Leishmania Donovani Cell Surface Sialoglycans Regulate Susceptibility for Siglec Mediated Macrophage Invasion and Parasite Survival

Glycoconjugates play a pivotal role in the survival of Leishmania parasites in destructive surroundings. An important constituent present on many glycoconjugates is sialic acid. By virtue of their peripheral position on oligosaccharide chains of glycoconjugates, sialic acids are well suited as molecular determinants of specific biological processes, including the interaction of pathogenic microorganisms with sialylated cellular receptors. Differences in a2,3- and a2,6-sialoglycan patterns detected in clonal virulent Leishmania donovani promastigotes, correlated with the level of a2,3- and a2,6-sialyltransferase activity present in these parasites. The role of macrophage sialic acid-receptors in uptake and survival of L.donovani was studied in the murine macrophage cell line raw 264.7. Macrophage invasion was dependent on the binding to Siglec-1, while suppression of MAPK signaling was mediated through Siglec-5. Sialic acid removal by neuraminidase treatment reduced parasite infectivity. The presence of trypsin resistant sialic acid residues in the neuraminidase treated parasites grown in a serum free medium in presence of sialoglycoconjugates indicated that the parasites could salvage sialic acid from exogenous sialoglycans and reutilize it for de novo glycoprotein sialylation in L.donovani parasites. Thus, our results demonstrate the involvement of sialoglycans in the invasion as well as the survival process of L.donovani parasites

TLR4 and NKT Cell Synergy in Immunotherapy against Visceral Leishmaniasis

NKT cells play an important role in autoimmune diseases, tumor surveillance, and infectious diseases, providing in most cases protection against infection. NKT cells are reactive to CD1d presented glycolipid antigens. They can modulate immune responses by promoting the secretion of type 1, type 2, or immune regulatory cytokines. Pathogen-derived signals to dendritic cells mediated via Toll like Receptors (TLR) can be modulated by activated invariant Natural Killer T (iNKT) cells. The terminal β-(1–4)-galactose residues of glycans can modulate host responsiveness in a T helper type-1 direction via IFN-γ and TLRs. We have attempted to develop a defined immunotherapeutic, based on the cooperative action of a TLR ligand and iNKT cell using a mouse model of visceral leishmaniasis. We evaluated the anti-Leishmania immune responses and the protective efficacy of the β-(1–4)-galactose terminal NKT cell ligand glycosphingophospholipid (GSPL) antigen of L. donovani parasites. Our results suggest that TLR4 can function as an upstream sensor for GSPL and provoke intracellular inflammatory signaling necessary for parasite killing. Treatment with GSPL was able to induce a strong effective T cell response that contributed to effective control of acute parasite burden and led to undetectable parasite persistence in the infected animals. These studies for the first time demonstrate the interactions between a TLR ligand and iNKT cell activation in visceral leishmaniasis immunotherapeutic

Insurgencies in India's northeast : conflict, co-option & change

For more about the East-West Center, see http://www.eastwestcenter.org/</a

Vestibulo-rectal pull through in H-fistula in girls

Aims and Objectives: Vestibulo-rectal pull-through (VRPT) in H-fistula in girls was first described by Chatterjee et al. We are presenting our experience with this approach in 47 cases. Materials and Methods: We have total 47 cases of which one is a recurrent fistula operated outside. A circumferential incision is made around the fistula in the vestibule; fistula tract is dissected liberally and delivered by invagination into the bowel. Then, the fistula tract is excised adequately and closed from within the bowel lumen so that no anterior outpouching of the rectum remains. The perineal body is repaired through the vestibular incision. In no cases, protective colostomy was performed. Only the recurrent fistula case had colostomy done in another institution. Results: Complete cure was obtained in 45 out of 47 cases. Two of our earlier cases had recurrences perhaps due to inadequate mobilization, but in later cases, we had no recurrence. Conclusion: VRPT yields good result without the need for colostomy. Incisions on the perineal skin or the anal verge are avoided, thus improving the cosmetic outcome

Effect of thermal and thermo-mechanical cycling on the microstructure of Ni-rich NiTi shape memory alloys

Microstructural changes of Ni-rich NiTi shape memory alloy during thermal and thermo-mechanical cycling have been investigated using Electron Back Scattered Diffraction. A strong dependence of the orientation of the prior austenite grain on the misorientation development has been observed during thermal cycling and thermo-mechanical cycling. This effect is more pronounced at the grain boundaries compared to grain interior. At a larger applied strain, the volume fraction of stabilized martensite phase increases with increase in the number of cycling. Deformation within the martensite leads to stabilization of martensitic phase even at temperatures slightly above the austenite finish temperature. Modulus variation with respect to temperature has been explained on the basis of martensitic transformation

TLR4 and MyD88 are required for GSPL induced anti-leishmanial response.

<p>(A) Splenic adherent cells were transfected with siRNAs specific to TLR2, 4 or MyD88 (specific). A control group was transfected with control siRNA (control). Twenty-four hours after transfection, cells were recovered and their TLR2, 4 and MyD88 levels assessed in Western blots. GAPDH in total proteins was used as loading controls. Blots are representative of three separate experiments. (B) Splenic adherent cells transfected with TLR2, TLR4, MyD88 or control siRNA (con), were infected with <i>LD</i> (APC/parasite 1∶20) for 12 h. Non-ingested promastigotes were removed by washing, and adherent cells were cultured for another 36 h. Infected APCs were then treated with GSPL (100 µg/mL) for 24 h. Intracellular parasite number was determined by Giemsa staining. Each experiment was conducted in triplicate and repeated at least three times each and one set of representative data is shown. Error bars represent mean ± SD. * p<0.0001; paired two-tailed Student's t-test. (C,D) Antileishmanial effect of GSPL on parasite growth in TLR4 deficient C3H/HeJ mice. Sixty days <i>LD</i> infected C3H/HeJ mice were given two subcutaneous injections of GSPL (100 µg each) at 15 days apart. The parasite burdens in liver (C), spleen (C), and bone marrow (D) of individual animals were then determined at 15 days after the last treatment. The results are representative of three independent experiments and data shown are means ± SD; n=5. *p<0.0001 versus corresponding infected control; paired two-tailed Student's t-test. (E,F) <i>In vivo</i> parasite load in liver, spleen and bone marrow of <i>LD</i> infected BALB/c mice treated with LPS. Sixty days <i>LD</i> infected BALB/c mice were given three intraperitoneal injections of LPS on alternate days (5 µg/injection or 10 µg/injection). The parasite burdens in liver (E), spleen (E), and bone marrow (F) of individual animals were then determined at days 1,3 and 12 after the last treatment. The results are representative of three independent experiments and data shown are means ± SD; n=3. *p<0.0001 versus corresponding infected control; paired two-tailed Student's t-test.</p

Induction of iNKT cell mediated IL-12 production by splenic adherent cells.

<p>Sixty days <i>LD</i> infected animals were treated with GSPL as described in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002646#ppat-1002646-g001" target="_blank">Figure 1</a>. (A) Animals were sacrificed 15 days after the last treatment and iNKT cells isolated from individual experimental animals (WT BALB/c and C3H/HeJ mice) were mixed with 1∶10 of autologous splenic adherent cells (1×10⁶ adherent cells) and were cultured in 24-well culture plates containing 100 µg/mL GSPL for 24 h. Culture supernatants were assayed for the concentration of IL-12p70 and IL-12p40 in ELISA. The results are representative of three independent experiments and data shown are means ± SD; *p<0.0001 versus corresponding infected control; Student's t-test. (B) mRNA expression of IL-12p40, IL-12p35, and IL-23p19 in the spleen of each GSPL treated <i>LD</i> infected animals was evaluated individually by real-time PCR. The fold up-regulation of mRNA post-GSPL treatment was calculated by normalizing the amount of cytokine mRNA with the housekeeping gene GAPDH, and comparing results from treated to infected; *p<0.0001; paired two-tailed Student's t-test. (means ± SD (n=3) of one from three independent experiments are shown).</p