Phosphatase inhibitors BVT-948 and alexidine dihydrochloride inhibit sexual development of the malaria parasite Plasmodium berghei.

BACKGROUND: With the emergence of resistance to front-line antimalarials, there is an urgent need to develop new medicines, including those targeting sexual development. This study aimed to assess the activity of a panel of phosphatase inhibitors against the sexual development of Plasmodium berghei and evaluate their potential as transmission-blocking agents. METHODS: Twenty-five compounds were screened for transmission-blocking activity in vitro using the P. berghei ookinete culture assay. The inhibitory effects on male gametogenesis, gamete-ookinete, and zygote-ookinete formation were evaluated. The transmission-blocking activity of two compounds was evaluated using an in vivo mosquito feeding assay. Their cytotoxic effects were assessed on the human cell line HepG2. RESULTS: Twelve compounds inhibited P. berghei ookinete formation with an IC50 < 10 µM. Two compounds, BVT-948 and alexidine dihydrochloride, significantly inhibited different developmental stages from gametogenesis through ookinete maturation. They also showed a substantial in vivo transmission-blocking activity by the mosquito feeding assay. CONCLUSIONS: Some phosphatase inhibitors effectively inhibited Plasmodium sexual development and exhibited evident transmission-blocking activity, suggesting that phosphatases are valid targets for antimalarial development.

Evaluation of two Plasmodium vivax sexual stage antigens as transmission-blocking vaccine candidates.

BACKGROUND: Plasmodium vivax transmission-blocking vaccines (TBVs) are receiving increasing attention. Based on excellent transmission-blocking activities of the PbPH (PBANKA_0417200) and PbSOP26 (PBANKA_1457700) antigens in Plasmodium berghei, their orthologs in P. vivax, PVX_098655 (PvPH) and PVX_101120 (PvSOP26), were selected for the evaluation of their potential as TBVs. METHODS: Fragments of PvPH (amino acids 22-304) and PvSOP26 (amino acids 30-272) were expressed in the yeast expression system. The recombinant proteins were used to immunize mice to obtain antisera. The transmission-reducing activities of these antisera were evaluated using the direct membrane feeding assay (DMFA) using Anopheles dirus mosquitoes and P. vivax clinical isolates. RESULTS: The recombinant proteins PvPH and PvSOP26 induced robust antibody responses in mice. The DMFA showed that the anti-PvSOP26 sera significantly reduced oocyst densities by 92.0 and 84.1% in two parasite isolates, respectively, whereas the anti-PvPH sera did not show evident transmission-reducing activity. The variation in the DMFA results was unlikely due to the genetic polymorphisms of the two genes since their respective sequences were identical in the clinical P. vivax isolates. CONCLUSION: PvSOP26 could be a promising TBV candidate for P. vivax, which warrants further evaluation.

Antitumor effects and mechanisms of CpG ODN combined with attenuated Salmonella-delivered siRNAs against PD-1.

Numerous studies have focused on the treatment of melanoma, but the current therapies for melanoma have limited therapeutic effects. To find a more effective therapy for melanoma, we combined artificially designed CpG ODN (cytosine-phosphate-guanine oligodeoxynucleotides) and siRNAs (small-interfering ribonucleic acids) targeting PD-1 (programmed cell death protein 1), which were delivered by attenuated Salmonella to treat melanoma in mice, and explored the underlying antitumor mechanisms. We found that mice receiving the combination therapy had the smallest tumor size and the longest survival time. The possible mechanisms underlying this phenomenon include pathways mediated by cyclin D1, p-STAT3 (phosphorylated signal transducers and activators of transcription protein 3), MMP2 (matrix metallopeptidase 2) and cleaved caspase 3, since after treatment, the expression of cyclin D1, p-STAT3, and MMP2 decreased but that of cleaved caspase 3 increased; additional mechanisms include increases in the recruitment of immune cells to tumor sites and in the number of immune cells in mouse spleens and the upregulation of TNF-α (tumor necrosis factor) and IL-6 (interleukin 6). We demonstrated that the combination therapy composed of CpG ODN and PD-1-siRNA delivered by attenuated Salmonella exhibited a strong ability to inhibit melanoma and improve the antitumor immune responses of tumor-bearing mice.

Neddylation of M1 negatively regulates the replication of influenza A virus.

Post-translational modification plays a critical role in viral replication. Previously we reported that neddylation of PB2 of influenza A virus (IAV) can inhibit viral replication. However, we found that NEDD8 overexpression can still inhibit the replication of PB2 K699R mutant viruses, implying that other viral protein(s) can be neddylated. In this study, we revealed that M1 of IAV can also be modified by NEDD8. We found that the E3 ligase HDM2 significantly promotes M1 neddylation. Furthermore, we identified M1 K187 as the major neddylation site. We generated an IAV M1 K187R mutant (WSN-M1 K187R) and compared the growth of wild-type and mutant viruses in Madin-Darby canine kidney (MDCK) cells. The data showed that the replication of WSN-M1 K187R was more efficient than that of wild-type WSN. More importantly, we observed that overexpression of NEDD8 inhibited the replication of the wild-type WSN more effectively than that of WSN-M1 K187R. In addition, we found that the neddylation-deficient M1 mutant (M1 K187R) had a longer half-life than that of wild-type M1, indicating that the neddylation of M1 reduces stability. Then we performed a viral infection assay and found that WSN-M1 K187R exhibited greater virulence in mice than wild-type WSN, suggesting that the neddylation of M1 reduced IAV replication in vivo. In conclusion, we uncovered that neddylation of M1 by HDM2 negatively regulates the stability of M1, which in turn inhibits viral replication.

Liraglutide ameliorates lipotoxicity-induced inflammation through the mTORC1 signalling pathway.

Lipotoxicity has been implicated in many disease processes, and prolonged exposure to high lipid levels often leads to the activation of a variety of abnormal signals, which in turn leads to the induction of inflammation. The aim of our study was to explore the correlation between mammalian target of rapamycin (mTOR) and inflammation by studying high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in rats and palmitate (PA)-induced inflammation (lipotoxicity) in HepG2 cells. In addition, we investigated whether the glucagon-like peptide-1 (GLP-1) analogue liraglutide can protect rats and HepG2 cells from lipotoxicity. Our results showed that an HFD and PA significantly increased inflammation by activating the mTORC1 pathway in vitro and in vivo. Treatment with rapamycin (an mTOR inhibitor) inhibited some effects of PA on inflammation. Furthermore, we observed that liraglutide inhibited PA-induced inflammation by inactivating mTORC1 signalling molecules. Overall, our findings demonstrated that mTORC1 signalling pathways were involved primarily in high lipid level-induced inflammation. Importantly, liraglutide may protect against lipotoxicity-induced inflammation by regulating mTORC1-dependent pathways.

Characterization of a Sulfhydryl Oxidase From Plasmodium berghei as a Target for Blocking Parasite Transmission.

Quiescin sulfhydryl oxidase (QSOX), present in a wide variety of eukaryotic species, catalyzes the insertion of disulfide bonds into unfolded, reduced proteins. Here we characterized the QSOX protein from the rodent malaria parasite Plasmodium berghei (PbQSOX), which is conserved in all sequenced malaria parasite species. The PbQSOX protein was not expressed in asexual erythrocytic stages, but was most abundantly expressed in ookinetes. Indirect immunofluorescence assays revealed PbQSOX was not only localized in cytoplasm of gametocytes, gametes and ookinetes, but also expressed on the surface of gametes and ookinetes. Western blot identified extracellular presence of PbQSOX in the culture medium of ookinetes suggestive of secretion. Pbqsox deletion (Δpbqsox) did not affect asexual intraerythrocytic development, but reduced exflagellation of male gametocytes as well as formation and maturation of ookinetes. Pbqsox deletion also led to a significant increase in the reduced thiol groups of ookinete surface proteins, suggesting that it may play a role in maintaining the integrity of disulfide bonds of surface proteins, which might be needed for ookinete development. Mosquitoes that fed on Δpbqsox-infected mice showed a significant reduction in ookinete and oocyst numbers compared to those fed on wild-type parasite-infected mice. Further, both polyclonal mouse antisera and a monoclonal antibody against the recombinant PbQSOX exhibited substantial transmission-blocking activities in in vitro and mosquito feeding assays, suggesting QSOX is a potential target for blocking parasite transmission.

Knockdown of PAK1 Inhibits the Proliferation and Invasion of Non-Small Cell Lung Cancer Cells Through the ERK Pathway.

The p21-activated kinase (PAK) family of serine/threonine kinases plays a pivotal role in various human tumors, as supported by our previous report on the overexpressed PAK isoforms in non-small cell lung cancer (NSCLC). To better understand the role of PAKs in tumorigenesis, the authors examined PAK1 expression patterns and its significance in NSCLC. It was demonstrated by immunohistochemical staining that PAK1 was increased and localized in the cytoplasm in 151 of 207 cases. High levels of PAK1 expression correlated with a histologic type of tumor (squamous cell carcinoma), tumor node metastasis stage, and lymph nodal status. We also examined the biological role of PAK1 in lung cancer cell lines transfected with PAK1-small interfering RNA. Decreased expression of PAK1 inhibited lung cancer cell proliferation and invasion, which is the major cause of lung cancer malignancy. Downregulated expression of PAK1 hampered rapidly accelerated fibrosarcoma/mitogen-activated extracellular signal-regulated kinase/extracellular signal-regulated kinase pathway activity but did not affect Wnt/ß-catenin signaling. Our findings suggest that PAK1 is an important oncogene in NSCLC, as decreased expression of PAK1 inhibited the proliferation and invasion of NSCLC cells by blocking the ERK pathway. These results provide evidence for using PAK1 inhibition as potential anticancer therapy.

Telbivudine antagonizes TLR4 to inhibit the epithelial-to-mesenchymal transition in human proximal tubular epithelial cells in vitro.

The antiviral drug Telbivudine (LdT) has an extrahepatic pharmaceutical effect that improves renal inflammation and tubulointerstitial fibrosis. However, the exact mechanism of action requires further investigation. Toll-like receptor 4 (TLR4) is involved in several physiological processes, including inflammation, fibrosis, innate immunity, and hepatitis B virus-associated glomerulonephritis. The epithelial-to-mesenchymal transition (EMT) is the characteristic pathological change in tubulointerstitial fibrosis. In this study, we used transforming growth factor-ß (TGF-ß) to stimulate human proximal tubular epithelial (HK-2) cells to investigate the effects of LdT in EMT. In addition, we treated HK-2 cells with a TLR4 agonist, lipopolysaccharide, to determine the effect of LdT on TLR4. The results indicated that LdT inhibited the expression of TLR4 and its downstream proteins. It also decreased the release of inflammatory factors, downregulated the TGF-ß/Smad signaling pathway, and reversed the EMT changes seen in HK-2 cells. In conclusion, LdT antagonized TLR4 to inhibit EMT in proximal tubular epithelial cells.

Characterization of Plasmodium berghei Homologues of T-cell Immunomodulatory Protein as a New Potential Candidate for Protecting against Experimental Cerebral Malaria.

The pathogenesis of cerebral malaria is biologically complex and involves multi-factorial mechanisms such as microvascular congestion, immunopathology by the pro-inflammatory cytokine and endothelial dysfunction. Recent data have suggested that a pleiotropic T-cell immunomodulatory protein (TIP) could effectively mediate inflammatory cytokines of mammalian immune response against acute graft-versus-host disease in animal models. In this study, we identified a conserved homologue of TIP in Plasmodium berghei (PbTIP) as a membrane protein in Plasmodium asexual stage. Compared with PBS control group, the pathology of experimental cerebral malaria (ECM) in rPbTIP intravenous injection (i.v.) group was alleviated by the downregulation of pro-inflammatory responses, and rPbTIP i.v. group elicited an expansion of regulatory T-cell response. Therefore, rPbTIP i.v. group displayed less severe brain pathology and feverish mice in rPbTIP i.v. group died from ECM. This study suggested that PbTIP may be a novel promising target to alleviate the severity of ECM.

Characterization of Pb51 in Plasmodium berghei as a malaria vaccine candidate targeting both asexual erythrocytic proliferation and transmission.

BACKGROUND: A vaccine that targets multiple developmental stages of malaria parasites would be an effective tool for malaria control and elimination. METHODS: A conserved gene in Plasmodium, the Plasmodium berghei gene (PBANKA_020570) encoding a 51 kDa protein (pb51 gene), was identified through search of the PlasmoDB database using a combination of expression and protein localization criteria. A partial domain of the Pb51 protein was expressed in a prokaryotic expression system (rPb51) and used for immunization in mice. The protein expression profile and localization were studied by Western blot and indirect immunofluorescence assay (IFA), respectively. The inhibitory effect of the anti-rPb51 antibodies on parasite proliferation was evaluated in erythrocytes in vivo. The transmission-blocking activity of the immune sera was determined by in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). RESULTS: The rPb51 elicited specific antibodies in mice. Western blot confirmed Pb51 expression in schizonts, gametocytes and ookinetes. IFA showed localization of Pb51 on the outer membranes of schizonts, gametocytes, zygotes, retorts, ookinetes and sporozoites of P. berghei. Mice immunized with the rPb51 protein significantly reduced parasite proliferation and gametocyte conversion in vivo. Moreover, the rPb51 antisera also significantly reduced the in vitro ookinete conversion when added into the ookinete culture medium. In DFA, mice immunized with the rPb51 reduced the prevalence of mosquito infection by 21.3% and oocyst density by 54.8%. CONCLUSIONS: In P. berghei, P51 was expressed in both asexual erythrocytic and sexual stages and localized on the surface of these stages with the exception of the ring stage. The anti-rPb51 antibodies inhibited both P. berghei proliferation in mice and transmission of the parasite to mosquitoes.

Functional characterization of Plasmodium berghei PSOP25 during ookinete development and as a malaria transmission-blocking vaccine candidate.

BACKGROUND: Plasmodium ookinete surface proteins as post-fertilization target antigens are potential malaria transmission-blocking vaccine (TBV) candidates. Putative secreted ookinete protein 25 (PSOP25) is a highly conserved ookinete surface protein, and has been shown to be a promising novel TBV target. Here, we further investigated the TBV activities of the full-length recombinant PSOP25 (rPSOP25) protein in Plasmodium berghei, and characterized the potential functions of PSOP25 during the P. berghei life-cycle. METHODS: We expressed the full-length P. berghei PSOP25 protein in a prokaryotic expression system, and developed polyclonal mouse antisera and a monoclonal antibody (mAb) against the recombinant protein. Indirect immunofluorescence assay (IFA) and Western blot were used to test the specificity of antibodies. The transmission-blocking (TB) activities of antibodies were evaluated by the in vitro ookinete conversion assay and by direct mosquito feeding assay (DFA). Finally, the function of PSOP25 during Plasmodium development was studied by deleting the psop25 gene. RESULTS: Both polyclonal mouse antisera and anti-rPSOP25 mAb recognized the PSOP25 proteins in the parasites, and IFA showed the preferential expression of PSOP25 on the surface of zygotes, retorts and mature ookinetes. In vitro, these antibodies significantly inhibited ookinetes formation in an antibody concentration-dependent manner. In DFA, mice immunized with the rPSOP25 and those receiving passive transfer of the anti-rPSOP25 mAb reduced the prevalence of mosquito infection by 31.2 and 26.1%, and oocyst density by 66.3 and 63.3%, respectively. Genetic knockout of the psop25 gene did not have a detectable impact on the asexual growth of P. berghei, but significantly affected the maturation of ookinetes and the formation of midgut oocysts. CONCLUSIONS: The full-length rPSOP25 could elicit strong antibody response in mice. Polyclonal and monoclonal antibodies against PSOP25 could effectively block the formation of ookinetes in vitro and transmission of the parasites to mosquitoes. Genetic manipulation study indicated that PSOP25 is required for ookinete maturation in P. berghei. These results support further testing of the PSOP25 orthologs in human malaria parasites as promising TBV candidates.

Limited genetic diversity in the PvK12 Kelch protein in Plasmodium vivax isolates from Southeast Asia.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged as a major threat for malaria control and elimination worldwide. Mutations in the Kelch propeller domain of PfK13 are the only known molecular markers for artemisinin resistance in this parasite. Over 100 non-synonymous mutations have been identified in PfK13 from various malaria endemic regions. This study aimed to investigate the genetic diversity of PvK12, the Plasmodium vivax ortholog of PfK13, in parasite populations from Southeast Asia, where artemisinin resistance in P. falciparum has emerged. METHODS: The PvK12 sequences in 120 P. vivax isolates collected from Thailand (22), Myanmar (32) and China (66) between 2004 and 2008 were obtained and 353 PvK12 sequences from worldwide populations were retrieved for further analysis. RESULTS: These PvK12 sequences revealed a very low level of genetic diversity (π = 0.00003) with only three single nucleotide polymorphisms (SNPs). Of these three SNPs, only G581R is nonsynonymous. The synonymous mutation S88S is present in 3% (1/32) of the Myanmar samples, while G704G and G581R are present in 1.5% (1/66) and 3% (2/66) of the samples from China, respectively. None of the mutations observed in the P. vivax samples were associated with artemisinin resistance in P. falciparum. Furthermore, analysis of 473 PvK12 sequences from twelve worldwide P. vivax populations confirmed the very limited polymorphism in this gene and detected only five distinct haplotypes. CONCLUSIONS: The PvK12 sequences from global P. vivax populations displayed very limited genetic diversity indicating low levels of baseline polymorphisms of PvK12 in these areas.

Effects of transmission-blocking vaccines simultaneously targeting pre- and post-fertilization antigens in the rodent malaria parasite Plasmodium yoelii.

BACKGROUND: Transmission-blocking vaccine (TBV) is a promising strategy for interrupting the malaria transmission cycle. Current TBV candidates include both pre- and post-fertilization antigens expressed during sexual development of the malaria parasites. METHODS: We tested whether a TBV design combining two sexual-stage antigens has better transmission-blocking activity. Using the rodent malaria model Plasmodium yoelii, we pursued a DNA vaccination strategy with genes encoding the gametocyte antigen Pys48/45 and the major ookinete surface protein Pys25. RESULTS: Immunization of mice with DNA constructs expression either Pys48/45 or Pys25 elicited strong antibody responses, which specifically recognized a ~45 and ~25 kDa protein from gametocyte and ookinete lysates, respectively. Immune sera from mice immunized with DNA constructs expressing Pys48/45 and Pys25 individually and in combination displayed evident transmission-blocking activity in in vitro ookinete culture and direct mosquito feeding experiments. With both assays, the Pys25 sera had higher transmission-blocking activity than the Pys48/45 sera. Intriguingly, compared with the immunization with the individual DNA vaccines, immunization with both DNA constructs produced lower antibody responses against individual antigens. The resultant immune sera from the composite vaccination had significantly lower transmission-blocking activity than those from Pys25 DNA immunization group, albeit the activity was substantially higher than that from the Pys48 DNA vaccination group. CONCLUSIONS: This result suggested that vaccination with the two DNA constructs did not achieve a synergistic effect, but rather caused interference in inducing antigen-specific antibody responses. This result has important implications for future design of composite vaccines targeting different sexual antigens.

Characterization of a Plasmodium berghei sexual stage antigen PbPH as a new candidate for malaria transmission-blocking vaccine.

BACKGROUND: Transmission-blocking vaccines (TBVs) are a promising strategy for malaria control and elimination. However, candidate TBV antigens are currently limited, highlighting the urgency of identifying new antigens for TBV development. METHODS: Using a combination of bioinformatic analysis and functional studies in the rodent malaria model Plasmodium berghei, we identified a conserved Plasmodium protein PbPH (PBANKA_041720) containing a pleckstrin homology (PH) domain. The expression of PbPH was detected by Western blot and indirect immunofluorescence assay (IFA). The function of PbPH was tested by genetic knockout. The TB activity was confirmed by in vitro ookinete conversion assay and mosquito feeding. RESULTS: PbPH was detected in Western blot as highly expressed in sexual stages (gametocytes and ookinetes). IFA revealed localizations of PbPH on the surface of gametes, zygotes, and ookinetes. Deletion of the pbph gene did not affect asexual growth, but significantly reduced the formation of gametocytes, ookinetes, and oocysts, indicating that PbPH protein is required for parasite sexual development. Recombinant PbPH expressed and purified from bacteria elicited strong antibody responses in mice and the antibodies significantly inhibited exflagellation of male gametocytes and formation of ookinetes in a concentration-dependent manner. Mosquito feeding experiments confirmed that mosquitoes fed on mice immunized with PbPH had 13 % reduction in the prevalence of infection and almost 48 % reduction in oocyst density. CONCLUSIONS: Pbph is a highly conserved Plasmodium gene and is required for parasite sexual development. PbPH protein is expressed on the surface of gametes and ookinetes. Immunization of mice against the recombinant PbPH protein induced strong antibody responses that effectively reduced the formation of male gametes and ookinetes in vitro and blocked transmission of the parasites to mosquitoes. These results highlight PbPH as a potential TBV candidate that is worth future investigations in human malaria parasites.

Identification of three ookinete-specific genes and evaluation of their transmission-blocking potentials in Plasmodium berghei.

With a renewed hope for malaria elimination, interventions that prevent transmission of parasites from humans to mosquitoes have received elevated attention. Transmission-blocking vaccines (TBVs) targeting the sexual stages are well suited for this task. Here, through bioinformatic analysis, we selected two putative Plasmodium berghei ookinete-stage proteins (PBANKA_111920, and PBANKA_145770) and a previously characterized ookinete protein PBANKA_135340 (PSOP7) for evaluation of their transmission-blocking potentials. Fragments of these predicted proteins were expressed in bacteria and purified recombinant proteins were used to immunize mice. Antisera against these recombinant proteins recognized proteins of predicted sizes from ookinete lysates and localized their expression on the surface of ookinetes. Inclusion of these antisera in in vitro ookinete culture significantly inhibited ookinete formation. Mosquitoes fed on mice immunized with the recombinant proteins also showed significantly reduced oocyst densities (60.0-70.7%) and modest reductions of oocyst prevalence (10.7-37.4%). These data, together with the conservation of these genes in Plasmodium, suggest that these three ookinete proteins could be new promising targets for TBVs and are worth of future investigations in the human malaria parasites.

Immunogenicity and immunizing protection effect of GAMA gene DNA vaccine on Plasmodium berghei.

OBJECTIVE: To explore the effect of immunogenicity and immunizing protection of GAMA gene DNA vaccine, which was related with merozoite, ookinete and sporozoite invasion. METHODS: Gene fragments were obtained using PCR technique and eukaryotic expression vector (containing immunostimulatory sequence) was built. BALB/c mice were divided into PBS control group, empty vector control group and study group and were immunized at week 0, 3 and 6 respectively. Blood was collected 2 weeks after each immunization and serum was separated to detect the IgG, IgG1 and IgG2a levels. Spleen of mice was obtained for preparation of splenic mononuclear cell and the cytokine IL-4 and IFN-γ levels were detected. Indirect immunofluorescence and western blot were employed to verify the specificity of antiserum. Sporozoite and merozoite invasion were used respectively to detect the immune protective effect 2 weeks after the third immunization. Ookinete conversion rate in vitro and oocyst numbers of mosquito stomach were observed to evaluate the transmission-blocking levels. RESULTS: In GAMA DNA vaccine group: antiserum could be combined with recombinant protein specifically and green fluorescence signals of merozoite, ookinete and sporozoite were observable, while specific fragments and fluorescence signals were not observable in empty vector group. Compared with control group, specific IgG in DNA vaccine immunity group significantly increased (P < 0.01), and IgG1 and IgG2a all increased (P < 0.01). IL-4, IFN-γ content in study group significantly increased, compared with control group (P < 0.01). GAMA DNA vaccine immunity could not obviously block the erythrocyte-stage infection (caused by sporozoite invasion); compared with control group, liver worm load was slightly reduced (P < 0.05), and antiserum ookinete numbers (cultured in vitro) had no significant difference with oocyst numbers of mosquito stomach in DNA vaccine group. CONCLUSIONS: GAMA has good antigenicity, which could stimulate the body to produce specific immune responses; while DNA vaccine immunity could not play a good protective effect, the effect of which is only limited to the slight reduction of liver worm load, and has no obvious erythrocyte-stage protective effect and transmission-blocking effect. Therefore, trying other immunization strategies for further research on the value of GAMA (as multi-stage antigen vaccine and multi-stage combined vaccine components of the life-cycle of plasmodium) is necessary.

Genetic diversity of transmission-blocking vaccine candidate Pvs48/45 in Plasmodium vivax populations in China.

BACKGROUND: The male gamete fertilization factor P48/45 in malaria parasites is a prime transmission-blocking vaccine (TBV) candidate. Efforts to develop antimalarial vaccines are often thwarted by genetic diversity of the target antigens. Here we evaluated the genetic diversity of Pvs48/45 gene in global Plasmodium vivax populations. METHODS: We determined 200 Pvs48/45 sequences collected from temperate and subtropical parasite populations in China. Population genetic and evolutionary analyses were performed to determine the levels of genetic diversity, potential signature of selection, and population differentiation. RESULTS: Analysis of the Pvs48/45 sequences from 200 P. vivax parasites collected in a temperate and a tropical region revealed a low level of genetic diversity (π = 0.0012) with 14 single nucleotide polymorphisms, of which 11 were nonsynonymous. Analysis of 344 Pvs48/45 sequences from nine worldwide P. vivax populations detected a total of 38 haplotypes, of which 13 haplotypes were present only once. Multiple tests for selection confirmed a signature of positive selection on Pvs48/45 with selection skewed to the second cysteine domain. Haplotype network analysis and Wright's fixation index showed large geographical differentiation with the presence of continent-or region-specific mutations in this gene. CONCLUSIONS: Pvs48/45 displays low levels of genetic diversity with the presence of region-specific mutations. Some of the mutations may be potential epitope targets based on their positions in the predicted structure, highlighting the need for future evaluation of these mutations in designing Pvs48/45-based TBV.

Vitamin D inhibits the occurrence of experimental cerebral malaria in mice by suppressing the host inflammatory response.

In animal models of experimental cerebral malaria (ECM), neuropathology is associated with an overwhelming inflammatory response and sequestration of leukocytes and parasite-infected RBCs in the brain. In this study, we explored the effect of vitamin D (VD; cholecalciferol) treatment on host immunity and outcome of ECM in C57BL/6 mice during Plasmodium berghei ANKA (PbA) infection. We observed that oral administration of VD both before and after PbA infection completely protected mice from ECM. VD administration significantly dampened the inducible systemic inflammatory responses with reduced circulating cytokines IFN-γ and TNF and decreased expression of these cytokines by the spleen cells. Meanwhile, VD also resulted in decreased expression of the chemokines CXCL9 and CXCL10 and cytoadhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain, leading to reduced accumulation of pathogenic T cells in the brain and ultimately substantial improvement of the blood-brain barriers of PbA-infected mice. In addition, VD inhibited the differentiation, activation, and maturation of splenic dendritic cells. Meanwhile, regulatory T cells and IL-10 expression levels were upregulated upon VD treatment. These data collectively demonstrated the suppressive function of VD on host inflammatory responses, which provides significant survival benefits in the murine ECM model.

Right atrial pressure alterations during echocardiography-guided-catheterization predict tricuspid valvular impairment: a novel method for the creation of a rabbit model of Staphylococcus aureus endocarditis.

BACKGROUND: We previously reported the use of a catheter system to damage the tricuspid valve and create infectious endocarditis (IE) in an animal model. The current study aims to create a faint IE model suitable for antibiotic prophylaxis using a low bacterial inoculum. We also aim to explore a way to quantitatively assess valvular impairment and to predict the success of the IE models during catheterization. METHODS: Ninety rabbits were assigned to two groups according to the density of bacteria inoculated (1 × 10(5) CFU for Group A and 1 × 10(4) CFU for Group B). A catheter system consisting of a polyethylene catheter and a guide wire were used to damage the valve. The catheter system was passed through the rabbits' tricuspid valves under echocardiographic guidance. A pressure transducer was used to assess right atrial pressure (P(RA)) before and just after valvular damage to calculate the pressure alterations (ΔP(RA)). The animals in group A and B were divided into 3 subgroups according to the ΔP(RA) (0-5 mmHg for Groups A1 and B1; 5-10 mmHg for Groups A2 and B2; 10-15 mmHg for Groups A3 and B3). Staphylococcus aureus (ATCC 29213) inoculation was performed 24 hr after cardiac catheterization. RESULTS: Faint IE was confirmed in 20%, 93.3%, 26.7%, 6.7%, 20%, and 33.3% of the rabbits in Groups A1, A2, A3, B1, B2, and B3, respectively. There was no difference in the LV/RV ratio and VTR of the No-IE, faint-IE, and severe IE animals. Faint IE rabbits had a larger ΔPRA than No-IE rabbits (7.81 ± 1.21 vs. 2.48 ± 1.0, P < 0.01, for Group A; 7.60 ± 1.32 vs. 2.98 ± 1.08, P < 0.01, for Group B). The ΔPRA of severe IE and faint IE rabbits was significantly different (13.11 ± 1.31 vs. 7.81 ± 1.21, P < 0.01, for Group A; 12.73 ± 1.44 vs.7.60 ± 1.32, P < 0.01, for Group B). CONCLUSION: ΔP(RA) could be used to assess valvular impairment. Controlling the value of ΔP(RA) during catheterization and inoculating of an appropriate dose of bacteria was associated with a successful IE model.