A rapid and accurate method to detect and quantify Leishmania parasite is urgently needed to facilitate early diagnosis of Leishmaniasis and monitoring of antileishmania therapy. In this study, real-time assay was applied to estimate parasite load in clinical samples of visceral leishmaniasis (VL) and post kala-azar dermal leishmaniasis (PKDL) patients. The mean parasite load in blood of VL patients (n = 31) was 8,372 parasites/ml, while the mean parasite load in bone marrow aspirate (BMA) was 194,962 parasites/million nucleated cells (n = 12). Parasite load was undetectable after treatment with amphotericin B (n = 9) in VL, while a residual parasite burden was detected in 2 of 6 patients following treatment with sodium antimony gluconate. Further, circulating levels of IFN-γ, TNF-α, IL-10, IL-6, IL-4 and IL-2 were analysed in VL patients (n = 29) by Cytometric Bead Array to evaluate correlation with parasitic load. Interestingly, IL-10 levels correlated significantly with parasite load (r = 0.82, P<0.0001). The mean parasite load in dermal lesions of PKDL patients was 9,502 parasites/µg tissue DNA at pre-treatment stage (n = 25), with no detectable parasites after therapy (n = 5). Parasite burden was distinctly higher (P<0.0001) in nodular lesions (n = 12) (19,586 parasites/µg tissue DNA) compared to papular/macular lesions (n = 13, 193 parasites/µg tissue DNA). Further, chronic PKDL lesions showed significantly (P = 0.0166) higher parasite load in comparison with acute lesions. Results indicate that chronic, nodular cases constitute the major parasite reservoir for anthroponotic transmission. Our results establish that the high parasite load in VL is strongly correlated with a high level of IL-10, implicating IL-10 as a marker of disease severity. The assay is applicable for diagnosis as well as prognosis of both VL and PKDL, providing a simple molecular tool to monitor the efficacy of antileishmanial drugs or vaccines.

The Vacuolar ATPase (V-ATPase) is a proton pump responsible for controlling the intracellular and extracellular pH of cells. The structure of V-ATPase has been highly conserved among all eukaryotic cells and is involved in diverse functions across species. V-ATPase is best known for its acidification of endosomes and lysosomes and is also important for luminal acidification of specialized cells. Several reports have suggested the involvement of V-ATPase in maintaining an alkaline intracellular and acidic extracellular pH thereby aiding in proliferation and metastasis of cancer cells respectively. Increased expression of V-ATPase and relocation to the plasma membrane aids in cancer modulates key tumorigenic cell processes like autophagy, Warburg effect, immunomoduation, drug resistance and most importantly cancer cell signaling. In this review, we discuss the direct role of V-ATPase in acidification and indirect regulation of signaling pathways, particularly Notch Signaling.

Tumor associated vacuolar H+-ATPases (V-ATPases) are multi-subunit proton pumps that acidify tumor microenvironment, thereby promoting tumor invasion. Subunit ‘a’ of its V0 domain is the major pH sensing unit that additionally controls sub-cellular targeting of V-ATPase and exists in four different isoforms. Our study reports an elevated expression of the V-ATPase-V0a2 isoform in ovarian cancer(OVCA) tissues and cell lines(A2780, SKOV-3 and TOV-112D). Among all V0’a’ isoforms, V0a2 exhibited abundant expression on OVCA cell surface while normal ovarian epithelia did not. Sub-cellular distribution of V-ATPase-V0a2 confirmed its localization on plasma-membrane, where it was also co-associated with cortactin, an F-actin stabilizing protein at leading edges of cancer cells. Additionally, V0a2 was also localized in early and late endosomal compartments that are sites for modulations of several signaling pathways in cancer. Targeted inhibition of V-ATPase-V0a2 suppressed matrix metalloproteinase activity(MMP-9 & MMP-2) in OVCA cells. In conclusion, V-ATPase-V0a2 isoform is abundantly expressed on ovarian tumor cell surface in association with invasion assembly related proteins and plays critical role in tumor invasion by modulating the activity of matrix-degrading proteases. This study highlights for the first time, the importance of V-ATPase-V0a2 isoform as a distinct biomarker and possible therapeutic target for treatment of ovarian carcinoma.

BackgroundPost kala-azar dermal leishmaniasis (PKDL), a sequel to visceral leishamaniasis (VL) in 5–15% cases, constitutes a parasite reservoir important in disease transmission. The precise immunological cause of PKDL outcome remains obscure. However, overlapping counter regulatory responses with elevated IFN-γ and IL-10 are reported.Methodology/Principal FindingsPresent study deals with ex-vivo mRNA and protein analysis of natural regulatory T cells (nTreg) markers (Foxp3, CD25 and CTLA-4) and IL-10 levels in lesion tissues of PKDL patients at pre and post treatment stages. In addition, correlation of nTreg markers and IL-10 with parasite load in tissue lesions was investigated. mRNA levels of nTreg markers and IL-10 were found significantly elevated in pre-treatment PKDL cases compared to controls (Foxp3, P = 0.0009; CD25 & CTLA-4, P<0.0001; IL-10, P<0.0001), and were restored after treatment. Analysis of nTreg cell markers and IL-10 in different clinical manifestations of disease revealed elevated levels in nodular lesions compared to macules/papules. Further, Foxp3, CD25 and IL-10 mRNA levels directly correlated with parasite load in lesions tissues.Conclusion/SignificanceData demonstrated accumulation of nTreg cells in infected tissue and a correlation of both IL-10 and nTreg levels with parasite burden suggesting their role in disease severity in PKDL.

In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma as well as vesicular membranes and critically influences metastatic behavior. The soluble, cleaved N-terminal domain of V-ATPase a2 isoform is associated with in vitro induction of tumorigenic characteristics in macrophages. This activity led us to further investigate its in vivo role in cancer progression by inhibition of a2 isoform (a2V) in tumor cells and the concomitant effect on tumor microenvironment in the mouse 4T-1 breast cancer model. Results showed that macrophages cocultivated with a2V knockdown (sh-a2) 4T-1 cells produce lower amounts of tumorigenic factors in vitro and have reduced ability to suppress T-cell activation and proliferation compared with control 4T-1 cells. Data analysis showed a delayed mammary tumor growth in Balb/c mice inoculated with sh-a2 4T-1 cells compared with control. The purified CD11b(+) macrophages from sh-a2 tumors showed a reduced expression of mannose receptor-1 (CD206), interleukin-10, transforming growth factor-β, arginase-1, matrix metalloproteinase and vascular endothelial growth factor. Flow cytometric analysis of tumor-infiltrated macrophages showed a significantly low number of F4/80(+)CD11c(+)CD206(+) macrophages in sh-a2 tumors compared with control. In sh-a2 tumors, most of the macrophages were F4/80(+)CD11c(+) (antitumor M1 macrophages) suggesting it to be the reason behind delayed tumor growth. Additionally, tumor-infiltrating macrophages from sh-a2 tumors showed a reduced expression of CD206 compared with control whereas CD11c expression was unaffected. These findings demonstrate that in the absence of a2V in tumor cells, the resident macrophage population in the tumor microenvironment is altered which affects in vivo tumor growth. We suggest that by involving the host immune system, tumor growth can be controlled through targeting of a2V on tumor cells.

Macrophage polarization contributes to distinct human pathologies. In tumors, a polarized M2 phenotype called tumor-associated macrophages (TAMs) are associated with promotion of invasion and angiogenesis. In cancer cells, vacuolar ATPase (V-ATPase), a multi-subunit enzyme, is expressed on the plasma/vesicular membranes and critically influences the metastatic behavior. In addition, the soluble, cleaved N-terminal domain of a2 isoform of V-ATPase (a2NTD) is associated with in vitro induction of pro-tumorigenic properties in monocytes. This activity of a2 isoform of V-ATPase (a2V) caused us to investigate its role in cancer progression through the evaluation of the immunomodulatory properties of a2NTD. Here, we present direct evidence that surface expression of V-ATPase is associated with macrophage polarization in tumor tissue. Macrophages from BALB/c mice (peritoneal/bone marrow derived) were stimulated with recombinant a2NTD in both ex vivo and in vivo systems and evaluated for TAM characteristics. a2V was highly expressed in tumor tissues (breast and skin) as well as on the surface of tumor cell lines. The a2NTD-stimulated macrophages (a2MΦ) acquired TAM phenotype, which was characterized by elevated expression of mannose receptor-1, Arginase-1, interleukin-10 and transforming growth factor-β. a2MΦ also exhibited increased production of other tumorigenic factors including matrix metalloproteinase-9 and vascular endothelial growth factor. Further, a2MΦ were cocultured with mouse B-16F0 melanoma cells for their functional characterization. The coculture of these a2MΦ subsequently increased the invasion and angiogenesis of less invasive B-16F0 cells. When cocultured with naive T cells, a2MΦ significantly inhibited T-cell activation. The present data establish the role of V-ATPase in modulating a macrophage phenotype towards TAMs through the action of a2NTD, suggesting it to be a potential therapeutic target in cancer.

Cellular organelles and proteins are degraded and recycled through autophagy, a process during which vesicles known as autophagosomes fuse with lysosomes. Altered autophagy occurs in various diseases, but its role in preterm labor (PTL) is unknown. We investigated the role of autophagic flux in two mouse models of PTL compared to controls: 1) inflammation-induced PTL (IPTL), induced by toll-like receptor agonists; and 2) non-inflammation (hormonally)-induced PTL (NIPTL). We demonstrate that the autophagy related genes Atg4c and Atg7 (involved in the lipidation of microtubule-associated protein 1 light chain 3 (LC3) B-I to the autophagosome-associated form, LC3B-II) decrease significantly in uterus and placenta during IPTL but not NIPTL. Autophagic flux is altered in IPTL, as shown by the accumulation of LC3B paralogues and diminishment of lysosome associated membrane protein (LAMP)-1, LAMP-2 and the a2 isoform of V-ATPase (a2V, an enzyme involved in lysosome acidification). These alterations in autophagy are associated with increased activation of NF-κB and proinflammatory cytokines/chemokines in both uterus and placenta. Similar changes are seen in macrophages exposed to TLR ligands and are enhanced with blockade of a2V. These novel findings represent the first evidence of an association between altered autophagic flux and hyper-inflammation and labor in IPTL.

Oral miltefosine, 50mg thrice daily for 60 days or twice daily for 90 days, could be an effective treatment for PKDL.