Olive pollen allergy: searching for immunodominant T-cell epitopes on the Ole e 1 molecule

BACKGROUND: The amino-acid and nucleotide sequence of Ole e 1 (the major antigen of olive pollen) has been described and the IgE antibody response to this major allergen was associated with DR7/DQ2 antigens. With this previous data we try to define the T-cell epitopes implicated in Ole e 1 reactivity. OBJECTIVES: To study the recognition of T cells (derived from allergic and non-allergic Ole e 1 patients) to Ole e 1 synthetic peptides in order to define immunodominant T-cell epitopes. METHODS: We have compared the proliferative response of the peripheral blood mononuclear cells from Ole e 1 sensitized patients vs. non-sensitized controls, induced by 14 Ole e 1 synthetic peptides. Thirty subjects were classified in two groups: group 1 (non-responders against Ole e 1, n=16) and group 2 (Ole e 1 responders, n=14), according to their clinical parameters and the presence or not in their sera of the significant Ole e 1 IgE antibody levels. RESULTS: Our results shown that it is possible to find T cells reactive to Ole e 1 peptides in patients with and without significant levels of Ole e 1 IgE antibodies. However, the percentage of response was higher in patients with IgE antibodies 71.4% vs 25%), and the recognition profile was different: the control group showed a broad reactivity pattern, in contrast, the response by the 'Ole e 1 responders' group was mainly directed against three peptides of the carboxi-terminal region, peptides 10 (91-102), 12 (109-120) and 13 (119-130), with a response frequency of 35.7, 28.5 and 28.5%, respectively. By direct and inhibition test no antibody response was found against the synthetic peptides. CONCLUSIONS: Our data suggest that the regions between 91 and 102 and 109-130 aminoacids on the Ole e 1 molecule are immunodominant T-cell epitopes. These epitopes are not recognized by IgE antibodies.     

Report of the first workshop on the genetic map of bovine chromosome 1

Allergic bronchopulmonary aspergillosis (ABPA), occurring primarily in patients with asthma or cystic fibrosis (CF), is a hypersensitivity reaction to Aspergillus fumigatus (Af), and is characterized by increased serum IgE levels and peripheral blood and pulmonary eosinophilia. We evaluated the IgE and cytokine profile in ABPA through enzyme-linked immunosorbent assay (ELISA), and evaluated eosinophil activity with the eosinophil peroxidase (EPO) assay. IgE and cytokines were measured in supernatants from cultures of peripheral blood mononuclear cells (PBMC) from three subject groups: ABPA patients, patients with asthma, and healthy individuals. All cultures for the three subject groups were studied in the presence and absence of two purified Af antigens (the 35-kD antigen and heat shock protein 1). We found that increased in vitro levels of IgE in unstimulated PBMC culture supernatants correlated significantly with serum IgE concentrations in ABPA patients. We measured a decrease in IgE levels of up to 75% of baseline values in supernatants from PBMC cultured with Af antigens. Interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) concentrations in cultures with Af were increased in ABPA, whereas concentrations of IL-4 did not differ in the three subject groups. An inverse relation was noted between the changes in IgE and IFN-gamma measured in 4 of 5 ABPA patients. The PBMC supernatants also promoted EPO activity in purified eosinophils from ABPA patients, and to a lesser extent in purified eosinophils from healthy subjects. These results show that the 35-kD antigen and HSP1 from Af downregulate IgE in vitro but are capable of inducing eosinophilia in ABPA. Further studies could result in the characterization of epitopes leading to these disparate effects. An identification of the IgE-down-regulating epitopes in Af antigens might have therapeutic significance.     

Delineation of PLA2 epitopes using short or long overlapping synthetic peptides: interest for specific immunotherapy

BACKGROUND: Venom immunotherapy is definitely indicated in severe systemic anaphylactic reactions to bee stings, but is not devoided of risks of anaphylaxis. Safer methods of immunotherapy need to be developed. OBJECTIVE: To delineate phospholipase A2 T-cell epitopes using short 15mer vs long 40-60mer overlapping peptides, and to approach the potential interest of a venom immunotherapy based on the use of long peptides (1-60, 51-99, 90-134) mapping the whole phospholipase A2 molecule vs a restricted number of immunodominant epitopes. METHODS: Proliferation of a CD8+ T cell depleted peripheral blood mononuclear cell fraction and short-term T-cell lines from unselected bee venom hypersensitive patients in response to phospholipase A2 synthetic peptides. RESULTS: Whereas T-cell proliferation to 15mer overlapping peptides was weak, T-cell response to long overlapping peptides was in contrast vigorous in all patients, mostly directed to C-terminal peptide 90-134. Our results did not support the concept of rare dominant T-cell epitopes, and disclosed T-cell responses to multiple epitopes in several patients. No significant IgE-binding to long overlapping peptides was detected except in one patient against peptide 90-134. CONCLUSION: 15mer peptides might not be sensitive enough to fully delineate all potential T-cell epitopes scattered along the allergen. Since they do not bind IgE in vitro or only weakly, and taking into account a T-cell response frequently directed to multiple epitopes, long overlapping peptides may represent ideal tools for immunotherapy.     

Immunological characterization of Asp f 2, a major allergen from Aspergillus fumigatus associated with allergic bronchopulmonary aspergillosis

The 37-kDa recombinant protein Asp f 2, encoding an allergen of Aspergillus fumigatus, was expressed in a prokaryotic expression system and immunologically evaluated for its functional and structural properties. The open reading frame for a 310-amino-acid-long protein was shown to encode a signal peptide of 31 amino acids. A native 37-kDa culture filtrate protein and a 55-kDa mycelial glycoprotein (gp55) exhibited complete N-terminal sequence homology to Asp f 2. A GenBank search for homologous proteins revealed 60 and 44% sequence homologies to the cytosolic protein ASPND1 from Aspergillus nidulans and fibrinogen binding protein from Candida albicans, respectively. The glycosylation sites and cysteine molecules are conserved in all the three proteins. The extracellular matrix protein laminin showed a dose-dependent interaction with Asp f 2. This protein, expressed as a major cell-associated protein within 24 h of in vitro fungal culture, comprises 20 to 40% of total fungal protein. Furthermore, both native and recombinant Asp f 2 exhibited specific immunoglobulin (IgE) binding with allergic bronchopulmonary aspergillosis (ABPA) and cystic fibrosis-ABPA patients, whereas A. fumigatus-sensitized allergic asthma and normal control subjects failed to show IgE binding with Asp f 2. These results indicate that Asp f 2 is a major allergen of A. fumigatus exhibiting IgE antibody binding with sera from patients with ABPA. The antigen should be explored further for its potential role in the differential diagnosis of A. fumigatus-associated allergic diseases.     

Determinant analysis of IgE and IgG4 antibodies and T cells specific for bovine alpha(s)1-casein from the same patients allergic to cow's milk: existence of alpha(s)1-casein-specific B cells and T cells characteristic in cow's-milk allergy

In an effort to clarify the etiology of milk allergy from the standpoint of allergen-specific immune reactions, we investigated the determinants of IgE, IgG4, and T cells specific for bovine alpha(s)1-casein from the same individual patients by using its synthetic peptides and cyanogen bromide-digested fragments. Alpha(s)1-casein is a major allergen in cow's milk, and its unique conformation enabled us to investigate the determinants of antibodies without consideration about missing the reactivities because of conformational changes. Nine patients were selected as subjects from among 129 milk-sensitive infants screened by ELISA to assess the anti-alpha(s)1-casein IgE levels in their sera. By using ELISA for epitope mapping, a C-terminal region of alpha(s)1-casein was identified as a common binding site for IgE from all of these patients, whereas those for anti-alpha(s)1-casein IgG4 were located in multiple regions of alpha(s)1-casein. We determined the specificities of seven alpha(s)1-casein-specific T-cell lines established from peripheral blood mononuclear cells of two of the patients. These T cells have been shown to secrete IL-4. All of the T-cell lines had different specificities to alpha(s)1-casein. However, a common amino acid residue use was found among the determinants of various T-cell lines from each patient. The results suggest that patients allergic to cow's milk have characteristic B cells recognizing a limited region of alpha(s)1-casein and secreting alpha(s)1-casein-specific IgE. These B cells may interact particularly with T cells recognizing determinants with a common structure.     

Responses of human birch pollen allergen-reactive T cells to chemically modified allergens (allergoids)

BACKGROUND: Allergoids are widely used in specific immunotherapy for the treatment of IgE-mediated allergic diseases. OBJECTIVE: The aim of this study was to analyse whether a modification of birch pollen allergens with formaldehyde affects the availability of T-cell epitopes. METHODS: Efficient modification of the allergens was verified by determining IgE and IgG binding activity using ELISA inhibition tests. T-cell responses to birch pollen allergoids were analysed in polyclonal systems, using peripheral blood mononuclear cells (PBMC) of five birch pollen-allergic individuals, as well as birch pollen extract-reactive T-cell lines (TCL), established from the peripheral blood of 14 birch pollen-allergic donors. To determine whether the modification of natural (n)Bet v 1 with formaldehyde or maleic anhydride results in epitope-specific changes in T-cell reactivities, 22 Bet v 1-specific T-cell clones (TCC), established from nine additional birch pollen-allergic individuals, were tested for their reactivity with these products. RESULTS: The majority of PBMC and TCL showed a reduced response to the birch pollen extract allergoid. Bet v 1-specific TCC could be divided into allergoid-reactive and -non-reactive TCC. No simple correlation between possible modification sites of formaldehyde in the respective T-cell epitopes and the stimulatory potential of the allergoid was observed. Mechanisms of suppression or of anergy induction were excluded as an explanation for the non-reactivity of representative TCC. All TCC could be stimulated by maleylated and unmodified nBet v 1 to a similar extent. CONCLUSION: These results demonstrate differences in the availability of T-cell epitopes between allergoids and unmodified allergens, which are most likely due to structural changes within the allergen molecule.     

Efficient class II major histocompatibility complex presentation of endogenously synthesized hepatitis C virus core protein by Epstein-Barr virus-transformed B-lymphoblastoid cell lines to CD4(+) T cells

The induction of an efficient CD4(+) T-cell response against hepatitis C virus (HCV) is critical for control of the chronicity of HCV infection. The ability of HCV structural protein endogenously expressed in an antigen-presenting cell (APC) to be presented by class II major histocompatibility complex molecules to CD4(+) T cells was investigated by in vitro culture analyses using HCV core-specific T-cell lines and autologous Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs) expressing structural HCV antigens. The T- and B-cell lines were generated from peripheral blood mononuclear cells derived from HCV-infected patients. Expression and intracellular localization of core protein in transfected cells were determined by immunoblotting and immunofluorescence. By stimulation with autologous B-LCLs expressing viral antigens, strong T-cell proliferative responses were induced in two of three patients, while no substantial stimulatory effects were produced by B-LCLs expressing a control protein (chloramphenicol acetyltransferase) or by B-LCLs alone. The results showed that transfected B cells presented mainly endogenously synthesized core peptides. Presentation of secreted antigens from adjacent antigen-expressing cells was not enough to stimulate a core-specific T-cell response. Only weak T-cell proliferative responses were generated by stimulation with B-LCLs that had been pulsed beforehand with at least a 10-fold-higher amount of transfected COS cells in the form of cell lysate, suggesting that presentation of antigens released from dead cells in the B-LCL cultures had a minimal role. Titrating numbers of APCs, we showed that as few as 10(4) transfected B-LCL APCs were sufficient to stimulate T cells. This presentation pathway was found to be leupeptin sensitive, and it can be blocked by antibody to HLA class II (DR). In addition, expression of a costimulatory signal by B7/BB1 on B cells was essential for T-cell activation.     

Effects of reconstructive surgery for left ventricular anterior aneurysm on ventriculoarterial coupling

Microbial agents are known to play a significant role in aggravating allergic diseases. Recently described viral and bacterial superantigens represent one important strategy by which infectious agents can stimulate the immune response. In previous work, we reported that the staphylococcal toxin toxic shock toxin-1 (TSST-1), a prototypic superantigen, induces in vitro total IgE synthesis after cross-linking T and B cells. This study was carried out to establish a potential link between superantigens and the enhanced IgE response to specific allergens in allergic patients. Peripheral blood mononuclear cells from atopic patients were isolated during and outside the pollen allergen season and stimulated with TSST-1, a prototypic superantigen. Total IgE and interferon-gamma production were measured in supernatants of these cultures. Outside the pollen season, TSST-1 significantly increased total IgE production only in the presence of exogenous interleukin-4, whereas during the pollen season IgE production was significantly enhanced without the need of exogenous interleukin-4. This increase in the absence of exogenous interleukin-4 was associated with significantly lower interferon-gamma production by peripheral blood mononuclear cells stimulated by TSST-1 during the pollen season. Moreover, TSST-1 stimulation of peripheral blood mononuclear cells from inhalant allergic patients was followed by an increased production of allergen-specific IgE that was restricted to the allergen to which the patient was allergic and recently exposed. In addition, TSST-1 induced on B cells the expression of B7.2, a molecule that has recently been demonstrated to enhance T helper 2 responses and to be involved in IgE regulation. This study, by demonstrating that superantigens can augment allergen-specific IgE synthesis and B7.2 expression, provides a mechanism by which microbial superantigens may modulate allergic responses.     

Leishmania pifanoi amastigote antigen P-4: epitopes involved in T-cell responsiveness in human cutaneous leishmaniasis

In experimental murine cutaneous leishmaniasis, the purified Leishmania pifanoi amastigote protein P-4 has been shown to induce significant protection against infection. Further, recent studies examining the response of peripheral blood mononuclear cells (PBMC) from Leishmania braziliensis-infected human patients have demonstrated that the P-4 protein selectively elicits a significant TH1-like response. Because a TH1-like response is associated with cure, epitope studies were conducted to further evaluate the human response to P-4. PBMC from confirmed cutaneous leishmaniasis patients infected with L. braziliensis in Rio de Janeiro, Brazil, an area where the disease is endemic, were examined for T-cell proliferation and/or cytokine production in response to whole-parasite homogenate, isolated P-4 protein, and/or P-4 peptides. Twenty of the 22 patients (91%) examined responded to the native P-4 protein by proliferation and/or gamma interferon (IFN-gamma) production. According to the proliferation data, PBMC from 14 patients (64%) were found to respond to the intact P-4 protein (stimulation index of >/=2.5). Fifty-seven percent of the P-4-responsive patients studied responded to at least one of the P-4 peptides; 11 individual peptides were found to elicit a proliferative response. Of 17 patients examined for cytokine production, no PBMC produced detectable interleukin-4 in response to P-4 protein or peptides. However, PBMC from 14 patients (82%) produced significant levels of IFN-gamma (>/=20 pg/ml) in response to native P-4 protein. Nineteen of the 23 peptides were found to elicit an IFN-gamma response from at least two patients. These data indicate that multiple epitopes spanning the entire P-4 molecule are responsible for the TH1-like immune response observed, indicating that the intact P-4 amastigote molecule, rather than selected peptides, may prove to be the most useful for leishmaniasis vaccine development.     

The association of herpes simplex virus type 2 (HSV-2), Haemophilus ducreyi, and syphilis with HIV infection in young men in northern Thailand

BACKGROUND: Protein antigens are presented to cytotoxic T lymphocytes as small peptides (approximately 9-10 amino acids long) bound to class I molecules of the major histocompatibility complex. The identification of tumor-associated antigens and specific peptide epitopes (i.e., antigenic determinants) may be useful in the development of anticancer vaccines. The generation of a cytotoxic T-cell response to one peptide epitope (amino acids 146-154) of human prostate-specific antigen (PSA) has been reported. PURPOSE: Our aim was to identify novel PSA peptides capable of eliciting specific cytotoxic T-cell responses. METHODS: Candidate peptides were identified on the basis of the following criteria: 1) they contained consensus amino acid motifs for binding to HLA-A2, which is the most common type of class I molecule; 2) they lacked strong homology with PSA-related kallikrein proteins; and 3) they were capable of stabilizing HLA-A2 class I molecules on the surface of human T2 (transport deletion mutant) cells, which are defective in antigen presentation. T-cell lines capable of killing (i.e., lysing) T2 target cells that had been pulsed with specific PSA peptides were generated from three different males (two disease-free individuals and one patient with prostate cancer) by incubating peripheral blood mononuclear cells with the peptides and interleukin 2. Specific cell lysis was monitored by the release of radioactivity from target cells that had been labeled with [111In]oxyquinoline. RESULTS: Two novel PSA peptides capable of eliciting cytotoxic T-cell responses were identified; these peptides were designated PSA-1 (amino acids 41-150) and PSA-3 (amino acids 154-163). Four different cytotoxic T-cell lines were generated in response to these peptides-three against PSA-3 and one against PSA-1. Specific lysis of peptide-pulsed T2 cells by the T-cell lines was blocked by the addition of a monoclonal antibody directed against class I molecules. The T-cell lines were also capable of lysing PSA-positive, HLA-A2-positive LNCaP cells (human prostate carcinoma cells). The specificity of LNCaP cell lysis was shown by the following: 1) the inability of added human K562 (chronic myelogenous leukemia) cells to inhibit it, 2) the ability of added anti-HLA-A2 antibodies to block it, and 3) the inability of the T-cell lines to induce substantial lysis of PSA-negative, HLA-A2-positive human cancer cells. IMPLICATIONS: Our studies form a rational basis for the use of PSA peptides or recombinant vectors encoding PSA in the development of anticancer vaccine immunotherapy protocols for patients with prostate cancer.     

Human CD4+ T-cell response to hepatitis delta virus: identification of multiple epitopes and characterization of T-helper cytokine profiles

The T-cell-mediated immune response plays a crucial role in defense against hepatotropic viruses as well as in the pathogenesis of viral chronic hepatitides. However, very little is known about the role of specific T cells during hepatitis delta virus (HDV) infection in humans. In this study, the T-cell response to HDV in chronic hepatitis B virus (HBV) carriers with HDV superinfection was investigated at different levels. Analysis of peripheral blood mononuclear cell (PBMC) proliferation in response to a recombinant form of large hepatitis delta antigen (HDAg) revealed that 8 of 30 patients studied (27%) specifically responded to HDAg. By employing synthetic peptides spanning the entire HDAg sequence, we found that T-cell recognition was directed against different antigenic determinants, with patient-to-patient variation in the pattern of response to peptides. Interestingly, all responders had signs of inactive HDV-induced disease, while none of the patients with active disease and none of the control subjects showed any significant proliferation. More accurate information about the specific T-cell response was obtained at the clonal level. A panel of HDAg-specific CD4+ T-cell clones from three HDV-infected individuals and fine-specificity analysis revealed that the clones tested individually recognized four epitopes corresponding to amino acids (aa) 26 to 41, 50 to 65, 66 to 81, or 106 to 121 of HDAg sequence. The study of human leukocyte antigen (HLA) restriction revealed that peptides 50 to 65 and 106 to 121 were presented to specific T cells in association with multiple class II molecules. In addition, peptide 26 to 41 was efficiently generated after processing of HDAg through the endogenous processing pathway. Cytokine secretion analysis showed that all the CD4+ T-cell clones assayed were able to produce high levels of gamma interferon (IFN-gamma), belonging either to T helper-1 (Th1) or Th0 subsets and that some of them were cytotoxic in a specific assay. This study provides the first evidence that detection of a specific T-cell response to HDAg in the peripheral blood of individuals with hepatitis delta is related to the decrease of HDV-induced disease activity. The HDAg epitopes identified here and particularly those recognized by CD4+ T cells in association with multiple major histocompatibility complex class II molecules may be potentially exploited for the preparation of a vaccine for prophylaxis and therapy of HDV infection.     

Identification of T-cell epitopes on the Rhesus polypeptides in autoimmune hemolytic anemia

We have shown previously that the Rhesus (Rh) polypeptides are the commonest targets for pathogenic anti-red blood cell (RBC) autoantibodies in patients with autoimmune hemolytic anemia (AIHA). The aim of the current work was to determine whether activated T cells from such patients also mount recall responses to epitopes on these proteins. Two panels of overlapping 15-mer peptides, corresponding to the sequences of the 30-kD Rh proteins associated with expression of the D and Cc/Ee blood group antigens, were synthesized and screened for the ability to stimulate the in vitro proliferation of mononuclear cells from the peripheral blood or spleen of nine AIHA cases. Culture conditions were chosen that favor recall proliferation by previously activated T cells, rather than primary responses. In seven of the patients, including all four cases with autoantibody to the Rh proteins, two or more peptides elicited proliferation, but cells from eight of nine patients with other anemias and seven of nine healthy donors failed to respond to the panels. Multiple peptides were also stimulatory in two positive control donors who had been alloimmunized with Rh D-positive RBCs. Six different profiles of peptides elicited responses in the AIHA patients, and this variation may reflect the different HLA types in the group. Stimulatory peptides were identified throughout domains shared between, or specific to, each of the related 30-kD Rh proteins, but T cells that responded to nonconserved regions did not cross-react with the alternative sequences. Anti-major histocompatibility complex class II antibodies blocked the responses and depletion experiments confirmed that the proliferating mononuclear cells were T cells. Notably, splenic T cells that proliferated against multiple Rh peptides also responded to intact RBCs. We propose that pathogenic autoantibody production in many cases of AIHA is driven by the activation of T-helper cells specific for previously cryptic epitopes on the Rh proteins.     

Nicotine does not modulate IL-4 and interferon-gamma release from peripheral blood mononuclear cells and T cell clones activated by phorbol myristate acetate and calcium ionophore

Tobacco smoking induces an increased nonspecific IgE response. IgE synthesis is controlled by IL-4 and IFN-gamma. The effect of nicotine (10(-10) to 10(-5) M), one of the major components of tobacco smoke, were studied on IL-4 and IFN-gamma release by peripheral blood mononuclear cells from 12 allergic patients and 12 nonallergic subjects and 16 T cell clones stimulated by nonspecific agonists (phorbol myristate acetate and calcium ionophore). The release of IL-4 and IFN-gamma was measured by ELISA in supernatants after a 48-hour culture. Nicotine did not modify IL-4 and IFN-gamma release by peripheral blood mononuclear cells or T cell clones. The effects of tobacco smoke on IgE production are unlikely to change in the T cell phenotype by nicotine.     

Immune responses in patients with T-cell lymphoma treated with an anti-idiotype antibody mimicking a highly restricted T-cell antigen

We generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) to a highly restricted T-cell antigen designated glycoprotein (gp) 37 that is found on T-cell malignancies but not on normal cells. gp37 is identified by the murine monoclonal antibody SN2 (Ab1) against which the Ab2 was raised. Each of four patients with T-cell lymphoma predominantly confined to the skin received a minimum of four intracutaneous injections of aluminum hydroxide precipitated anti-idiotype murine monoclonal antibody (1 mg/injection) given every 2 weeks. For responding patients, injections were continued on a monthly basis. All tumors were measured along orthogonal major and minor axes, using a ruler and/or calipers, by the same observer. Tumor sizes were documented photographically. Three of the four patients developed specific idiotypic humoral immune responses, and two of the four patients also demonstrated idiotypic cell-mediated responses. Humoral responses included binding of the patients' sera to the anti-idiotype antibody as well as specific inhibition of binding of the SN2 antibody (Ab1) to the anti-idiotype antibody (Ab2). Anti-anti-idiotypic (Ab3) antibody from one patient's serum bound specifically to the gp37-positive cell line MOLT-4 and also to semipurified gp37 antigen. Cell-mediated responses were demonstrated by specific proliferative response to the aluminum hydroxide precipitated anti-idiotype antibody by patients' peripheral blood mononuclear cells. While three of the four patients had extensive disease and did not have clinical responses, one of the patients who had nine discrete skin tumors and peripheral blood involvement without other detectable disease had virtually complete disappearance of the tumors lasting over 11 months. Our results demonstrate that this particular anti-idiotype antibody can induce humoral and cellular immune responses, and at least in one patient led to a meaningful therapeutic response. Future trials should focus on immunocompetent patients with minimal disease.     

Relationship between interferon-gamma, interleukin-4 and IgE production of lymphocytes from hen's egg-sensitive patients

We measured in vitro interferon-gamma, interleukin-4 and IgE production of peripheral blood mononuclear cells stimulated with ovalbumin. Interferon-gamma in culture supernatants of ovalbumin-stimulated peripheral blood mononuclear cells from hen's egg- sensitive patients with atopic dermatitis was significantly higher than that of healthy children or hen's egg-sensitive patients with immediate symptoms. Furthermore, in patients with atopic dermatitis who were sensitive to hen's egg and patients with immediate symptoms, there was an inverse relationship between interferon-gamma and IgE production (r = -0.535, p <0.05), and significant correlation was found between interleukin-4 and IgE production (r = 0.802, p <0.05). For the above reasons, IgE synthesis of ovalbumin-stimulated peripheral blood mononuclear cells may be suppressed by interferon-gamma in patients with atopic dermatitis. In contrast, in patients with immediate symptoms, interleukin-4 may play a major role in the pathogenesis of increased IgE production.     

Therapeutic potential of peptides in allergic disease

Immunotherapy with crude allergens prevents allergic symptoms in many patients, but its effects are temporary and variable. This type of intervention provokes a transient increase in IgE antibody synthesis that may produce untoward side effects. Recent research has suggested that such immunotherapy downregulates T-cell activity, indicating that regulation of proinflammatory T cells may be a critical mechanism of the therapeutic response. Animal studies have shown that T cells can be rendered anergic by the administration of nonimmunogenic, T-cell-active peptides. Peptides prepared by urea denaturation of purified allergens and by pepsin digestion of crude allergens have been evaluated in humans. Although evidence of specific immunosuppression was noted, allergic reactions occurred as well. Subsequently, researchers synthesized peptides representing short sequences from the protein chains of principal allergens, such as Amb a I of ragweed and Fel d I of cat. Assays of proliferation of T-cell lines from ragweed- and cat-sensitive patients have shown that relatively short sequences from these proteins are responsible for a major portion of the activity of the whole protein. One such cat peptide has shown no reactivity with human IgE. The characteristics of these peptides suggest they should be evaluated further in clinical trials of allergic patients. The anticipated outcome would be prolonged T-cell downregulation, which might result in suppression of late-phase allergic inflammation and IgE antibody synthesis. The question whether such changes will reduce clinical reactivity sufficiently to be clinically useful remains to be answered in future studies.     

Cell-mediated autoimmunity in patients with Wegener's granulomatosis (WG)

Despite the well described infiltration of cells of the cellular immune system in vasculitic lesions and the granuloma formation in patients with WG, the role of T cell-mediated autoimmunity in WG is not clear. Reports of T cell proliferation in response to neutrophil azurophilic granule proteins are contradictory. In this study we have assessed the proliferation of T cells of WG patients to purified proteinase 3 (PR3) and to total azurophilic granule proteins in two different assays. In addition to the classical proliferation assay with isolated peripheral blood mononuclear cells, we have used a whole blood proliferation assay. In both assays we found proliferative responses to PR3 in patients with WG. The number of patients reacting to the azurophilic granule extract was higher than the patients reacting to the purified PR3, suggesting that other autoantigens may also be involved. We have identified epitopes of PR3 that may be potential targets of class I-restricted T cell responses in the context of HLA-A*0201, the most common MHC class I molecule. These epitopes were determined by the binding of synthetic PR3 peptides to HLA-A*0201 on the antigen-processing defective cell line, T2. In addition, T cell lines were established from tissue biopsies, obtained from WG patients, and assessed for cytolytic reactivity against T2 cells, preloaded with synthetic PR3 peptides. We conclude that T lymphocytes of WG patients have increased proliferative responses to purified PR3 and to a larger extent to non-fractionated proteins of azurophilic granules of polymorphonuclear neutrophilic leucocytes (PMN).     

Clinical applications of registration and fusion of multimodality brain images from PET, SPECT, CT, and MRI

The aim was to assess the specificity and functional significance of liver-infiltrating and peripheral blood T cells in chronic hepatitis C. Peripheral blood mononuclear cells hepatitis C virus from 50 of 58 (86.2%) patients with chronic hepatitis C virus infection and 6 of 28 (21.4%) controls showed a proliferative T cell response to at least one of 16 synthetic peptides covering highly conserved regions of the core, envelope (El) and non-structural regions (NS4) of hepatitis C virus. However, six immunodominant peptides were exclusively recognized by the proliferating blood mononuclear cells from 46 patients with chronic hepatitis C virus infection (79.3%). Fine specificity and HLA-restriction were studied with 15 peptide-specific CD4+ T cell lines and 23 T cell clones isolated from liver tissue and peripheral blood of 12 patients with chronic hepatitis C. It was demonstrated that the peptide-specific response of CD4+ T cells was restricted to the presence of autologous accessory cells and HLA-DR and -DP molecules. Eight peptide-specific T cell lines and five T cell clones derived from liver tissue and peripheral blood, released interferon-gamma (200-6600 pg/ml) and tumor necrosis factor-alpha (100-400 pg/ml) and no or little interleukin-4 (< 140 pg/ml) after peptide-specific or mitogeneic stimulation, thus resembling a Th1-like cytokine profile. Patients with active liver disease showed significantly higher proliferative responses to hepatitis C virus core peptides than asymptomatic hepatitis C virus carriers or complete responders to interferon therapy. In conclusion, class II-restricted CD4+ T cell responses to some immunodominant epitopes within the hepatitis core region correlated with disease activity in chronic hepatitis C virus infection. Functionally, liver-infiltrating and peripheral blood T cells released Th1-like cytokines in response to the specific stimulus. Thus, it can be suggested that CD4+ T cells can mediate the pathogenesis of chronic hepatitis C virus induced liver disease.     

Effects of IL-4 and IL-13 on total and allergen specific IgE production by cultured PBMC from allergic patients determined with recombinant pollen allergens

BACKGROUND: Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells. OBJECTIVE: In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood mononuclear cells (PBMC) from pollen allergic patients and healthy control individuals. METHODS: Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profilin (Bet v II) and recombinant timothy grass pollen allergens, Phl p I, Phl p II, and Phl p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. RESULTS: PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected. CONCLUSION: Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.