Bcl-2 mediates coelomocytes apoptosis by suppressing cytochrome c release in Vibrio splendidus challenged Apostichopus japonicus.

Apoptosis is an evolutionarily conserved immune response and plays a fundamental role in many physiological processes. In this study, the important apoptosis regulator of Bcl-2 homolog from economic marine animal Apostichopus japonicus (AjBcl-2) was cloned and its roles in V. splendidus infection explored. The AjBcl-2 gene contains 3263 nucleotides, with a 5' UTR of 519 bp, an ORF of 660 bp encoding 219 aa sequences, and a 3' UTR of 2084 bp. The AjBcl-2 protein shared a conserved Bcl domain and three Bcl-2 homology domains by SMART program. In healthy sea cucumbers, AjBcl-2 mRNA was expressed in all examined tissues with the peak expression in coelomocytes. The mRNA and protein levels of AjBcl-2 in coelomocytes were depressed at 12 h and 24 h, and induced at 48 h post V. splendidus challenge. In the same conditions, coelomocytes apoptosis rates were significantly increased at 24 h and decreased at 48 h. Moreover, siRNA-mediated AjBcl-2 knockdown significantly increased the coelomocytes apoptosis rates, which could be partially recovered by recombinant AjBcl-2 administration. Furthermore, there was an increase in the AjCyt c protein expression coupled with the downregulation expression of AjBcl-2 post AjBcl-2 silencing. Our results suggested that AjBcl-2 suppressed apoptosis by preventing the AjCyt c release in coelomocytes, and thus mediating V. splendidus infection in sea cucumbers.

SpBcl2 promotes WSSV infection by suppressing apoptotic activity of hemocytes in mud crab, Scylla paramamosain.

White spot syndrome virus (WSSV) is one of the most virulent and widespread pathogens that infect almost all marine crustaceans and therefore cause huge economic losses in aquaculture. The Bcl2 protein plays a key role in the mitochondrial apoptosis pathway, which is a crucial immune response in invertebrates. However, the role of Bcl2 in apoptosis and immunoregulation in mud crab, Scylla paramamosain, is poorly understood. Here, the Bcl2 homolog (SpBcl2) in S. paramamosain was cloned and its role in WSSV infection explored. The expression of SpBcl2 increased at both the transcriptional level and post-transcriptional level after WSSV infection, while the hemocytes apoptosis decreased significantly. Furthermore, there was increase in the level of cytochrome c coupled with an upregulation in the expression of SpBcl2. These results indicated that SpBcl2 suppressed apoptosis by preventing the release of cytochrome c from mitochondria, thereby promoting WSSV replication in mud crab. The findings here therefore provide novel insight into the immune response of mud crabs to WSSV infection.

Oriented surface epitope imprinted polymer-based quartz crystal microbalance sensor for cytochrome c.

A quartz crystal microbalance (QCM) sensor for detecting cytochrome c based on an oriented surface epitope imprinted polymer was fabricated in this paper. By using the palmitic acid-modified epitope of cytochrome c as the template and the 3-aminopropyltriethoxysilane as the monomer, we prepared a new oriented surface epitope imprinted polymer by the reverse microemulsion polymerization. The prepared oriented imprinted polymer had better imprinting effect than the non-oriented imprinted polymer. And compared to previous studies, this polymerization method is simple and could be carried out at room temperature in the presence of oxygen, under regular atmospheric conditions. Then, by combining the advantages of molecularly imprinted polymers and QCM sensors, we used the prepared polymer to establish a QCM sensor. The described sensor showed good sensitivity and selectivity towards cytochrome c. The linear range was from 0.005 µg mL-1 to 0.050 µg mL-1 and the detection limit was 3.6 ng mL-1 which is lower than most of previous works. Besides, it could be used for real sample analysis and had satisfactory reproducibility and accuracy. This work proposed a new way of fabricating oriented surface epitope imprinted polymers-based QCM sensors for selectively detecting proteins at very low concentrations.

Therapeutic effects of vaccine derived from amastigote surface protein-2 (ASP-2) against Chagas disease in mouse liver.

This study investigated the efficacy of the vaccine in liver of mice infected with the Trypanosoma cruzi (T. cruzi) and immunized with AdASP-2. For this purpose, histopathological analysis and gene expression of COX-2, TNF-alpha, TNFR, iNOS, cytochrome C, caspase-3, TLR4, IL-6 and IL10 were evaluated. The following groups were used in this study: Group 1 - Control Group (CTRL) animals received AdßGal vehicle; Group 2 - Infected Group (TC) animals were infected with T. cruzi; Group 3 - Immunized Group (AdASP-2): animals were immunized by AdASP-2 vaccine; Group 4 - Immunized and Infected Group (AdASP-2+TC) animals were infected with T. cruzi and immunized by AdSP-2 vaccine. A significant decrease of amastigote nests was noticed in the group of animals that were immunized with AdASP-2 and infected on the same day. COX-2 and TNF-alpha gene expressions increased in TC group, whereas TNF-alpha decreased in the TC+AdASP-2 group. TNFR expression was high in AdASP-2+TC group. iNOS expression was high for all experimental groups whereas cytochrome C decreased for all experimental groups. Caspase 3 increased in TC and TC+AdASP-2 groups. The gene expression of TLR4 and IL-10 showed an increase in AdASP-2+TC group. Finally, hepatic fibrosis was noticed to TC and AdASP-2 + TC groups. Taken together, our results demonstrated that vaccination with AdASP-2 was effective against the acute phase of experimental Chagas disease as a result of a more powerful and rapid immune response closely related to expression of some inflammatory genes, such as iNOS, TNF-alpha, TLR 4, and IL-10.

BST2 inhibits infection of influenza A virus by promoting apoptosis of infected cells.

BST2 is an antiviral factor that inhibits the release of enveloped virus at the plasma membrane via an unusual topology in which its N-terminal is in the cytosol while its C-terminal is anchored by glycophosphatidylinositol (GPI). BST2-deficient cells showed substantially higher release of virions than wild type cells. Influenza-infected BST2-deficient cells showed greatly reduced cytopathic effect (CPE) than wild type cells despite their generally robust virus production. This finding prompted us to determine whether BST2 was involved in the apoptotic process of virus-infected host cells. Our results revealed that BST2 might be involved in IRE1α-mediated ER stress pathway by increasing spliced form XBP-1. Consequently, levels of cytochrome C, caspase-3, caspase-9, and PARP as representative molecules of apoptosis were significantly increased in wild type cells than those in BST2-deficient cells. These results suggest that BST2 might participate in innate host defense by augmenting ER-stress-induced apoptotic signaling to inhibit the replication and spread of virus.

Rv3033, as an Emerging Anti-apoptosis Factor, Facilitates Mycobacteria Survival via Inhibiting Macrophage Intrinsic Apoptosis.

Apoptosis inhibition is a critical strategy of mycobacteria facilitating its survival in macrophages, but the underlying mechanism is not completely understood. In this study, we found that Rv3033, a secreted virulence factor of mycobacteria, played an important role in bacillary survival within macrophages. Forced over-expressed of Rv3033 in macrophages could efficiently resist mycobacteria-induced early and late apoptosis, accompanied with the obvious increased cellular bacterial burden. By exploring the underlying mechanism, we found that Rv3033 efficiently repressed the intrinsic (caspase-9 meditated), but not the extrinsic (caspase-8 mediated) apoptotic pathway in mycobacteria-infected macrophages. And this repression relied on the orchestrating blockade of both mitochondrial cytochrome c release and endoplasmic reticulum (ER) stress PERK branch activation. Our study uncovered a novel function of mycobacterial virulence factor Rv3033 as an anti-apoptotic protein, which may provide a new target for tuberculosis (TB) treatment.

Surfactant protein D regulates caspase-8-mediated cascade of the intrinsic pathway of apoptosis while promoting bleb formation.

Surfactant-associated protein D (SP-D) is a soluble innate immune collectin present on many mucosal surfaces. We recently showed that SP-D suppresses the extrinsic pathway of apoptosis by downregulating caspase-8 activation. However, the effects of SP-D on the intrinsic pathway of apoptosis are not clearly understood. In the intrinsic pathway, cytochrome c is released by mitochondria into the cytoplasm. Oxidation of cytochrome c by cytochrome c oxidase activates the apoptosome and caspase-9 cascade. Both caspase-8- and caspase-9-mediated branches are activated in the intrinsic pathway of apoptosis; however, little is known about the relevance of the caspase-8 pathway in this context. Here we studied the effects of SP-D on different branches of the intrinsic pathway of apoptosis using UV-irradiated Jurkat T-cells. We found that SP-D does not inhibit the caspase-9 branch of apoptosis and the relevance of the caspase-8-related branch became apparent when the caspase-9 pathway was inhibited by blocking cytochrome c oxidase. Under these conditions, SP-D reduces the activation of caspase-8, executioner caspase-3 and exposure of phosphatidylserine (PS) on the membranes of dying cells. By contrast, SP-D increases the formation of nuclear and membrane blebs. Inhibition of caspase-8 confirms the effect of SP-D is unique to the caspase-8 pathway. Overall, SP-D suppresses certain aspects of the intrinsic pathway of apoptosis via reduction of caspase-8 activation and PS flipping while at the same time increasing membrane and nuclear bleb formation. This novel regulatory aspect of SP-D could help to regulate intrinsic pathway of apoptosis to promote effective blebbing and breakdown of dying cells.

Divergent roles of three cytochrome c in CTSB-modulating coelomocyte apoptosis in Apostichopus japonicus.

Cytochrome c plays crucial roles in apoptosis and the immune response. We previously demonstrated that cathepsin B from Apostichopus japonicus (AjCTSB) induces coelomocyte apoptosis. However, the mechanistic explanation and the regulation of this process have not been investigated. In the present study, we identified three cytochrome c cDNAs from A. japonicus (designated Ajcytc1, Ajcytc-1, and Ajcytc-2) using expressed sequence tag- (EST) and RACE-based approaches. The deduced amino acid sequences of the three cytochrome isoforms contained conserved CXXCH motifs, which are involved in binding heme and maintaining proteolytic activity. Time course expression analysis in vitro and in vivo revealed that the three cytochrome isoforms were induced upon pathogen challenge and LPS exposure. More importantly, AjCTSB knockdown by siRNA dramatically increased mitochondrial membrane potential (ΔΨm) in a time-dependent manner based on JC-1 fluorescent probe staining. AjCTSB knockdown also resulted in decreased expression of these three cytochromes 24 h after siAjCTSB transfection. Functional analysis using isoform-specific siRNAs revealed that Ajcytc-1, but not Ajcytc1 or Ajcytc-2, is involved in coelomocyte apoptosis. Moreover, the transcript level of Ajcaspase-3, an apoptosis executioner, was also consistently down-regulated upon silencing of Ajcytc-1 but not Ajcytc1 or Ajcytc-2. Collectively, these results indicate that Ajcytc1, Ajcytc-1, and Ajcytc-2 play distinct roles in mediating the immune response to bacteria according to AjCTSB expression. Moreover, Ajcytc-1 could be released upon dissipation of the ΔΨm, which could further trigger coelomocyte apoptosis through the activation of Ajcaspase-3.

Early markers for myocardial ischemia and sudden cardiac death.

The post-mortem diagnosis of acute myocardial ischemia remains a challenge for both clinical and forensic pathologists. We performed an experimental study (ligation of left anterior descending coronary artery in rats) in order to identify early markers of myocardial ischemia, to further apply to forensic and clinical pathology in cases of sudden cardiac death. Using immunohistochemistry, Western blots, and gene expression analyses, we investigated a number of markers, selected among those which are currently used in emergency departments to diagnose myocardial infarction and those which are under investigation in basic research and autopsy pathology studies on cardiovascular diseases. The study was performed on 44 adult male Lewis rats, assigned to three experimental groups: control, sham-operated, and operated. The durations of ischemia ranged between 5 min and 24 h. The investigated markers were troponins I and T, myoglobin, fibronectin, C5b-9, connexin 43 (dephosphorylated), JunB, cytochrome c, and TUNEL staining. The earliest expressions (≤30 min) were observed for connexin 43, JunB, and cytochrome c, followed by fibronectin (≤1 h), myoglobin (≤1 h), troponins I and T (≤1 h), TUNEL (≤1 h), and C5b-9 (≤2 h). By this investigation, we identified a panel of true early markers of myocardial ischemia and delineated their temporal evolution in expression by employing new technologies for gene expression analysis, in addition to traditional and routine methods (such as histology and immunohistochemistry). Moreover, for the first time in the autopsy pathology field, we identified, by immunohistochemistry, two very early markers of myocardial ischemia: dephosphorylated connexin 43 and JunB.

Superresolution imaging reveals nanometer- and micrometer-scale spatial distributions of T-cell receptors in lymph nodes.

T cells become activated when T-cell receptors (TCRs) recognize agonist peptides bound to major histocompatibility complex molecules on antigen-presenting cells. T-cell activation critically relies on the spatiotemporal arrangements of TCRs on the plasma membrane. However, the molecular organizations of TCRs on lymph node-resident T cells have not yet been determined, owing to the diffraction limit of light. Here we visualized nanometer- and micrometer-scale TCR distributions in lymph nodes by light sheet direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). This dSTORM and SIM approach provides the first evidence, to our knowledge, of multiscale reorganization of TCRs during in vivo immune responses. We observed nanometer-scale plasma membrane domains, known as protein islands, on naïve T cells. These protein islands were enriched within micrometer-sized surface areas that we call territories. In vivo T-cell activation caused the TCR territories to contract, leading to the coalescence of protein islands and formation of stable TCR microclusters.

Thermo-sensitive imprinted polymer embedded carbon dots using epitope approach.

A new type of thermo-sensitive receptor carbon dots/SiO2/molecularly imprinted polymer (CDs/SiO2/MIP) was prepared by surface imprinting procedure and the epitope approach. The synthetic CDs/SiO2/MIP was able to selectively capture target protein with fluorescence quenching via the special interaction between them and the recognition cavities. The receptor exhibited the linear fluorescence quenching to cytochrome c (cyt c) in the range of 0.1-40 µM, and the detection limit was 89 nM. The precision for five replicate detection of cyt c at 20 µM was 3.11%. Moreover, the receptor owned the temperature-sensitive element that allowed for swelling and shrinking in response to temperature changes to realize recognition of the target cytochrome c. The proposed strategy revealed the feasibility of fabrication of a thermo-sensitive imprinted polymer based on CDs and surface imprinting procedure and the epitope approach.

A universal polysaccharide conjugated vaccine against O111 E. coli.

E. coli O111 strains are responsible for outbreaks of blood diarrhea and hemolytic uremic syndrome throughout the world. Because of their phenotypic variability, the development of a vaccine against these strains which targets an antigen that is common to all of them is quite a challenge. Previous results have indicated, however, that O111 LPS is such a candidate, but its toxicity makes LPS forbidden for human use. To overcome this problem, O111 polysaccharides were conjugated either to cytochrome C or to EtxB (a recombinant B subunit of LT) as carrier proteins. The O111-cytochrome C conjugate was incorporated in silica SBA-15 nanoparticles and administered subcutaneously in rabbits, while the O111-EtxB conjugate was incorporated in Vaxcine(TM), an oil-based delivery system, and administered orally in mice. The results showed that one year post-vaccination, the conjugate incorporated in silica SBA-15 generated antibodies in rabbits able to inhibit the adhesion of all categories of O111 E. coli to epithelial cells. Importantly, mice immunized orally with the O111-EtxB conjugate in Vaxcine(TM) generated systemic and mucosal humoral responses against all categories of O111 E. coli as well as antibodies able to inhibit the toxic effect of LT in vitro. In summary, the results obtained by using 2 different approaches indicate that a vaccine that targets the O111 antigen has the potential to prevent diarrhea induced by O111 E. coli strains regardless their mechanism of virulence. They also suggest that a conjugated vaccine that uses EtxB as a carrier protein has potential to combat diarrhea induced by ETEC.

T cell immunodominance is dictated by the positively selecting self-peptide.

Naive T cell precursor frequency determines the magnitude of immunodominance. While a broad T cell repertoire requires diverse positively selecting self-peptides, how a single positively selecting ligand influences naive T cell precursor frequency remains undefined. We generated a transgenic mouse expressing a naturally occurring self-peptide, gp250, that positively selects an MCC-specific TCR, AND, as the only MHC class II I-E(k) ligand to study the MCC highly organized immunodominance hierarchy. The single gp250/I-E(k) ligand greatly enhanced MCC-tetramer(+) CD4(+) T cells, and skewed MCC-tetramer(+) population toward V11α(+)Vß3(+), a major TCR pair in MCC-specific immunodominance. The gp250-selected V11α(+)Vß3(+) CD4(+) T cells had a significantly increased frequency of conserved MCC-preferred CDR3 features. Our studies establish a direct and causal relationship between a selecting self-peptide and the specificity of the selected TCRs. Thus, an immunodominant T cell response can be due to a dominant positively selecting self-peptide. DOI: http://dx.doi.org/10.7554/eLife.01457.001.

Mutations in subunit interface and B-cell epitopes improve antileukemic activities of Escherichia coli asparaginase-II: evaluation of immunogenicity in mice.

L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.

12/15-lipoxygenase expressed in non-epithelial cells causes airway epithelial injury in asthma.

The mechanisms underlying asthmatic airway epithelial injury are not clear. 12/15-lipoxygenase (an ortholog of human 15-LOX-1), which is induced by IL-13, is associated with mitochondrial degradation in reticulocytes at physiological conditions. In this study, we showed that 12/15-LOX expressed in nonepithelial cells caused epithelial injury in asthma pathogenesis. While 12/15-LOX overexpression or IL-13 administration to naïve mice showed airway epithelial injury, 12/15-LOX knockout/knockdown in allergic mice reduced airway epithelial injury. The constitutive expression of 15-LOX-1 in bronchial epithelia of normal human lungs further indicated that epithelial 15-LOX-1 may not cause epithelial injury. 12/15-LOX expression is increased in various inflammatory cells in allergic mice. Though non-epithelial cells such as macrophages or fibroblasts released 12/15-LOX metabolites upon IL-13 induction, bronchial epithelia didn't release. Further 12-S-HETE, arachidonic acid metabolite of 12/15-LOX leads to epithelial injury. These findings suggested 12/15-LOX expressed in non-epithelial cells such as macrophages and fibroblasts leads to bronchial epithelial injury.

Macroautophagy substrates are loaded onto MHC class II of medullary thymic epithelial cells for central tolerance.

Macroautophagy serves cellular housekeeping and metabolic functions through delivery of cytoplasmic constituents for lysosomal degradation. In addition, it may mediate the unconventional presentation of intracellular antigens to CD4(+) T cells; however, the physiological relevance of this endogenous MHC class II loading pathway remains poorly defined. Here, we characterize the role of macroautophagy in thymic epithelial cells (TECs) for negative selection. Direct presentation for clonal deletion of MHC class II-restricted thymocytes required macroautophagy for a mitochondrial version of a neo-antigen, but was autophagy-independent for a membrane-bound form. A model antigen specifically expressed in Aire(+) medullary TECs (mTECs) induced efficient deletion via direct presentation when targeted to autophagosomes, whereas interference with autophagosomal routing of this antigen through exchange of a single amino acid or ablation of an essential autophagy gene abolished direct presentation for negative selection. Furthermore, when this autophagy substrate was expressed by mTECs in high amounts, endogenous presentation and indirect presentation by DCs operated in a redundant manner, whereas macroautophagy-dependent endogenous loading was essential for clonal deletion at limiting antigen doses. Our findings suggest that macroautophagy supports central CD4(+) T cell tolerance through facilitating the direct presentation of endogenous self-antigens by mTECs.

Mapping of discontinuous conformational epitopes by amide hydrogen/deuterium exchange mass spectrometry and computational docking.

Understanding antigen-antibody interactions at the sub-molecular level is of particular interest for scientific, regulatory, and intellectual property reasons, especially with increasing demand for monoclonal antibody therapeutic agents. Although various techniques are available for the determination of an epitope, there is no widely applicable, high-resolution, and reliable method available. Here, a combination approach using amide hydrogen/deuterium exchange coupled with proteolysis and mass spectrometry (HDX-MS) and computational docking was applied to investigate antigen-antibody interactions. HDX-MS is a widely applicable, medium-resolution, medium-throughput technology that can be applied to epitope identification. First, the epitopes of cytochrome c-E8, IL-13-CNTO607, and IL-17A-CAT-2200 interactions identified using the HDX-MS method were compared with those identified by X-ray co-crystal structures. The identified epitopes are in good agreement with those identified using high-resolution X-ray crystallography. Second, the HDX-MS data were used as constraints for computational docking. More specifically, the non-epitope residues of an antigen identified using HDX-MS were designated as binding ineligible during computational docking. This approach, termed HDX-DOCK, gave more tightly clustered docking poses than stand-alone docking for all antigen-antibody interactions examined and improved docking results significantly for the cytochrome c-E8 interaction.

Potential mechanisms for hypoalgesia induced by anti-nerve growth factor immunoglobulin are identified using autoimmune nerve growth factor deprivation.

Nerve growth factor (NGF) antagonism has long been proposed as a chronic pain treatment. In 2010, the FDA suspended clinical trials using tanezumab, a humanized monoclonal anti-NGF antibody, to treat osteoarthritis due to worsening joint damage in 16 patients. Increased physical activity in the absence of acute pain which normally prevents self-harm was purported as a potential cause. Such an adverse effect is consistent with an extension of tanezumab's primary mechanism of action by decreasing pain sensitivity below baseline levels. In animal inflammatory pain models, NGF antagonism decreases intraepidermal nerve fiber (IENF) density and attenuates increases in expression of nociception-related proteins, such as calcitonin gene-related peptide (CGRP) and substance P (SP). Little is known of the effects of NGF antagonism in noninflamed animals and the hypoalgesia that ensues. In the current study, we immunized rats with NGF or cytochrome C (cytC) and examined (1) nocifensive behaviors with thermal latencies, mechanical thresholds, the hot plate test, and the tail flick test, (2) IENF density, and (3) expression of CGRP, SP, voltage-gated sodium channel 1.8 (Nav1.8), and glutaminase in subpopulations of dorsal root ganglion (DRG) neurons separated by size and isolectin B4 (IB4) labeling. Rats with high anti-NGF titers had delayed responses on the hot plate test but no other behavioral abnormalities. Delayed hot plate responses correlated with lower IENF density. CGRP and SP expression was decreased principally in medium (400-800 µm(2)) and small neurons (<400 µm(2)), respectively, regardless of IB4 labeling. Expression of Nav1.8 was only decreased in small and medium IB4 negative neurons. NGF immunization appears to result in a more profound antagonism of NGF than tanezumab therapy, but we hypothesize that decreases in IENF density and nociception-related protein expression are potential mechanisms for tanezumab-induced hypoalgesia.

Cur l 3, a major allergen of Curvularia lunata-derived short synthetic peptides, shows promise for successful immunotherapy.

Allergens with reduced IgE binding and intact T cell reactivity are required for safety and efficacy of immunotherapy (IT). Curvularia lunata is an important fungus for respiratory allergic disorders having cross-reactive and specific allergens. Previously, we have identified major allergens-namely, Cur l 1 (31 kD, serine protease), Cur l 2 (48 kD, enolase), and Cur l 3 (12 kD, cytochrome c)-from this fungus. Furthermore, Cur l 3 epitope-peptide, P6, showed immunogenicity and higher IgE binding, where cysteine and histidine were observed to be vital for IgE binding. Thus, this peptide and three derivatives with reduced IgE binding were selected for analysis in mice. In the present study, the effect of IT was assessed with Cur l 3, P6, its derivatives (P6.1-6.3), and P10 in a mouse model of allergy. IT with P6.2 and P10 reduced IgE and IgG1 levels significantly (P < 0.05), with increase in IgG2a levels as compared to other antigens. There was a significant reduction of IL-4 level associated with increased IFN-γ after IT. Airway inflammation was reduced significantly in terms of eosinophil counts in lung tissue and bronchoalveolar lavage fluid. IT with P6 and P6.2 induced significantly higher IL-10 secretion than baseline after 40 days of treatment. Generally, the effect of IT was more pronounced after 40 days than after 10 days of treatment. In summary, the modified peptide, P6.2, with reduced IgE binding, but intact immunogenicity, showed promise for successful IT.

Structural basis of specificity and cross-reactivity in T cell receptors specific for cytochrome c-I-E(k).

T cells specific for the cytochrome c Ag are widely used to investigate many aspects of TCR specificity and interactions with peptide-MHC, but structural information has long been elusive. In this study, we present structures for the well-studied 2B4 TCR, as well as a naturally occurring variant of the 5c.c7 TCR, 226, which is cross-reactive with more than half of possible substitutions at all three TCR-sensitive residues on the peptide Ag. These structures alone and in complex with peptide-MHC ligands allow us to reassess many prior mutagenesis results. In addition, the structure of 226 bound to one peptide variant, p5E, shows major changes in the CDR3 contacts compared with wild-type, yet the TCR V-region contacts with MHC are conserved. These and other data illustrate the ability of TCRs to accommodate large variations in CDR3 structure and peptide contacts within the constraints of highly conserved TCR-MHC interactions.