No vaccine is currently available for visceral leishmaniasis (VL) caused by Leishmania donovani. This study addresses whether a live attenuated centrin gene-deleted L. donovani (LdCen1−/−) parasite can persist and be both safe and protective in animals. LdCen1−/− has a defect in amastigote replication both in vitro and ex vivo in human macrophages. Safety was shown by the lack of parasites in spleen and liver in susceptible BALB/c mice, immune compromised SCID mice, and human VL model hamsters 10 wk after infection. Mice immunized with LdCen1−/− showed early clearance of virulent parasite challenge not seen in mice immunized with heat killed parasites. Upon virulent challenge, the immunized mice displayed in the CD4+ T cell population a significant increase of single and multiple cytokine (IFN-γ, IL-2, and TNF) producing cells and IFN-γ/IL10 ratio. Immunized mice also showed increased IgG2a immunoglobulins and NO production in macrophages. These features indicated a protective Th1-type immune response. The Th1 response correlated with a significantly reduced parasite burden in the spleen and no parasites in the liver compared with naive mice 10 wk post challenge. Protection was observed, when challenged even after 16 wk post immunization, signifying a sustained immunity. Protection by immunization with attenuated parasites was also seen in hamsters. Immunization with LdCen1−/− also cross-protected mice against infection with L. braziliensis that causes mucocutaneous leishmaniasis. Results indicate that LdCen1−/− can be a safe and effective vaccine candidate against VL as well as mucocutaneous leishmaniasis causing parasites.

In the present study we have demonstrated some features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral Leishmaniasis. We report that PCD is initiated in stationaryphaseculturesofpromastigotesandbothinactively growing cultures of axenic amastigotes and promastigotes upon treatment with anti Leishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antileishmanial drugs differently. The features of PCD in L. donovani promastigotes are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, increase in plasma membrane permeability, decrease in the mitochondrial membrane potential (DC m ) and induction of a PhiPhiLux (PPL)-cleavage activity. PCD in both stationary phase culture and upon induction by amphotericin B resulted first in the decrease of mitochondrial membrane potential followed by simultaneous change in plasma membrane permeability and induction of PPL-cleavage activity. Of the total PPL-cleavage activity, several caspase inhibitors inhibited a significant amount (21 ± 34%). Inhibitors of cathepsin or calpain did not inhibit PPL-cleavage activity. Taken together this study demonstrates that the characteristic features of PCD exist in unicellularprotozoanLeishmaniadonovani.Theimplicationof PCD on the Leishmania pathogenesis is discussed. Cell Death and Differentiation (2002) 9, 53 ± 64.

An endothelial cell inhibitor was purified from supernatant of an Epstein-Barr virus–immortalized cell line and identified as fragments of calreticulin. The purified recombinant NH2-terminal domain of calreticulin (amino acids 1–180) inhibited the proliferation of endothelial cells, but not cells of other lineages, and suppressed angiogenesis in vivo. We have named this NH2-terminal domain of calreticulin vasostatin. When inoculated into athymic mice, vasostatin significantly reduced growth of human Burkitt lymphoma and human colon carcinoma. Compared with other inhibitors of angiogenesis, vasostatin is a small, soluble, and stable molecule that is easy to produce and deliver. As an angiogenesis inhibitor that specifically targets proliferating endothelial cells, vasostatin has a unique potential for cancer treatment.

We have developed a PCR assay that is capable of amplifying kinetoplast DNA (kDNA) of Leishmania donovani in a species-specific manner among Old World leishmanias. With Indian strains and isolates of L. donovani the assay was sensitive enough to detect kDNA in an amount equivalent to a single parasite or less. The extreme sensitivity of the assay was reflected in its ability to detect parasite DNA from small volumes of peripheral blood of patients with kala-azar (KA) and from skin lesions from patients with post-KA dermal leishmaniasis (PKDL). A total of 107 clinical leishmaniasis samples were analyzed. Of these 102 (95.3%) were positive by PCR. The test provided a diagnosis of KA with 96% sensitivity using patient whole-blood samples instead of bone marrow or spleen aspirates that are obtained by invasive procedures. The assay was also successful in the diagnosis of 45 of 48 PKDL cases (93.8%). Cross-reactions with pathogens prevalent in the area of endemicity, viz., Mycobacterium tuberculosis, Mycobacterium leprae, and Plasmodium spp., could be ruled out. Eighty-one control samples, including dermal scrapings from healthy portions of skin from patients with PKDL were all negative. Two of twenty controls from the area of endemicity were found positive by PCR assay; however, there was a good possibility that these two were asymptomatic carriers since they were serologically positive for KA. Thus, this PCR assay represents a tool for the diagnosis of KA and PKDL in Indian patients in a noninvasive manner, with simultaneous species identification of parasites in clinical samples.