Molecular characterization of carboxypeptidase B-like (CPB) in Scylla paramamosain and its role in white spot syndrome virus and Vibrio alginolyticus infection.

Carboxypeptidase plays an important physiological role in the tissues and organs of animals. In this study, we cloned an entire 2316 bp carboxypeptidase B-like (CPB) sequence with a 1302 bp open reading frame encoding a 434 amino acid peptide from Scylla paramamosain. The CPB gene was expressed highly in hepatopancreas and decreased in crab hemocytes after challenges with white spot syndrome virus (WSSV) or Vibrio alginolyticus. After CPB gene knockdown using double-stranded RNA (CPB-dsRNA), the expression of JAK, STAT, C-type lectin, crustin antimicrobial peptide, Toll-like receptors, prophenoloxidase, and myosin II essential light chain-like protein were down-regulated in hemocytes at 24 h post dsRNA treatment. CPB knockdown decreases total hemocyte count in crabs indicated that CPB may negatively regulate crab hemocyte proliferation in crabs. CPB showed an inhibitory effect on hemocyte apoptosis in crabs infected with WSSV or V. alginolyticus. The phagocytosis rate of WSSV by hemocytes was increased after CPB-dsRNA treatment. After WSSV challenge, the mortality and WSSV copy number were both decreased but the rate of hemocyte apoptosis was increased in CPB-dsRNA-treated crabs. The results indicate that the antiviral activity of the crabs was enhanced when CPB was knocked down, indicating WSSV may take advantage of CPB to benefit its replication. In contrast, the absence of CPB in crabs increased mortality following the V. alginolyticus challenge. The phagocytosis rate of V. alginolyticus by hemocytes was increased after CPB-dsRNA treatment. It was revealed that CPB may play a positive role in the immune response to V. alginolyticus through increasing the phagocytosis rate of V. alginolyticus. This research further adds to our understanding of the CPB and identifies its potential role in the innate immunity of crabs.

Novel mechanisms underlying the immediate and transient global tolerization of splenic dendritic cells after vaccination with a self-antigen.

Dendritic cells (DCs) are important orchestrators of the immune response, ensuring that immunity against pathogens is generated, whereas immunity against healthy tissues is prevented. Using the tumor Ag MUC1, we previously showed that i.v. immunization of MUC1 transgenic mice, but not wild-type, with a MUC1 peptide resulted in transient tolerization of all splenic DCs. These DCs did not upregulate costimulatory molecules and induced regulatory T cells rather than effector T cells. They were characterized by suppressed expression of a cohort of pancreatic enzymes not previously reported in DCs, which were upregulated in DCs presenting the same MUC1 peptide as a foreign Ag. In this article, we examined the self-antigen-tolerized DC phenotype, function, and mechanisms responsible for inducing or maintaining their tolerized state. Tolerized DCs share some characteristics with immature DCs, such as a less inflammatory cytokine/chemokine profile, deficient activation of NF-κB, and sustained expression of zDC and CCR2. However, tolerized DCs demonstrated a novel inducible expression of aldehyde dehydrogenase 1/2 and phospho-STAT3. Suppressed expression of one of the pancreatic enzymes, trypsin, in these DC impeded their ability to degrade extracellular matrix, thus affecting their motility. Suppressed metallopeptidases, reflected in low expression of carboxypeptidase B1, prevented optimal Ag-specific CD4(+) T cell proliferation suggesting their role in Ag processing. Tolerized DCs were not refractory to maturation after stimulation with a TLR3 agonist, demonstrating that this tolerized state is not terminally differentiated and that tolerized DCs can recover their ability to induce immunity to foreign Ags.

Molecular characterization of the carboxypeptidase B1 of Anopheles stephensi and its evaluation as a target for transmission-blocking vaccines.

Malaria is one of the most important infectious diseases in the world, and it has many economic and social impacts on populations, especially in poor countries. Transmission-blocking vaccines (TBVs) are valuable tools for malaria eradication. A study on Anopheles gambiae revealed that polyclonal antibodies to carboxypeptidase B1 of A. gambiae can block sexual parasite development in the mosquito midgut. Hence, it was introduced as a TBV target in regions where A. gambiae is the main malaria vector. However, in Iran and neighboring countries as far as China, the main malaria vector is Anopheles stephensi. Also, the genome of this organism has not been sequenced yet. Therefore, in this study, carboxypeptidase B1 of A. stephensi was characterized by genomic and proteomic approaches. Furthermore, its expression pattern after ingestion of Plasmodium falciparum gametocytes and the effect of anti-CPBAs1 antibodies on sexual parasite development were evaluated. Our results revealed that the cpbAs1 expression level was increased after ingestion of the mature gametocytes of P. falciparum and that anti-CPBAs1 directed antibodies could significantly reduce the mosquito infection rate in the test group compared with the control group. Therefore, according to our findings and with respect to the high similarity of carboxypeptidase enzymes between the two main malaria vectors in Africa (A. gambiae) and Asia (A. stephensi) and the presence of other sympatric vectors, CPBAs1 could be introduced as a TBV candidate in regions where A. stephensi is the main malaria vector, and this will broaden the scope for the potential wider application of CPBAs1 antigen homologs/orthologs.

Immuno-spin trapping of a post-translational carboxypeptidase B1 radical formed by a dual role of xanthine oxidase and endothelial nitric oxide synthase in acute septic mice.

Post-translational modification of proteins due to exposure to radicals and other reactive species are markers of metabolic and inflammatory oxidative stress such as sepsis. This study uses the nitrone spin-trap DMPO and a combination of immuno-spin trapping and mass spectrometry to identify in vivo products of radical reactions in mice. We report the detection of dose-dependent production of DMPO-carboxypeptidase B1 (CPB1) adducts in the spleens of mice treated with lipopolysaccharide (LPS). Additionally, we report significant detection of DMPO-CPB1 adducts in mice experiencing normal physiological conditions. Treatments with inhibitors and experiments with knock-out mice indicate that xanthine oxidase and endothelial nitric oxide synthase are important sources of the reactive species that lead to CPB1 adduct formation. We also report a significant loss of CPB1 activity following LPS challenge in conjunction with an increase in CPB1 protein accumulation. This suggests the presence of a possible mechanism for CPB1 activity loss with compensatory protein production.

Carboxypeptidases B of Anopheles gambiae as targets for a Plasmodium falciparum transmission-blocking vaccine.

Anopheles gambiae is the major African vector of Plasmodium falciparum, the most deadly species of human malaria parasite and the most prevalent in Africa. Several strategies are being developed to limit the global impact of malaria via reducing transmission rates, among which are transmission-blocking vaccines (TBVs), which induce in the vertebrate host the production of antibodies that inhibit parasite development in the mosquito midgut. So far, the most promising components of a TBV are parasite-derived antigens, although targeting critical mosquito components might also successfully block development of the parasite in its vector. We previously identified A. gambiae genes whose expression was modified in P. falciparum-infected mosquitoes, including one midgut carboxypeptidase gene, cpbAg1. Here we show that P. falciparum up-regulates the expression of cpbAg1 and of a second midgut carboxypeptidase gene, cpbAg2, and that this up-regulation correlates with an increased carboxypeptidase B (CPB) activity at a time when parasites establish infection in the mosquito midgut. The addition of antibodies directed against CPBAg1 to a P. falciparum-containing blood meal inhibited CPB activity and blocked parasite development in the mosquito midgut. Furthermore, the development of the rodent parasite Plasmodium berghei was significantly reduced in mosquitoes fed on infected mice that had been immunized with recombinant CPBAg1. Lastly, mosquitoes fed on anti-CPBAg1 antibodies exhibited reduced reproductive capacity, a secondary effect of a CPB-based TBV that could likely contribute to reducing Plasmodium transmission. These results indicate that A. gambiae CPBs could constitute targets for a TBV that is based upon mosquito molecules.