Chizu Sanjoba

CD4+ cells are indispensable for ulcer development in murine cutaneous leishmaniasis

ABSTRACT One of the most characteristic clinical features in cutaneous leishmaniasis is the development of nodules followed by ulcerations at the site of infection. Leishmania amazonensis-infected mice show similar ulcerative lesions. Leishmania-infected severe combined immunodeficiency (SCID) mice, however, have been shown to develop nonulcerative nodules. In the present study, the roles of T cells in ulceration were examined using SCID mice in cell reconstitution experiments. After development of nonulcerative nodules, SCID mice were inoculated with splenocytes from eitherLeishmania-infected or naive immunocompetent mice, resulting in ulceration in all mice. When naive splenocytes were depleted of CD4+, CD8+, or B220+cell populations and the remaining cells were injected intoLeishmania-infected SCID mice after the development of nodules, only SCID mice inoculated with splenocytes depleted of CD4+ cells did not show ulceration. The evidence obtained in this study clearly shows that the CD4+ cell population is indispensable for ulceration in leishmaniasis lesions of SCID mice.

Distribution and ecological aspects of sand fly (Diptera: Psychodidae) species in Sri Lanka.

ABSTRACT: Human indigenous cutaneous leishmaniasis caused by Leishmania donovani complex is endemic in Sri Lanka. We performed an entomological survey to determine the distribution of probable vector species. Sand flies were collected in districts in the dry zone, in the wet zone highlands, and in the wet zone coastal belt of Sri Lanka using CDC light traps, sticky traps and cattle-baited net traps during July, 2005. The survey was reconducted in February, 2006. Overall, 584 sand flies belonging to Phlebotomus (266 specimens, 2 species) and Sergentomyia (318 specimens, 8 species) genera were collected. A total of 266 Phlebotomus was identified as P. argentipes (258/266; 97%) and P. stantoni (8/266; 3%). The identification studies of Sergentomyia specimens showed that there are at least 8 species in Sri Lanka. Higher number of Phlebotomus sand flies (76/266) were caught in the southern part of the country compared to the other parts probably due to different ecological aspects. P. argentipes were widely distributed throughout the island whereas P. stantoni were collected only in four districts. Since P. argentipes is known to be the vector of L. donovani responsible of visceral leishmaniasis in India, this species may be incriminated as the most possible vector of human cutaneous leishmaniasis in Sri Lanka.

The development of Leishmania turanica in sand flies and competition with L. major

BackgroundIn Central Asian foci of zoonotic cutaneous leishmaniases, mixed infections of Leishmania turanica and L. major have been found in a reservoir host (the great gerbil, Rhombomys opimus) as well as in the sand fly vector Phlebotomus papatasi, but hybrids between these two Leishmania species have never been reported. In addition, the role of sand fly species other than P. papatasi in L. turanica circulation is not clear.MethodsIn this work we compared the development of L. turanica in three sand fly species belonging to different subgenera. In addition, we studied experimental co-infections of sand flies by both Leishmania species using GFP transfected L. turanica (MRHO/MN/08/BZ18(GFP+)) and RFP transfected L. major (WHOM/IR/-/173-DsRED(RFP+)). The possibility of Leishmania genetic exchange during the vectorial part of the life cycle was studied using flow cytometry combined with immunofluorescent microscopy.ResultsLate-stage infections of L. turanica with frequent colonization of the stomodeal valve were observed in the specific vector P. (Phlebotomus) papatasi and in the permissive vector P. (Adlerius) arabicus. On the other hand, in P. sergenti (the specific vector of L. tropica), L. turanica promatigotes were present only until the defecation of bloodmeal remnants. In their natural vector P. papatasi, L. turanica and L. major developed similarly, and the spatiotemporal dynamics of localization in the sand fly gut was the same for both leishmania species. Fluorescence microscopy in combination with FACS analyses did not detect any L. major / L. turanica hybrids in the experimental co-infection of P. papatasi and P. duboscqi.ConclusionOur data provide new insight into the development of different leishmania parasite species during a mixed infection in the sand fly gut. Despite the fact that both Leishmania species developed well in P. papatasi and P. duboscqi and did not outcompete each other, no genetic exchange was found. However, the ability of L. turanica to establish late-stage infections in these specific vectors of L. major suggests that the lipophosphoglycan of this species must be identical or similar to that of L. major.

Adhesion of MRP8/14 to amastigotes in skin lesions of Leishmania major-infected mice

In murine experimental cutaneous leishmaniasis, parasite infection induces an accumulation of macrophages expressing migration inhibitory factor-related protein 8 (MRP8) and MRP14, two members of the S100 calcium-binding protein family. Although MRP8 and MRP14 are cytoplasmic proteins expressed by myeloid cells, recent studies have demonstrated that MRP8 and MRP14 have extracellular functions such as chemotactic activities. In this study, we examined whether extracellular MRP8 and MRP14 interact with Leishmania parasites during infection. By immunohistochemistry, positive staining by MRP8 and MRP14 was detected on amastigotes in skin lesions of Leishmania major-infected mice. Western blot analysis with amastigotes purified from the skin lesions demonstrated that both of these proteins adhered to amastigotes. The adhesion of MRP14 to amastigotes was reproduced in vitro and enhanced in the presence of Ca2+ and Zn2+. MRP14 adhered to not only amastigotes, but also promastigotes, suggesting receptor molecules for MRP14 are expressed commonly in both developmental stages.

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.

The accumulation of macrophages expressing myeloid-related protein 8 (MRP8) and MRP14 in the spleen of BALB/cA mice during infection with Plasmodium berghei.

Splenomegaly is one of the typical symptoms of malaria. However, the pathogenesis of splenic enlargement still remains unclear. Spleen is a major organ for clearance of malaria parasites, but excessive response to the parasites can lead to splenomegaly. Myeloid-related protein (MRP) 8 and MRP14 are expressed by myeloid cells and are regarded as marker proteins of an immature and inflammatory subtype of macrophage. Previous studies have demonstrated that accumulation of MRP8(+) and MRP14(+) macrophages is associated with the pathological changes associated with various inflammatory diseases. In order to elucidate whether MRP8(+) and MRP14(+) cells are also involved in splenomegaly during malaria, we investigated expression of MRP8 and MRP14 in the spleens of mice infected with Plasmodium berghei. The MRP8 and MRP14 levels in the serum were analyzed by western blot, which confirmed that these proteins were elevated during infection compared with uninfected controls. Enlargement of the spleen was prominent at 7days of infection, and histological analysis of the spleens demonstrated deposition of malaria pigments and accumulation of mononuclear cells. Immunohistochemical staining of the tissue revealed the accumulation of cells expressing MRP8 and MRP14. In addition, the locations of those cells overlapped with CD11b(+) cells in the red pulp. These results suggest that splenomegaly in malaria is partly due to the accumulation of MRP8(+) and MRP14(+) macrophages.

Elevation of Serum B-Cell Activating Factor Levels During Visceral Leishmaniasis

Elevation of serum B-cell activating factor (BAFF) is one of the characteristics of immunological disorders, including autoimmunity, but the levels of BAFF in infectious diseases have not been studied well. Here, we showed the elevation of serum BAFF in patients with visceral leishmaniasis (VL). The mean serum BAFF value in VL patients (4.65 ng/mL) was 4.3 times higher than that of healthy controls (1.08 ng/mL), and 90% of VL patients showed serum BAFF above the cutoff that was calculated as the mean + 3 SDs of the controls. This report is the first on elevation of serum BAFF during VL.

Hemophagocytosis in Experimental Visceral Leishmaniasis by Leishmania donovani

Hemophagocytosis is a phenomenon in which macrophages phagocytose blood cells. There are reports on up-regulated hemophagocytosis in patients with infectious diseases including typhoid fever, tuberculosis, influenza and visceral leishmaniasis (VL). However, mechanisms of infection-associated hemophagocytosis remained elusive due to a lack of appropriate animal models. Here, we have established a mouse model of VL with hemophagocytosis. At 24 weeks after infection with 1 x 107 Leishmania donovani promastigotes, BALB/cA mice exhibited splenomegaly with an average tissue weight per body weight of 2.96%. In the tissues, 28.6% of macrophages contained phagocytosed erythrocytes. All of the hemophagocytosing macrophages were parasitized by L. donovani, and higher levels of hemophagocytosis was observed in heavily infected cells. Furthermore, more than half of these hemophagocytes had two or more macrophage-derived nuclei, whereas only 15.0% of splenic macrophages were bi- or multi-nuclear. These results suggest that direct infection by L. donovani causes hyper-activation of host macrophages to engulf blood cells. To our knowledge, this is the first report on hemophagocytosis in experimental Leishmania infections and may be useful for further understanding of the pathogenesis.

Geographical Distribution and Ecological Aspect of Sand Fly Species in Bangladesh

Phlebotomine sand flies, which are biological vectors of Leishmania spp., are represented by around 400 species in the Old World and more than 600 species in the Americas. The vector sand fly species generally belong to the Phlebotomus genus in the Old World and the Lutzomyia genus in the New World. They are yellowish, long legged hairy insects and active after sunset until sunrise. Sand flies can transmit Leishmania parasites as well as some group of viruses called Phleboviruses and a bacterium, Bartonella bacilliformis. Visceral leishmaniasis (VL, Kala-azar) caused by Leishmania donovani is an important health problem in the Indian subcontinent including Bangladesh and Phlebotomus argentipes is a proven vector species of Leishmania donovani. In Bangladesh, a total of 13 sand fly species (3 Phlebotomus, 10 Sergentomyia spp.) were recorded so far. All studies showed the dominancy of P. argentipes especially in endemic areas for VL. In this chapter, besides P. argentipes and its biological and ecological features, other species constituting sand fly fauna and their geographical distribution in Bangladesh are discussed.

IL-25, IL-33 and TSLP receptor are not critical for development of experimental murine malaria

IL-25, IL-33 and TSLP, which are produced predominantly by epithelial cells, can induce production of Th2-type cytokines such as IL-4, IL-5 and/or IL-13 by various types of cells, suggesting their involvement in induction of Th2-type cytokine-associated immune responses. It is known that Th2-type cytokines contribute to host defense against malaria parasite infection in mice. However, the roles of IL-25, IL-33 and TSLP in malaria parasite infection remain unclear. Thus, to elucidate this, we infected wild-type, IL-25−/−, IL-33−/− and TSLP receptor (TSLPR)−/− mice with Plasmodium berghei (P. berghei) ANKA, a murine malaria strain. The expression levels of IL-25, IL-33 and TSLP mRNA were changed in the brain, liver, lung and spleen of wild-type mice after infection, suggesting that these cytokines are involved in host defense against P. berghei ANKA. However, the incidence of parasitemia and survival in the mutant mice were comparable to in the wild-type mice. These findings indicate that IL-25, IL-33 and TSLP are not critical for host defense against P. berghei ANKA.