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.

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.

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.

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.