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A new xenicane diterpenoid, cristaxenicin A (1), has been isolated from the deep sea gorgonian Acanthoprimnoa cristata. The structure of 1 was elucidated on the basis of spectral analysis including NMR and MS. The absolute configuration of 1 was determined on the basis of quantum chemical calculation of CD spectra. Cristaxenicin A (1) showed antiprotozoal activities against Leishmania amazonensis and Trypanosoma congolense with IC(50) values of 0.088 and 0.25 μM, respectively.

Background: In 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., Methods: In 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., Results: Late-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., Conclusion: Our 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.

Three lipopeptides, ciliatamides A-C ( 1- 3), were isolated from the deep-sea sponge Aaptos ciliata, and their structures were elucidated on the basis of spectroscopic and chemical methods. Ciliatamides A ( 1) and B ( 2) were found to be antileishmanial, while 2 also exhibited marginal cytotoxicity to HeLa cells.

Leishmania amazonensis recombinants expressing the enhanced green fluorescent protein (egfp) gene or beta-galactosidase gene (lacZ) were constructed for drug screening and histopathological analysis. The egfp or lacZ in a leishmanial transfection vector, p6.5, was introduced into L. amazonensis promastigotes, and egfp or lacZ-carrying recombinant L. amazonensis, La/egfp and La/lacZ, respectively, were obtained. Expression of egfp or lacZ in both promastigotes and amastigotes could be clearly visualized by fluorescence microscopy or by light microscopy with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), respectively. Fluorescence signal and beta-galactosidase activity measured by a colorimetric reaction with chlorophenol red beta-D-galactopyranoside (CPRG) were well correlated to the numbers of these parasites. The inhibitory concentration (IC50) of a leishmanicidal drug, amphotericin B, in L. amazonensis promastigotes measured using La/egfp and La/lacZ was similar to that measured by conventional methods such as cell counting, thymidine incorporation and colorimetric assay. Furthermore, the fluorescence signal and absorbance of CPRG correlated well with the numbers of La/egfp and La/lacZ amastigotes in macrophages, respectively, suggesting La/egfp and La/lacZ can be a convenient and useful tool for drug screening not only in promastigotes, but also in amastigotes of L. amazonensis. La/lacZ collected from mouse tissues four weeks after the parasite infection were stained well with X-Gal. La/lacZ allowed parasite detection at high sensitivity in the tissues of infected mice and will be useful for following infections in macrophages in vivo. Thus, the marker-transfected Leishmania parasites constructed in this study will be useful for analyses of Leishmania parasites, especially at the intracellular stage.

It has been reported that Leishmania promastigotes have ability to express foreign genes on drug selectable plasmids. To investigate further abilities of the recently described expression vector, P6.5, in the transfection of Leishmania organisms (Chen D-Q, Kolli BK, Yadava N et al. Episomal expression of specific sense and antisense mRNAs in Leishmania amazonensis: modulation of gp63 levels in promastigotes and their infection of macrophages in vitro. Infect Immun 2000;68:80--86), the constructed expression vector, which contains canine interleukin-8 (cIL-8) coding cDNA, was introduced by electroporation to promastigotes of four species of the genus Leishmania: Leishmania amazonensis, L. equatorensis, L. donovani and L. infantum. Extrachromosomal DNAs and total RNAs from the transfected promastigotes were subjected to polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively, using cIL-8 gene specific primers, and a predicted product of 330 bp was detected. Western blot analysis using a mouse monoclonal antibody raised against cIL-8 demonstrated the successful expression of cIL-8 in the transfectants and culture supernatants. Culture supernatants of the transfected L. amazonensis and L. equatorensis promastigotes showed a high chemotactic activity to both dog and mouse polymorphonuclear leukocytes. These results indicate that Leishmania promastigotes transfected with the expression vector P6.5 containing cIL-8 cDNA are capable of producing biologically active cIL-8. The Leishmania expression system using the P6.5 vector might be a useful alternative for the production of biologically active recombinant cytokines.

Visceral leishmaniasis (VL) is a major problem worldwide and causes significant morbidity and mortality. Existing drugs against VL have limitations, including their invasive means of administration long duration of treatment regimens. There are also concerns regarding increasing treatment relapses as well as the identification of resistant clinical strains with the use of miltefosine, the sole oral drug for VL. There is, therefore, an urgent need for new alternative oral drugs for VL. In the present study, we show the leishmanicidal effect of a novel, oral antimalarial endoperoxide N-251. In our In vitro studies, N-251 selectively and specifically killed Leishmania donovani D10 amastigotes with no accompanying toxicity toward the host cells. In addition, N-251 exhibited comparable activities against promastigotes of L. donovani D10, as well as other L. donovani complex parasites, suggesting a wide spectrum of activity. Furthermore, even after a progressive infection was established in mice, N-251 significantly eliminated amastigotes when administered orally. Finally, N-251 suppressed granuloma formation in mice liver through parasite death. These findings indicate the therapeutic effect of N-251 as an oral drug, hence suggest N-251 to be a promising lead compound for the development of a new oral chemotherapy against VL. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Migration inhibitory factor-related protein 8 (MRP8) and MRP14 are expressed by myeloid cells and especially known as marker proteins of an immature and inflammatory subtype of macrophages. In this study, we immunohistochemically examined an accumulation of MRP8+ and MRP14+ macrophages in skin lesions during Leishmania major infection in susceptible BALB/c and RAG-2−/− mice. L. major infection caused the development of a nodular type of skin lesion at the infection site in mice and a massive accumulation of macrophages was observed in the lesions at four weeks after the infection. Immunohistochemical analyses showed MRP8+ and MRP14+ macrophages are predominant cell types in the skin lesions in both mouse strains. In contrast, F4/80+ cells, which correspond to mature macrophages, were rarely found in the skin lesions. These data suggest that the accumulation of inflammatory subtype of macrophages in BALB/c mice during L. major infection can be induced without acquired immune responses. [Copyright &y& Elsevier]

Susceptibility to infection with Leishmania amazonensis promastigotes was examined in six B10 congenic mouse strains, including C57BL/10J (H2b), B10.BR (H2k), B10.M (H2f), B10.S (H2s), B10.RIII (H2r), and B10.D2 (H2d). All strains of mice developed skin nodules with punch-out ulcers by 8 weeks post-infection, but B10.M and B10.S mice showed resolution of cutaneous leishmaniasis lesions by 16 weeks post-infection. In addition, the skin lesions were much larger in BALB congenic mice than in B10 and C3H mice, even though these mice share the same H2 haplotypes. These results suggest that H2 complex controls the growth of L. amazonensis in cutaneous lesions, and that non-H2 genes inherited by BALB congenic mice have a more potent role than the H2 complex in lesion progression. [Copyright &y& Elsevier]

B-cell activating factor (BAFF) is known as a cytokine responsible for survival and activation of B cells. However, involvement of the molecule in IgG antibody production during infection remains elusive. In this study, dependency of antibody production in Leishmania infection on BAFF was examined by using BAFF-knockout (BAFF-KO) mice. When BAFF-KO mice were infected with L. major , there was no significant difference in lesion development or parasite burden from those in infected wildtype mice. In contrast, levels of IgG antibodies to Leishmania crude antigen were lower in BAFF-KO mice, suggesting that antibody production during L. major infection is BAFF-dependent. ELISA using defined leishmanial antigens demonstrated that the influence of BAFF on antibody production during L. major varies depending on antigens; IgG production to tandem repeat proteins were more affected by BAFF than non-repeat antigens. On the contrary, all of the defined antigens tested were strongly affected by BAFF for IgG antibody production during L. donovani infection. These results suggest degree of BAFF contribution to antibody production during infection is variable depending on the type of infection and even on the type of antigen in a given infection. These results may explain contradictory roles of BAFF in antibody production in previous works. • BAFF was thought to be indispensable for humoral response. • BAFF has variable effects on IgG antibody production during L. major infection. • IgG antibody production is broadly affected by BAFF in L. donovani infection. • Thus, role of BAFF in IgG response is affected by the type of antigen/infection. [ABSTRACT FROM AUTHOR]