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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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 AdβGal 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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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-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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

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.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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.

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(+)Vβ3(+), a major TCR pair in MCC-specific immunodominance. The gp250-selected V11α(+)Vβ3(+) 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.

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.

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.

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.

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., (Copyright © 2012 John Wiley & Sons, Ltd.)

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.

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., (Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

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.

Although a direct causative pathway from the gene mutation to the selective neostriatal neurodegeneration remains unclear in Huntington's disease (HD), one putative pathological mechanism reported to play a prominent role in the pathogenesis of this neurological disorder is mitochondrial dysfunction. We examined mitochondria in preferentially vulnerable striatal calbindin-positive neurons in moderate-to-severe grade HD patients, using antisera against mitochondrial markers of COX2, SOD2 and cytochrome c. Combined calbindin and mitochondrial marker immunofluorescence showed a significant and progressive grade-dependent reduction in the number of mitochondria in spiny striatal neurons, with marked alteration in size. Consistent with mitochondrial loss, there was a reduction in COX2 protein levels using western analysis that corresponded with disease severity. In addition, both mitochondrial transcription factor A, a regulator of mtDNA, and peroxisome proliferator-activated receptor-co-activator gamma-1 alpha, a key transcriptional regulator of energy metabolism and mitochondrial biogenesis, were also significantly reduced with increasing disease severity. Abnormalities in mitochondrial dynamics were observed, showing a significant increase in the fission protein Drp1 and a reduction in the expression of the fusion protein mitofusin 1. Lastly, mitochondrial PCR array profiling in HD caudate nucleus specimens showed increased mRNA expression of proteins involved in mitochondrial localization, membrane translocation and polarization and transport that paralleled mitochondrial derangement. These findings reveal that there are both mitochondrial loss and altered mitochondrial morphogenesis with increased mitochondrial fission and reduced fusion in HD. These findings provide further evidence that mitochondrial dysfunction plays a critical role in the pathogenesis of HD.

T cell receptor (TCR) ligation is required for the extrathymic differentiation of forkhead box p3(+) (Foxp3(+)) regulatory T cells. Several lines of evidence indicate that weak TCR stimulation favors induction of Foxp3 in the periphery; however, it remains to be determined how TCR ligand potency influences this process. We characterized the density and affinity of TCR ligand favorable for Foxp3 induction and found that a low dose of a strong agonist resulted in maximal induction of Foxp3 in vivo. Initial Foxp3 induction by weak agonist peptide could be enhanced by disruption of TCR-peptide major histocompatibility complex (pMHC) interactions or alteration of peptide dose. However, time course experiments revealed that Foxp3-positive cells induced by weak agonist stimulation are deleted, along with their Foxp3-negative counterparts, whereas Foxp3-positive cells induced by low doses of the strong agonist persist. Our results suggest that, together, pMHC ligand potency, density, and duration of TCR interactions define a cumulative quantity of TCR stimulation that determines initial peripheral Foxp3 induction. However, in the persistence of induced Foxp3(+) T cells, TCR ligand potency and density are noninterchangeable factors that influence the route to peripheral tolerance.

A total on-line analysis of a target protein from a plasma sample was made using a selective immunoextraction step coupled on-line to an immobilized enzymatic reactor (IMER) for the protein digestion followed by LC-MS/MS analysis. For the development of this device, cytochrome c was chosen as model protein due to its well-known sequence. An immunosorbent (IS) based on the covalent immobilization of anti-cytochrome c antibodies on a solid support was made and an immunoextraction procedure was carefully developed to assess a selective extraction of the target protein from plasma. For the first time, IS was easily coupled on-line with a laboratory-made IMER based on pepsin. The whole on-line device (IS-IMER-LC-MS/MS) allowed the quantification of cytochrome c from 8.5pmol to 1.7nmol in buffer medium. Finally, this device was applied to the analysis of only 85pmol of cytochrome c from plasma with a RSD value lower than 10% (n=3)., (2009 Elsevier B.V. All rights reserved.)

Distinct dendritic cell (DC) subsets differ with respect to pathways of Ag uptake and intracellular routing to MHC class I or MHC class II molecules. Murine studies suggest a specialized role for CD8alpha(+) DC in cross-presentation, where exogenous Ags are presented on MHC class I molecules to CD8(+) T cells, while CD8alpha(-) DC are more likely to present extracellular Ags on MHC class II molecules to CD4(+) T cells. As a proportion of CD8alpha(+) DC have been shown to express langerin (CD207), we investigated the role of langerin(+)CD8alpha(+) DC in presenting Ag and priming T cell responses to soluble Ags. When splenic DC populations were sorted from animals administered protein i.v., the ability to cross-present Ag was restricted to the langerin(+) compartment of the CD8alpha(+) DC population. The langerin(+)CD8alpha(+) DC population was also susceptible to depletion following administration of cytochrome c, which is known to trigger apoptosis if diverted to the cytosol. Cross-priming of CTL in the presence of the adjuvant activity of the TLR2 ligand N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-Cys-[S]-Serl-[S]-Lys4-trihydrochloride or the invariant NKT cell ligand alpha-galactosylceramide was severely impaired in animals selectively depleted of langerin(+) cells in vivo. The production of IL-12p40 in response to these systemic activation stimuli was restricted to langerin(+)CD8alpha(+) DC, and the release of IL-12p70 into the serum following invariant NKT cell activation was ablated in the absence of langerin(+) cells. These data suggest a critical role for the langerin(+) compartment of the CD8alpha(+) DC population in cross-priming and IL-12 production.

During the germinal centre reaction (GC), B cells with non-functional or self-reactive antigen receptors are negatively selected by apoptosis to generate B cell repertoire with appropriate antigen specificities. We studied the molecular mechanism of Fas/CD95- and B cell receptor (BCR)-induced apoptosis to shed light on the signalling events involved in the negative selection of GC B cells. As an experimental model, we used human follicular lymphoma (FL) cell line HF1A3, which originates from a GC B cell, and transfected HF1A3 cell lines overexpressing Bcl-x(L), c-FLIP(long) or dominant negative (DN) caspase-9. Fas-induced apoptosis was dependent on the caspase-8 activation, since the overexpression of c-FLIP(long), a natural inhibitor of caspase-8 activation, blocked apoptosis induced by Fas. In contrast, caspase-9 activation was not involved in Fas-induced apoptosis. BCR-induced apoptosis showed the typical characteristics of mitochondria-dependent (intrinsic) apoptosis. Firstly, the activation of caspase-9 was involved in BCR-induced DNA fragmentation, while caspase-8 showed only marginal role. Secondly, overexpression of Bcl-x(L) could block all apoptotic changes induced by BCR. As a novel finding, we demonstrate that caspase-9 can enhance the cytochrome-c release and collapse of mitochondrial membrane potential (DeltaPsi(m)) during BCR-induced apoptosis. The requirement of different signalling pathways in apoptosis induced by BCR and Fas may be relevant, since Fas- and BCR-induced apoptosis can thus be regulated independently, and targeted to different subsets of GC B cells.

Full T cell activation requires TCR engagement (signal 1) in the context of costimulation (signal 2). Costimulation is required for maximal expression of effector cytokines and prevention of T cell anergy. It has become increasingly clear that another major function of costimulation is to up-regulate the metabolic machinery necessary for T cell function. In this report we demonstrate that anergic T cells are metabolically anergic, in that upon full stimulation (signals 1 plus 2) they fail to up-regulate the machinery necessary to support increased metabolism. These findings suggest that one mechanism responsible for the maintenance of T cell anergy is failure to up-regulate the metabolic machinery. Furthermore, we demonstrate that by blocking leucine, glucose, and energy metabolism, T cell activation is mitigated. Additionally, inhibition of these metabolic pathways during T cell activation leads to anergy in Th1-differentiated cells. Overall, our findings extend the role of T cell metabolism in regulating T cell function.

Yersinia species during infection adhere to host immune cells primarily to macrophages and employ its secretary proteins known as Yersinia outer proteins to trigger death in infected cells. In the present study, it is shown that recombinant Yersinia outer protein J (rYopJ) could induce apoptosis in murine peritoneal macrophages in vitro as assessed by morphological features, internucleosomal DNA fragmentation, change in mitochondrial membrane potential (MMP) (Deltapsim), activation of caspases and Annexin V binding. rYopJ-induced cell death was dose and time dependent. Pre-treatment with broad-spectrum caspase inhibitor Z-VAD-FMK, caspase-3 inhibitor Ac-DEVD-CHO and caspase-8 inhibitor Z-IETD-FMK prevented the change in MMP and DNA fragmentation, suggesting caspase-dependent apoptosis of rYopJ-treated macrophages. Blocking the endocytosis by pre-treatment of cells with cytochalasin B did not prevent the rYopJ-induced macrophages apoptosis. The data further suggest that rYopJ-induced apoptosis is mediated by molecules upstream of caspase-8 and relay through mitochondrial pathway involving Bax, Bcl-2, activation of caspase-8 and caspase-3, Bid and polyadenosine diphosphate-ribose polymerase cleavage, cytochrome c release and DNA fragmentation.

This paper proposes a novel perfusion-based micro opto-fluidic system (PMOFS) as a reusable immunosensor for in-situ and continuous protein detection. The PMOFS includes a fiber optic interferometry (FOI) sensor housed in a micro-opto-fluidic chip covered with a microdialysis membrane. It features a surface regeneration mechanism for continuous detection. Gold nanoparticles (GNPs) labeled anti-rabbit IgG were used to enhance the immune conjugation signal by the elongated optical path from GNPs conjugation. Surface regeneration of the sensor was achieved through local pH level manipulation by means of a photoactive molecule, o-Nitrobenzaldehyde (o-NBA), which triggered the elution of immune complexes. Experimental results showed that the pH level of the o-NBA solution can be reduced from 7 to 3.5 within 20 seconds under UV irradiation, sufficient for an effective elution process. The o-NBA molecules, contained within poly(ethylene glycol) diacrylate (PEG) complexes, were trapped within the sensing compartment by the microdialysis membrane and would not leak into the outside environment. The pH variation was also limited in the neighborhood of the sensor surface, resulting in a self-contained sensing system. In-situ immune detection and surface regeneration of the sensing probe has been successfully carried out for two identical cycles by the same sensing probe, and the cycle time can be less than 8 minutes, which is so far the fastest method for continuous monitoring on protein/peptide molecules. In addition, the interference fringe shift of the sensor is linearly related to the concentration of anti-cytochrome C antibody solution and the detection limit approaches 10 ng/ml.

The knowledge on epitopes of proteins can help in devising new therapeutic modalities for allergic disorders. In the present study, five B (P1-P5) and five T cell (P6-P10) epitopes were predicted in silico based on sequence homology model of Cur l 3, a major allergen of Curvularia lunata. Peptides (epitopes) were synthesized and assessed for biological activity by ELISA, competitive ELISA, lymphoproliferation and cytokine profiling using Curvularia allergic patients' sera. B cell peptides showed higher IgE binding by ELISA than T cell epitopes except P6. Peptides P1-P6 achieved EC(50) at 100 ng, whereas P7-P10 required 10 mug in inhibition assays. Peripheral blood mononuclear cells from Curvularia allergic patients (n = 20) showed higher lymphoproliferation for T cell epitopes than B cell epitopes except P6 confirming the properties of B and T cell prediction. The supernatant from these patients show highest interleukin-4 release on stimulation with P6 followed by B cell peptides. P4 and P6 together identified 35/37 of Curvularia positive patients by skin tests. In summary, experimental analysis confirmed in silico predicted epitopes containing important antigenic regions of Cur l 3. P6, a predicted T cell epitope, showed the presence of a cryptic B cell epitope. Peptides P4 and P6 have potential for clinical application. The approach used here is relevant and may be used to delineate epitopes of other proteins.

We previously elucidated an important role for gangliosides in renal cell carcinoma-mediated T lymphocyte apoptosis, although the mechanism by which they mediated lymphocyte death remained unclear. Here, we show that when added in purified form, GD3 is internalized by activated T cells, initiating a series of proapoptotic events, including the induction of reactive oxygen species (ROS), an enhancement of p53 and Bax accumulation, an increase in mitochondrial permeability, cytochrome c release, and the activation of caspase-9. GD3-induced apoptosis of activated T cells was dose dependent and inhibitable by pretreating the lymphocytes with N-acetylcysteine, cyclosporin A, or bongkrekic acid, emphasizing the essential role of ROS and mitochondrial permeability to the process. Ganglioside-induced T-cell killing was associated with the caspase-dependent degradation of nuclear factor-kappaB-inducible, antiapoptotic proteins, including RelA; this suggests that their loss is initiated only after the cascade is activated and that their disappearance amplifies but not triggers GD3 susceptibility. Resting T cells did not internalize appreciable levels of GD3 and did not undergo any of the proapoptotic changes that characterize activated T lymphocytes exposed to the ganglioside. RelA overexpression endows Jurkat cells with resistance to GD3-mediated apoptosis, verifying the role of the intact transcription factor in mediating protection from the ganglioside.

Recent studies have implicated the cell surface receptor Programmed Death-1 (PD-1) in numerous models of T cell anergy, though the specific mechanisms by which the PD-1 signal maintains tolerance is not clear. We demonstrate that the depletion of PD-1 with siRNA results in a complete reversal of clonal anergy in the A.E7 T cell model, suggesting that the mechanism by which PD-1 maintains the anergic phenotype is a T-cell-intrinsic phenomenon, and not one dependent on other cell populations in vivo. We have also shown that the neutralization of IL-2 during restimulation abrogates the effect of PD-1 depletion, suggesting that tolerance mediated by PD-1 is wholly IL-2 dependent, and likewise intrinsic to the tolerized cells.

Effective subunit vaccines must elicit strong CD4(+) T cell responses. In this issue of Immunity, Malherbe et al. (2008) find that the ability of adjuvants to stimulate high-avidity T cell responses correlates with Toll-like-receptor engagement.

How T cell receptor (TCR) specificity evolves in vivo after protein vaccination is central to the development of helper T (Th) cell function. Most models of clonal selection in the Th cell compartment favor TCR affinity-based thresholds. Here, we demonstrated that depot-forming vaccine adjuvants did not require Toll-like receptor (TLR) agonists to induce clonal dominance in antigen-specific Th cell responses. However, readily dispersible adjuvants using TLR-9 and TLR-4 agonists skewed TCR repertoire usage by increasing TCR selection thresholds and enhancing antigen-specific clonal expansion. In this manner, vaccine adjuvants control the local accumulation of Th cells expressing TCR with the highest peptide MHC class II binding. Clonal composition was altered by mechanisms that blocked the local propagation of clonotypes independently of antigen dose and not as a consequence of interclonal competition. This capacity of adjuvants to modify antigen-specific Th cell clonal composition has fundamental implications for the design of future protein subunit vaccines.

Cross-presentation as a fundamental pathway of activating CD8(+) T cells has been well established. So far the application of this concept in vivo is limited, and the mechanisms that specialize CD8(+) dendritic cells (DCs) for this task are not fully understood. Here we take advantage of the specific cytosolic export feature of cross-presenting DCs together with the property of cytosolic cytochrome c (cyt c) in initiating Apaf-1-dependent apoptosis selectively in cross-presenting DCs. A single i.v. injection of cyt c in B6 mice produced a 2- to 3-fold reduction in splenic CD8(+) DCs but not in Apaf-1-deficient mice. Functional studies both in vivo and in vitro showed that cyt c profoundly abrogated OVA-specific CD8(+) T cell proliferation through its apoptosis-inducing effect on cross-presenting DCs. More importantly, in vivo injection of cyt c abolished the induction of cytotoxic T lymphocytes to exogenous antigen and reduced subsequent immunity to tumor challenge. In addition, only a proportion of CD8(+) DCs that express abundant IL-12 and Toll-like receptor 3 were efficient cross-presenters. Our data support the hypothesis that cross-presentation in vivo requires cytosolic diversion of endocytosed proteins, conferring cross-presentation specialization to a proportion of CD8(+) DCs. We propose that DCs incapable of such transfer, even within the CD8(+) DC subset, are unable to cross-present. Our model opens an avenue to specifically target cross-presenting DCs in vivo for manipulating cytotoxic T lymphocyte responses toward infections, tumors, and transplants.

Background: Recombinant allergens are required for component-resolved diagnosis/therapy of allergic disorders. The study was aimed to express and characterize an allergenic protein from Curvularia lunata and study its cross-reactivity., Methods: A clone encoding a 12-kDa protein screened from the cDNA library of C. lunata was sequenced and expressed in pET22b+ vector. The purified protein was characterized by biophysical and immunological methods., Results: The sequence of gene encoding a 12-kDa protein showed homology to cytochrome c. It was expressed in Escherichia coli yielding 0.5 mg protein/l culture and designated as Cur l 3. The absorption and circular dichroism spectrum of Cur l 3 were similar to horse cytochrome c and the protein reacted with cytochrome c antibody. ELISA with different fungal-positive patients' sera showed > or = 3 times specific IgE to Cur l 3 compared with healthy controls. Mice anti-Cur l 3 reacted with tropical and temperate grass extracts. Protein also reacted with grass-positive patients' sera. In vitro stimulation of peripheral blood mononuclear cells from C. lunata, fungi or grass-positive patients with it released significant levels of Th2 cytokines. In vivo testing of this protein in allergic patients showed marked positive skin reactivity in 60% fungal and 43% grass-positive cases. Cross inhibition assays (EC(50)) demonstrated allergenic cross-reactivity of Cur l 3 with fungi and grasses., Conclusions: Cytochrome c, a major allergen from C. lunata was cloned, sequenced and expressed. It was identified as a cross-reactive allergen among fungi and grasses and has potential for clinical application.

Glucocorticoid hormone (GC) production by thymic epithelial cells influences TcR signalling in DP thymocytes and modifies their survival. In the present work, we focused on exploring details of GC effects on DP thymocyte apoptosis with or without parallel TcR activation in AND transgenic mice, carrying TcR specific for pigeon cytochrome C, in vivo. Here we show that the glucocorticoid receptor (GR) protein level was the lowest in DP thymocytes, and it was slightly down-regulated by GC analogue, anti-CD3, PCC and combined treatments as well. Exogenous GC analogue treatment or TcR stimulation alone lead to marked DP cell depletion, coupled with a significant increase of early apoptotic cell ratio (AnnexinV staining), marked abrogation of the mitochondrial function in DP cells (CMXRos staining), and significant decrease in the Bcl-2(high) DP thymocyte numbers, respectively. On the other hand, the simultaneous exposure to these two proapototic signals effectively reversed all the above-described changes. The parallel analysis of CD4 SP cell numbers, AnnexinV, CMXRos, Bcl-2 and GR stainings revealed, that the GR and TcR signals were not antagonistic on the mature thymocytes. These data provide experimental evidence in TcR transgenic mice, in vivo, that when TcR activation and GR signals are present simultaneously, they rescue double positive thymocytes from programmed cell death. The two separate signalling pathways merge in DP thymocytes at such important apoptosis regulating points as the Bcl-2 and GR, showing that their balanced interplay is essential in DP cell survival.

Natural killer (NK) cells play an important role in innate immunity against virally infected or transformed cells as the first defense line. Granzyme M (GzmM) is an orphan granzyme that is constitutively highly expressed in NK cells and is consistent with NK cell-mediated cytolysis. We recently demonstrated that GzmM induces caspase-dependent apoptosis with DNA fragmentation through direct cleavage of inhibitor of caspase-activated DNase (ICAD). However, the molecular mechanisms for GzmM-induced apoptosis are unclear. We found GzmM causes mitochondrial swelling and loss of mitochondrial transmembrane potential. Moreover, GzmM initiates reactive oxygen species (ROS) generation and cytochrome c release. Heat shock protein 75 (HSP75, also known as TRAP1) acts as an antagonist of ROS and protects cells from GzmM-mediated apoptosis. GzmM cleaves TRAP1 and abolishes its antagonistic function to ROS, resulting in ROS accumulation. Silencing TRAP1 through RNA interference increases ROS accumulation, whereas TRAP1 overexpression attenuates ROS production. ROS accumulation is in accordance with the release of cytochrome c from mitochondria and enhances GzmM-mediated apoptosis.

How vaccines control the development of antigen-specific effector and memory T helper cells is central to protective immunity but remains poorly understood. Here we found that protein vaccination selected high-affinity, CXCR5+ICOS(hi) follicular B-helper T cells (T(FH) cells) that developed in draining lymphoid tissue to regulate B cell responses. In the memory phase, reservoirs of antigen-specific CXCR5+ICOS(lo) T(FH) cells persisted with less effector activity but accelerated antigen-recall ability. This new compartment of memory T(FH) cells was retained in draining lymphoid sites with antigen-specific memory B cells, persistent complexes of peptide and major histocompatibility complex class II and continued expression of CD69. Thus, protein vaccination promotes B cell immunity by selecting high-affinity effector T(FH) cells and creating lymphoid reservoirs of antigen-specific memory T(FH) cells in vivo.

T cell sensitivity to antigen is intrinsically regulated during maturation to ensure proper development of immunity and tolerance, but how this is accomplished remains elusive. Here we show that increasing miR-181a expression in mature T cells augments the sensitivity to peptide antigens, while inhibiting miR-181a expression in the immature T cells reduces sensitivity and impairs both positive and negative selection. Moreover, quantitative regulation of T cell sensitivity by miR-181a enables mature T cells to recognize antagonists-the inhibitory peptide antigens-as agonists. These effects are in part achieved by the downregulation of multiple phosphatases, which leads to elevated steady-state levels of phosphorylated intermediates and a reduction of the T cell receptor signaling threshold. Importantly, higher miR-181a expression correlates with greater T cell sensitivity in immature T cells, suggesting that miR-181a acts as an intrinsic antigen sensitivity "rheostat" during T cell development.

The development of immune tolerance is dependent on the expression of self-peptides in the thymus and bone marrow during lymphocyte development. However, not all self-antigens are expressed in the thymus, particularly for proteins that become post-translationally modified during other biological processes in a cell. We have found that one such post-translational modification, the spontaneous conversion of an aspartic acid to isoaspartic acid (isoAsp), causes ignored self-antigens to become immunogenic. In order to determine the mechanism for this autoimmune response, pigeon cytochrome c peptide 88-104 (PCC p88-104) was synthesized with and without an isoaspartyl residue. Each form was digested with cathepsin D, an enzyme involved in antigen processing. The products of cathepsin digestion were dramatically different between the two forms of self-protein suggesting that cryptic self-peptides may be revealed to the immune system by natural modifications to self-proteins. This observation also held true if whole PCC protein contained isoaspartyl residues was digested with cathespsin D. Additionally, AND transgenic TCR T cells (recognizing PCC 88-104) proliferated to a greater extent in response to isoaspartyl PCC as compared to the normal form of PCC. These finding demonstrate the importance of post-translational modifications in shaping autoimmune responses in and the development of tolerance to self-proteins.

We reported previously that pigeon cytochrome c-derived peptides (Pan-IA), which bind broad ranges of MHC class II molecules efficiently, activate T helper (Th) function in mice. In an experimental model, Pan-IA DNA vaccines augmented antitumor immunity in tumor antigen-immunized mice. To elicit more potent antitumor immunity and to eradicate tumors in a therapeutic setting, Pan-IA-loaded dendritic cells (DCs) were inoculated in combination with vaccines including ovalbumin (OVA) antigen DNA in tumor-bearing mice. Seventy percent of the immunized mice survived tumor-free for at least 4 months after treatment. In contrast, mice vaccinated with OVA DNA, either with or without naïve DCs, did not eliminate the tumors and died within 5 weeks. Only in mice vaccinated with OVA DNA and Pan-IA-loaded DCs were both cytotoxic and helper responses specific for OVA induced at the spleen and tumor sites as well as at the vaccination sites. Furthermore, accumulation of OVA-specific CD4(+) and CD8(+) T lymphocytes and interferon-gamma-mediated anti-angiogenesis were observed in the tumors of these mice. Thus, the combined vaccination primed both tumor-specific cytotoxicity and helper immunity resulting in augmented tumor lysis ability and anti-angiogenic effects. This is the first report to show that most established tumors were successfully eradicated by collaboration of potent antitumor immunity and anti-angiogenic effects by vaccination with tumor antigens and helper-activating analogs. This novel vaccination strategy is broadly applicable, regardless of identifying helper epitopes in target molecules, and contributes to the development of therapeutic cancer vaccines.

Remarkably, apoptosis was induced by exposing peritoneal resident macrophages (PRM) of C3H mice, but not other strains of mice, to ionizing radiation. The molecular mechanism of this strain-specific apoptosis in PRM was studied. The apoptosis elicited in C3H mouse PRM 4 h after exposure was effectively blocked by proteasome inhibitors. Irradiation-induced disruption of mitochondrial transmembrane potential and the release of cytochrome c into the cytosol were also suppressed by a proteasome inhibitor but not by a caspase inhibitor. To determine whether the apoptosis occurred due to a depletion of antiapoptotic proteins, Bcl-2 family proteins were examined. Irradiation markedly decreased the level of Mcl-1, but not Bcl-2, Bcl-X(L), Bax, A1, or cIAP1. Mcl-1's depletion was suppressed by a proteasome inhibitor but not by a caspase inhibitor. The amount of Mcl-1 was well correlated with the rate of apoptosis in C3H mouse PRM exposed to irradiation and not affected by irradiation in radioresistant B6 mouse PRM. Irradiation increased rather than decreased the Mcl-1 mRNA expression in C3H mouse PRM. On the other hand, Mcl-1 protein synthesis was markedly suppressed by irradiation. Global protein synthesis was also suppressed by irradiation in C3H mouse PRM but not in B6 mouse PRM. The down-regulation of Mcl-1 expression with Mcl-1-specific small interfering RNA or antisense oligonucleotide significantly induced apoptosis in both C3H and B6 mouse PRM without irradiation. It was concluded that the apoptosis elicited in C3H mouse PRM by ionizing radiation was attributable to the depletion of Mcl-1 through radiation-induced arrest of global protein synthesis.

CD4(+) T cells are central in mediating granuloma formation and limiting growth and dissemination of mycobacterial infections. To determine whether T cells responding to influenza infection can interact with T cells responding to Mycobacterium bovis bacille Calmette-Guérin (BCG) infection and disrupt granuloma formation, we infected mice containing two monoclonal T cell populations specific for the model Ags pigeon cytochrome c (PCC) and hen egg lysozyme (HEL). These mice were chronically infected with PCC epitope-tagged BCG (PCC-BCG) and acutely infected with HEL epitope-tagged influenza virus (HEL-flu). In these mice, PCC-BCG infection is much more abundant in the liver than the lung, whereas HEL-flu infection is localized to the lung. We observe that both T cells have access to both inflammatory sites, but that PCC-specific T cells dominate the PCC-BCG inflammatory site in the liver, whereas HEL-specific T cells dominate the HEL-flu inflammatory site in the lung. Influenza infection, in the absence of an influenza-specific T cell response, is able to increase the activation state and IFN-gamma secretion of PCC-BCG-specific T cells in the granuloma. Activation of HEL-specific T cells allows them to secrete IFN-gamma and contribute to protection in the granuloma. Ultimately, infection with influenza has little effect on bacterial load, and bacteria do not disseminate. In summary, these data illustrate complex interactions between T cell responses to infectious agents that can affect effector responses to pathogens.

Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ gammadelta TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor alpha chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of gammadelta T cells from birth to approximately 5 months of age. However, the absolute numbers of gammadelta T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of gammadelta T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute gammadelta T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life.

Antrodia cinnamomea is well known in Taiwan as a traditional medicine for treating cancer and inflammation. The purpose of this study was to evaluate the apoptotic effects of ethylacetate extract from A. cinnamomea (EAC) fruiting bodies in Hep 3B, a liver cancer cell line. EAC decreased cell proliferation of Hep 3B cells by inducing apoptotic cell death. EAC treatment increased the level of calcium (Ca2+) in the cytoplasm and triggered the subsequent activation of calpain and caspase-12. EAC also initiated the mitochondrial apoptotic pathway through regulation of Bcl-2 family proteins expression, release of cytochrome c, and activation of caspase-9 in Hep 3B cells. Furthermore, the mitochondrial apoptotic pathway amplified the calpain pathway by Bid and Bax interaction and Ca2+ translocation. We have therefore concluded that the molecular mechanisms during EAC-mediated proliferation inhibition in Hep 3B cells were due to: (1) apoptosis induction, (2) triggering of Ca2+/calpain pathway, (3) disruption of mitochondrial function, and (4) apoptotic signaling being amplified by cross-talk between the calpain/Bid/Bax and Ca2+/mitochondrial apoptotic pathways.

Background: Cytochrome c release from mitochondria to cytosol is a hallmark of apoptosis and is used to characterize the mitochondria-dependent pathway of this type of cell death. Techniques currently used to measure cytochrome c release, Western blot and fluorescence microscopy of immunolabeled cells, are time-consuming and inaccurate, and the latter is still limited by sample size., Methods: We developed a rapid and reliable technique to detect cytochrome c release during drug-induced apoptosis, using flow cytometry. Plasma membrane of apoptotic HL-60 cells and thymocytes, treated with staurosporine and dexamethasone, respectively, were selectively permeabilized by digitonin at a low concentration. The released cytochrome c was quickly washed out from cells and that which remained in the mitochondria was immunolabeled after fixing the cells., Results: The fraction of cells that retained their mitochondrial cytochrome c, or the highly fluorescent cells, gradually decreased so that after 4-8 h of drug treatment almost all the cells lost their cytochrome c and emerged as a population of low fluorescent cells. This was confirmed by parallel fluorescence microscopy of cells immunolabeled for cytochrome c., Conclusions: This technique allows the analysis of cytochrome c release from mitochondria of a large number of apoptotic cells in a short period of time and is proposed as an alternative to the methods currently used for this same purpose., (Copyright 2006 International Society for Analytical Cytology.)

The innate immune system is composed of neutrophils and monocyte/macrophages. As a cell type, bone marrow-derived macrophage (BMM) are easier to study than neutrophils since they are still capable of cell division and have a longer life span. However, in comparison with neutrophils, few methodological studies on the production of reactive oxygen species (ROS) by such macrophages have been reported. Here we present studies on ROS production of this cell type under various conditions including the use of different priming and stimulating agents. In addition, we report that the de novo adhesion of BMM to tissue culture plates induces superoxide anion production and this can be further enhanced by stimulation with PMA. BMM are able to adhere to endothelial cells that have been activated by TNF-alpha exposure, and under these circumstances also generate ROS. We explored different methods to introduce gene products into BMM without activating them to avoid complicating subsequent studies of ROS production. Infection with lentiviral vectors was very efficient, allowed long-term expression and did not activate the BMM. We conclude that BMM are very suitable for the biochemical study of the oxidative burst.

The mitochondrial inner membrane typically shows a condensed structure when examined by electron microscopy. However, this typical structure is known to disappear upon induction of the mitochondrial permeability transition (PT). This change in the appearance of the mitochondrial membrane structure that accompanies the induction of PT is thought to reflect changes in the permeability of inner mitochondrial membrane; however, its molecular basis has remained uncertain. In the present study, changes in membrane status were examined by immuno-electron microscopy using antibodies against the voltage-dependent anion channel (VDAC), beta-subunit of F1-ATPase (F1beta), and cytochrome c (cyt. c). In control mitochondria, antibody against VDAC was observed at the rim of the mitochondria, whereas antibodies against F1beta and cytochrome c bound these molecules inside of the mitochondria. However, in PT-induced mitochondria, all three antibodies were observed at the mitochondrial rim. These results strongly suggest that the inner mitochondrial membrane is shoved to the rim region of mitochondria upon induction of mitochondrial PT.

The current study examines how responsiveness of T cells is affected by the avidity of the peptide/MHC engaged during positive selection of their thymocyte precursors. We used a thymus reaggregate culture system in which CD4(+)CD8(+) thymocytes from AND TCR transgenic mice were induced to undergo positive selection by pigeon cytochrome c (PCC) peptide or its analogs presented by I-E(k) class II MHC on a thymic epithelial cell line. When low-affinity peptide analogs drove positive selection, up to 100 microM was needed to produce >50% CD4(+) T cells, and these cells were highly responsive to PCC. In contrast, <0.2 microM high-affinity peptides was required to achieve similar selection efficiency, but the resultant cells failed to respond to PCC. However, these cells were not dead based on dye exclusion and capacity to respond to phorbal ester and to agonist if IL-2 was also present, supporting the view that non-responsiveness of cells selected on high-affinity peptides is a form of central T cell tolerance distinct from deletion. Cells selected on intermediate-affinity peptides showed variable responsiveness which was suppressed 5- to 10-fold by addition during reaggregate culture of antibody to the IL-7R. Similarly, supplementary IL-7 in the reaggregate culture produced CD4(+) T cells that were promiscuously responsive. Overall, this study demonstrates that the responsiveness of T cells is not rigidly controlled and that the presence of IL-7 during T cell development has the potential to negate central T cell tolerance and produce autoreactive T cells.