccinia virus expressing the HIV-1 env gene. Priming of CTL was also successful when using 16-amino acid lipopeptides as 34-amino acid lipopeptides, suggesting that several epitopes might be included in a single construct. In vivo priming of CTL also requires CD4+ T cell help. We therefore searched for Th cell activation after priming with lipopeptides. Our results show that, as with CTL induction, Th cell activation with lipopeptides did not require mixing with adjuvant. In addition, lipopeptides were also efficient at stimulating antibody-mediated responses. Our results show that a single lipopeptidic construct can induce a total immune response, which is of importance in vaccine development.     

Conformation of magainin-2 and related peptides in aqueous solution and membrane environments probed by Fourier transform infrared spectroscopy.

The conformational properties of the magainin family of antimicrobial peptides in aqueous solution and in model membranes have been probed by Fourier transform infrared spectroscopy. The magainins were found to be structureless in aqueous solution at neutral pD, confirming other studies by Raman and circular dichroism spectroscopy. Increasing the pD to 10 induced the formation of predominantly alpha-helical secondary structures, with some beta-sheet. In the presence of negatively charged liposomes (dimyristoylphosphatidylglycerol), the peptides folded into alpha-helical secondary structures with some beta-sheet structure evident. On the other hand, in the presence of zwitterionic phospholipids (dimyristoylphosphatidylcholine), the spectra were identical to those in aqueous solution. For some magainins, the interaction with charged liposomes was modulated by the presence of cholesterol; cholesterol was found to promote the formation of beta-sheet structures, as evidenced by the appearance of amide I bands at 1614 and 1637 cm-1. Differences in structure were observed between the amidated and nonamidated forms of some peptides. From the data, a mechanism of antimicrobial action of the magainin family of peptides is proposed.     

Characterization of T cell responses to Hev b 3, an allergen associated with latex allergy in spina bifida patients

The prevalence of type I allergy to Hevea brasiliensis latex is particularly high among individuals with frequent exposure such as health care workers and patients with spina bifida (SB). Due to a birth defect of the spinal canal and the resulting neurological and orthopedic defects, these patients require multiple surgeries during childhood. SB patients display a unique pattern of sensitization: IgE-reactivity is preferentially directed against Hev b 3 and Hev b 1, two latex allergens with high sequence similarity. In this study, we analyzed the T cell response to Hev b 3 in latex-allergic SB patients using poly-, oligo-, and monoclonal T lymphocyte cultures. All T cell clones (TCC) were CD3/CD4-positive and expressed the alphabeta TCR. According to their cytokine production pattern (IL-4 vs IFN-gamma), 12 of 21 TCC were classified as Th2-like, 2 of 21 were Th1-like, and 7 of 21 belonged to a Th0-like subset. Using 11 T cell lines and 21 TCC, nine T cell stimulating fragments were determined out of 52 overlapping 12-mer peptides representing the complete amino acid sequence of Hev b 3. Ag presentation of one dominant T cell epitope could be associated with a four-amino acid binding motif (YSTS, position 11-13) in the beta 1 chain of HLA-DR molecules expressed by the respective patients. No reactivity was observed when Hev b 3-reactive T cell lines or TCC were incubated with peptides representing homologous parts of the Hev b 1 molecule, i.e., no cross-reactivity between Hev b 3 and Hev b 1 at the T cell level was evident.