Full-length extracellular region of the var2CSA variant of PfEMP1 is required for specific,high-affinity binding to CSA

Pregnancy-associated malaria (PAM) is a serious consequence of sequestration of Plasmodium falciparum-parasitized erythrocytes (PE) in the placenta through adhesion to chondroitin sulfate A (CSA) present on placental proteoglycans. Recent work implicates var2CSA, a member of the PfEMP1 family, as the mediator of placental sequestration and as a key target for PAM vaccine development. Var2CSA is a 350 kDa transmembrane protein, whose extracellular region includes six Duffy-binding-like (DBL) domains. Due to its size and high cysteine content, the full-length var2CSA extracellular region has not hitherto been expressed in heterologous systems, thus limiting investigations to individual recombinant domains. Here we report for the first time the expression of the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) from the 3D7 parasite strain using the human embryonic kidney 293 cell line. We show that the recombinant extracellular var2CSA region is correctly folded and that, unlike the individual DBL domains, it binds with high affinity and specificity to CSA (K_D = 61 nM) and efficiently inhibits PE from binding to CSA. Structural characterization by analytical ultracentrifugation and small-angle x-ray scattering reveals a compact organization of the full-length protein, most likely governed by specific interdomain interactions, rather than an extended structure. Collectively, these data suggest that a high-affinity, CSA-specific binding site is formed by the higher-order structure of the var2CSA extracellular region. These results have important consequences for the development of an effective vaccine and therapeutic inhibitors.     

Production and characterization of monoclonal antibodies against human IgG in Balb/c mouse

Immunoglobulin G (IgG) is the main immunoglobulin in natural human serum. It constitutes 70 to 75 percent of all immunoglobulins. Monoclonal antibodies have many applications in diagnosis, treatment and purification. The conjugated monoclonal antibodies against human IgG are used in most diagnostic kits. For production of monoclonal antibodies against human IgG, spleen cells of the most immune mouse were fused with SP2/0 (myeloma cells) using Poly Ethylene Glycol (PEG). Supernatant of hybridoma cells was screened for detection of antibody by ELISA. The suitable clones were selected for limiting dilution (L.D). Then, the supernatant of suitable monoclones were assessed for cross reactivity with IgM & IgA by ELISA and confirmed by immunobloting. The subclasses of the selected monoclonal antibodies were determined and the clones were frozen and kept in liquid nitrogen. Finally, suitable monoclone was injected into the mouse, intraperitoneally, that has been primed with Pristane. In this study, 127 clones were obtained of which 15 clones had absorbance more than 1 which two of them with absorbance about 1.5 were selected for limiting dilution. The yield of limiting dilution was 6 clones with absorbance about 1.8 which did not show cross reactivity with IgM & IgA. Among these clones, G2 monoclone with IgG1 subclass was selected as suitable one and it was reproduced in FCS free RPMI 1640. For large scale production in invivo, the suitable clone was implanted in the peritoneum of the Balb/c mouse and its titer was measured, which showed 1/100,000 dilution for ascitic fluid, having no cross reactivity with IgM & IgA. Monoclonal antibody was purified by chromatography, confirmed by SDS- PAGE, conjugated with enzyme and applied for diagnostic kits.     

Accumulation of a potent gammadelta T-cell stimulator after deletion of the lytB gene in Escherichia coli

Activation of human Vgamma9/Vdelta2 T cells by many pathogens depends on the presence of small phosphorylated non-peptide compounds derived from the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. We here demonstrate that in Escherichia coli mutants deficient in lytB, an essential gene of the MEP pathway, a potent Vgamma9/Vdelta2 T-cell activator accumulates by a factor of approximately 150 compared to wild-type E. coli. The compound responsible for the strong immunogenicity of this E. coli mutant was subsequently characterized and identified as a small pyrophosphorylated metabolite, with a molecular mass of 262 Da, derived from the MEP pathway. Stimulation of human peripheral blood mononuclear cells (PBMC) with extracts prepared from the lytB-deficient E. coli mutant led to upregulation of T-cell activation markers on the surface of Vgamma9/Vdelta2 T cells as well as proliferation and expansion of Vgamma9/Vdelta2 T cells. This response was dependent on costimulatory growth factors, such as interleukin (IL)-2, IL-15 and IL-21. Significant levels of interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) were secreted in the presence of IL-2 and IL-15, but not in the presence of IL-21, demonstrating that proliferating phosphoantigen-reactive Vgamma9/Vdelta2 T cells do not necessarily produce proinflammatory cytokines.     

Unusual presentation of rectal adenocarcinoma

Metastatic tumors in the gastrointestinal tract are rare with an overall prevalence of 1-4 per cent in the postmortem series. Lung cancer, renal cell carcinoma, breast carcinoma and malignant melanoma are considered the most common primary tumors metastatic to the small bowel. Local duodenal metastasis from colonic cancer and cecum have been reported, but metastasis to the duodenum from rectosigmoid adenocarcinoma has not been reported before. We report the first case of metastasis in the duodenum from an adenocarcinoma of the rectum presented as a recurrent acute prerenal azotemia caused by volume depletion which had resulted from duodenal obstruction.     

Resistance status of main malaria vector,Liston (Diptera:Culicidae) to insecticides in a malaria Endemic Area,Southern Iran

Objective: To evaluate the susceptibility of Anopheles stephensi(An. stephensi) Liston, the main malaria vector in southern Iran, to WHO recommended insecticides. Methods: Larvae of An. stephensi were collected from three different larval habitats in both urban and rural area of Bandar Abbas city and one rural area in Rudan county southern Iran. WHO standard method was used for evaluation of adult and larval mosquito susceptibility. Bendiocarb, permethrin, lambda-cyhalothrin, deltamethrin as insecticide and temephos and chlorpyriphos as larvicide were used at the diagnostic dosages recommended by WHO. Results: Findings of this study showed all larval populations of An. stephensi were completely susceptible to temephos and candidate for resistance to chlorpyriphos. Adult mosquitoes in rural areas of Bandar Abbas city were resistant to pyrethroid and carbamate insecticides. Conclusion: Comparison of the results of this survey with previous studies indicates that the resistance to pyrethroids and carbamates in this malaria endemic region is increasing. Wide use of pesticides in agriculture is certainly effective in increasing resistance. The inter-sectoral coordination and collaboration in health and agriculture seem to be necessary to manage insecticide resistance in malaria vectors.     

Latent tuberculosis infection among medical students in Malaysia

Objective: This study aimed to determine prevalence of latent tuberculosis infection among medical students and tuberculosis exposure at the health facilities. Methods: A cross-section of study year 1(n=68) and year 5(n=75) medical students in a local university were recruited for latent tuberculosis infection testing using QuantiFERON-TB Gold Plus and a questionnaire analyzed for multivariate risk. Results: The majority of the study were vaccinated with BCG. None of year 1 medical students were positive for latent tuberculosis infection, however, six(8.0%) year 5 students were tested positive for latent tuberculosis infection. A higher incidence of year 5 medical students claimed to be exposed to tuberculosis at health facility(65.3% vs. 4.4%) and a higher percentage reported contact with tuberculosis case over the preceding year compared to year 1 students(30.7% vs. 8.8%). Conclusion: We observed a higher incidence of latent tuberculosis infection and higher exposure to tuberculosis in health facilities among year 5 medical students. Baseline screening and monitoring for progression to tuberculosis infection may benefit tuberculosis management programs.