Refinement of techniques for the propagation of Leishmania donovani in hamsters

Improved animal models are urgently required for drug and vaccine development against visceral leishmaniasis. Here we report refinements to the hamster model of infection that reduce the severity of the disease as well as the number of animals required to maintain infection while improving parasite yields. A comparison between infection via the intracardiac and intraperitoneal routes showed that the less commonly used intraperitoneal route is the simpler and preferred method. The KAtex latex agglutination test for visceral leishmaniasis accurately detected Leishmania donovani antigen in hamster urine as early as 6 weeks post-inoculation. With modification, this assay could be an important tool in the evaluation of experimental drugs and vaccines.     

Interleukin 10 receptor blockade--pentavalent antimony treatment in experimental visceral leishmaniasis

Interleukin 10 (IL-10), a suppressive Th2 cell-type cytokine, promotes disease progression in experimental visceral leishmaniasis. To extend testing the therapeutic effects of applying IL-10 receptor (IL-10R) blockade with antileishmanial chemotherapy, BALB/c mice with established intracellular Leishmania donovani infection were injected once with anti-IL-10R mAb at the time low-dose, daily pentavalent antimony (Sb) therapy was initiated. In this treatment model, simultaneous administration of anti-IL-10R enhanced overall antileishmanial activity in the liver in an interferon-gamma-dependent fashion, and accelerated the kinetics of Sb-associated killing, induced a >10-fold Sb dose-sparing effect and shortened the required duration of Sb treatment. These results suggest the possibility of using mAb-induced IL-10R blockade to develop low-dose and/or short-course immunochemotherapeutic regimens in visceral leishmaniasis.     

Parasitic load and histopathology of cutaneous lesions,lymph node,spleen, and liver from BALB/c and C57BL/6 mice infected with Leishmania mexicana

The course of infection, parasitic loads, and histopathology of cutaneous lesions, draining lymph node, spleen, and liver were compared in BALB/c and C57BL/6 mice over a period of 34 weeks after inoculation in footpad with promastigotes of a Leishmania mexicana reference strain. The results show that the primary footpad lesions first present a 12-week phase that develops similarly in both strains of mice. Thereafter, a cutaneous and visceral dissemination of L. mexicana parasites occurs in BALB/c mice; the latter experience an extensive breakdown of the lymphoid organ microarchitecture, whereas C57BL/6 mice succeed in eliminating the parasite infection from the lymph nodes but not from the primary cutaneous lesion, which does not heal. These results highlight marked differences between responses of key anatomical compartments controlling L. mexicana infection in BALB/c and C57BL/6 mice.     

Experimental leishmaniasis in humans: review

Experimental infection of humans with Leishmania parasites has contributed significantly to the understanding of the etiology, transmission, and pathogenesis of leishmaniasis and the immunity associated with it. Leishmania organisms recovered from human and animal tissue, insect vectors, and in vitro cultures have all produced cutaneous or visceral leishmaniasis in human subjects who were voluntarily inoculated with them. Volunteers bitten by infected Phlebotomine sandflies also developed cutaneous or visceral disease. In these experiments, it appeared that the parasite must undergo certain developmental changes within the sandfly for it to become infective and that the parasites in sandflies were far more efficient in causing full-blown infection than were cultured Leishmania organisms. The clinical manifestations of these experimental infections did not differ from infections that were acquired naturally. Natural or experimental infections appeared to confer resistance to subsequent leishmanial infection. This immunity was best documented to be a species-specific phenomenon; however, a small number of studies have demonstrated cross protection between some Leishmania species. In this review article, data from human experimental infections are summarized and discussed in light of recent advances in the field.     

克拉玛依地区的利什曼病 Ⅸ.白蛉自然感染前鞭毛体的鉴定

本文报道新疆克拉玛依地区的大沙鼠洞、旷野及人房等不同场所内白蛉自然感染利什曼原虫的情况、蛉体内原虫对实验动物的致病特征以及用利什曼原虫McAb的dot-ELISA法对白蛉自然感染的利什曼前鞭毛体的检测结果;并结合4年来对当地白蛉的生态和大沙鼠体内的利什曼原虫对白蛉的感染性等一系列的调查研究,证实蒙古白蛉和安氏白蛉为克拉玛依大沙鼠体内利什曼原虫的传播媒介。     

Human placental extract offers protection against experimental visceral leishmaniasis: a pilot study for a phase-I clinical trial

An aqueous extract of human placenta (HPE) was found to offer protection against established experimental visceral leishmaniasis in BALB/c mice and hamsters, whether the Leishmania donovani strain involved was one that was sensitive or resistant to pentavalent antimony. Intraperitoneal administration of the extract, into mice or hamsters that had been infected 2 months previously, led to antileishmanial T-cell proliferation among splenic mononuclear cells, the generation of host-protective cytokines (interferon-gamma, tumour necrosis factor and interleukin-12) and the upregulation of the expression of inducible nitric oxide synthase (and subsequent NO generation) in splenocytes. Furthermore, splenic macrophages from the HPE-treated mice showed increased generation of reactive oxygen species and enhanced surface expression of antigens of major histocompatibility complex class II (MHCII), and the extract restored the otherwise-defective antigen-presenting ability of the macrophages. Thus, in mice and hamsters infected with L. donovani, HPE therapy can stimulate both arms of the host's immune system and favour the complete resolution of the leishmanial infection. Among five human cases of visceral leishmaniasis, 30 daily intramuscular injections of HPE, at doses much lower than those used in the experimental infections, also gave very promising results. Based on the results of this pilot study, a further evaluation of the efficacy of HPE therapy, which may offer a cost-effective way of improving the treatment of antimony-resistant cases of visceral leishmaniasis, is being undertaken.     

Th17 cells and neutrophils: Close collaborators in chronic Leishmania mexicana infections leading to disease severity

Cutaneous leishmaniasis caused by Leishmania mexicana is associated with an important inflammatory response. We here analysed the kinetics of Th17 cells and neutrophils in ear lobe lesions caused by Leishmania mexicana throughout 90 days of disease progression in susceptible BALB/c and semi‐resistant C57BL/6 mice infected with 1 × 10⁵ Leishmania mexicana promastigotes. Cells in the lesions were extracted and quantified by flow cytometry, whereas their distribution in the tissues in relation to the parasites was analysed by immunohistochemistry. Our results show that in BALB/c mice, both Th17 cells and neutrophils increase concomitantly and to significantly higher levels on day 90 post‐infection, as compared to C57BL/6 mice. Our results provide novel evidence on the cells causing chronic inflammation throughout Leishmania mexicana infections, resulting as a consequence of neutrophil recruitment together with Th17 cell differentiation and recruitment, both of which remain in the infection site throughout the late phase of the infection. We conclude that the more enhanced levels of Th17 cells and neutrophils during chronic inflammatory lesions in BALB/c mice participate in their enhanced susceptibility towards a progressive disease evolution, whereas the more controlled response of these cells in C57BL/6 mice possibly relates to the more resistant profile of this mouse strain.     

Kala-azar in a high transmission focus: an ethnic and geographic dimension

In 1994-1996, we studied a group of 58 game wardens stationed in an area known to be highly endemic for visceral leishmaniasis (kala-azar) for evidence of infection with Leishmania donovani. Leishmania DNA was detected by the polymerase chain reaction in the peripheral blood of cases of active kala-azar, former patients with visceral leishmaniasis, patients, and asymptomatic subjects. Using the cloned antigen rk39, antibodies were detected in 44.2% of the game wardens while leishmanin skin test result was positive in 77% of our sample. It was shown that certain tribes from northern Sudan were more likely to develop subclinical infections, while those of the Baria tribe from southern Sudan and those of the Nuba tribe from western Sudan were more likely to develop visceral leishmaniasis. Whether this is due to genetic factors or previous exposure to Leishmania parasites remains to be elucidated.     

Immunohistological features of visceral leishmaniasis in BALB/c mice

It has been reported that the level of protection provided by vaccines against murine visceral leishmaniasis (VL) is low and that progress in research on VL may be due to the lack of appropriate models to study protective immunity. We have analysed the immunohistological features occurring in BALB/c mice after intravenous administration of 10(3), 10(5) and 10(6) parasites of Leishmania infantum. Our results show that in all cases parasite administration leads to the establishment of infection and to the development of quantifiable immunohistological features which are dependent on the inoculum size. This study demonstrates that differences in the parasite challenge result in changes in the evolution of some of the parameters associated with the degree of the infection in the BALB/c model: level of anti-Leishmania antibodies, up-regulation of spleen arginase activity, balance between IFN-gamma and IL-10, extent of lymphoid follicle depletion in the splenic white pulp and ineffective development of hepatic granulomas. Also, and depending on the initial infectious inoculum, the absence of parasites in the bone marrow and the number of mature and empty type granulomas were parameters associated with protection. We think that in this model a challenge of the order of 10(5) parasites should prove useful for vaccine studies against VL.     

Factors influencing Leishmania major infection in IL-4-deficient BALB/c mice

The outcome of Leishmania major infection in IL-4-deficient BALB/c mice has been a controversial subject. We have shown that IL-4-deficient BALB/c mice infected with Leishmania major developed progressive lesions and could not contain the replication of the parasites, whereas other studies have reported that IL-4-deficient mice were able to resist infection. Therefore, we examined different factors that can influence the course of Leishmania major infection. We tested different lines of IL-4-deficient BALB/c mice and show that the reported differences in the outcome of infection were not due to the different genetic origin of the embryonic stem cells used to disrupt the IL-4 gene. In addition, we infected IL-4-deficient mice with different isolates of L. major parasites and show that none of the parasite strains tested were cleared, although some of them caused milder pathology. Interestingly, this milder pathology was paralleled by a reduced arginase activity of the parasites. We also tested the influence of age on the course of Leishmania major infection in IL-4-deficient BALB/c mice and show that older mice express a transient resistance. Thus, we conclude that differences in the age of the mice and in the arginase activity of the different isolates of parasites are factors that can influence the non-healing phenotype of IL-4-/- BALB/c mice.     

Liver involvement during visceral leishmaniasis

Visceral leishmaniasis is a life-threatening infection due to Leishmania parasites that has been reported in 62 countries. The Mediterranean area is endemic for Leishmania infantum and cases have been reported in Spain, France, Italy and Portugal. During the infection, parasites disseminate in the phagocyte cells of bone marrow, the spleen, liver and lymph nodes. In this paper, we review the clinical and biological signs observed in visceral cases of leishmaniasis with hepatic involvement. We also focus on experimental and pathophysiological data to clarify our understanding of liver involvement during this infection.     

Leishmania sp. isolated from human cases of cutaneous leishmaniasis in Brazil characterized as Leishmaniamajor-like

The identification and characterization of Leishmania are relevant to diagnosis, treatment, eco-epidemiology studies, prophylactic measures and control of the disease. Two strains of Leishmania (MHOM/BR/1971/BH49 and MHOM/BR/1971/BH121), isolated from human cutaneous leishmaniasis, were studied using biological and molecular characteristics, in comparison with WHO reference strains. These studies are important because both strains were incorporated in a vaccine against American cutaneous leishmaniasis, and one of these strains has been used to prepare specific and sensitive antigen for serodiagnosis of visceral leishmaniasis in Brazil. Studies were made on the growth rates of promastigotes in Grace's insect medium, infectivity to C57BL/6 and BALB/c mice, electrophoresic mobility patterns of isoenzymes, random amplification of polymorphic DNA (RAPD), simple sequence repeat-anchored PCR amplification (SSR-PCR) and DNA fingerprinting profiles, infectivity to murine macrophages and cellular immune response. Infections of mice and macrophages were significantly different among the strains studied. Attempts to infect mice with culture promastigotes were unsuccessful with BH121, but BH49 infected BALB/c and C57BL/6 mice. Isoenzyme electrophoretic mobility patterns, RAPD and SSR-PCR using DNA amplified by polymerase chain reaction (PCR) with nine arbitrary primers, as well as DNA fingerprinting studies with a biotin-labeled 33.15 fingerprinting probe showed similar profiles to those of the Leishmania major WHO reference strain.     

A limiting dilution assay for quantifying Leishmania major in tissues of infected mice

A limiting dilution assay for the quantification of Leishmania major in infected mouse tissue was developed. The assay was found to be both sensitive and reliable, and, due to its design, could be scored either visually or following the incorporation of 3H-thymidine by the growing parasites. Results are presented in which the assay was employed to enumerate L. major in the tissues of susceptible (BALB/c) and resistant (CBA) mice at intervals after infection with L. major. It was found that parasites could be detected at the site of injection with L. major as early as 3 days after infection. By day 8, a substantial increase in the number of parasites at the lesion site had occurred in both strains of mice. Subsequently, whereas the number of parasites decreased in the lesions of CBA mice, their number steadily increased in the lesions of BALB/c mice. Parasites were detected in lymph nodes draining the lesion site in both BALB/c and CBA mice by 28 days after infection. Interestingly, a low number of L. major was found in the lymph nodes of CBA mice at 100 days after infection, a time when no parasites could be detected at the lesion site. Previous results from this laboratory have demonstrated that the adoptive transfer of L. major-specific L3T4-positive T-cell populations exacerbated cutaneous lesions induced by L. major in BALB/c mice. Experiments presented here indicate that the adoptive transfer of L. major-specific T-cells also exacerbated cutaneous leishmaniasis in CBA mice. Using the sensitive limiting dilution assay presently described, it was found that this unexpected exacerbative effect of L. major-specific T-cells on lesion development was accompanied by a substantial increase in the number of parasites in the lesions of the adoptively transferred mice.     

The activity of nitrofurazone and furazolidone against Leishmania donovani, L. major and L. enriettii in vitro and in vivo

Furazolidone and nitrofurazone showed in vitro activity against amastigotes of Leishmania donovani, L. enriettii and L. major in macrophages, at concentrations which were also toxic to the macrophages. A low grade of activity was observed against L. donovani infections in BALB/c mice by furazolidone but not with nitrofurazone. Nitrofurazone, in two concentrations, was not active when applied to the lesions of cutaneous leishmaniasis due to L. enriettii (guinea-pig infection) or L. major strain P (BALB/c mouse infection). After systemic administration to BALB/c mice infected with L. major strain JISH 252 clone 1, low-grade activity was observed at the highest level tested.     

Endogenous IL-13 plays a crucial role in liver granuloma maturation during Leishmania donovani infection, independent of IL-4Rα-responsive macrophages and neutrophils

Previous studies comparing interleukin 4 receptor α (IL-4Rα)(-/-) and interleukin 4 (IL-4)(-/-) BALB/c mice have indicated that interleukin 13 (IL-13), whose receptor shares the IL-4Rα subunit with IL-4, plays a protective role during visceral leishmaniasis. We demonstrate that IL-13(-/-) BALB/c mice were less able to control hepatic growth of Leishmania donovani compared with wild-type mice. This correlated with significantly retarded granuloma maturation in IL-13(-/-) mice, defective interferon γ (IFN-γ) production, and elevated IL-4 and interleukin 10 (IL-10) levels. L. donovani-infected IL-13(-/-) mice also responded poorly to sodium stibogluconate-mediated chemotherapy compared with wild-type BALB/c mice. Because murine lymphocytes do not have IL-13 receptors, we examined the ability of macrophage/neutrophil-specific IL-4Rα(-/-) mice to control primary infection with L. donovani and to respond to chemotherapy. Macrophage/neutrophil-specific IL-4Rα(-/-) mice were as resistant to leishmaniasis as wild-type mice, and chemotherapy retained its efficacy. Consequently, in L. donovani infected BALB/c mice, IL-13 promotes hepatic granuloma formation and controls parasite burdens independently of direct effects on macrophages/neutrophils.     

Hyperbaric oxygen therapy reduces the size of Leishmania amazonensis-induced soft tissue lesions in mice

In this study we determined whether exposing mice to hyperbaric oxygen (HBO) would alter various disease parameters of a susceptible mouse strain infected with Leishmania amazonensis. BALB/c mice exposed to HBO (100% O2 at a pressure of 2.5 ATA, 1h before parasite inoculation and subsequently for 20 days) showed significant delay in lesion development and reduction in lesion parasite burdens compared with HBO-unexposed mice. Circulating levels of interferon gamma (IFN-gamma) and tumor necrosis factor (TNF-alpha) were significantly elevated in HBO-exposed as compared to HBO-unexposed mice. Concanavalin A-stimulated lymph nodes cultures from HBO-exposed mice released significantly more IFN-gamma and less interleukin 10 (IL-10) than cultures from HBO-unexposed mice, consistent with a skewed Th1 response. These results demonstrate, for the first time, that HBO can play a pathogen control role during leishmaniasis. Further studies are needed to elucidate whether hyperoxia alone or increased atmospheric pressure alone can exert a similar effect.     

Infectivity of five different types of macrophages by Leishmania infantum

Leishmania are intracellular parasites that multiply as the amastigote form in the macrophages of their vertebrate hosts. Since vaccines against leishmaniases are still under development, the control of these diseases relies on prompt diagnosis and chemotherapy in infected humans as well as in dogs, which are the main reservoir of Leishmania infantum, in Mediterranean countries. To establish the macrophage type to be used as an in vitro model for antileishmanial chemotherapeutic studies, we analysed the susceptibility of human peripheral blood derived macrophages, macrophages derived from mouse bone marrow, mouse peritoneal macrophages and macrophages differentiated from cell lines U-937 and DH82 to infection by two L. infantum strains, one obtained from a human leishmanial infection and other from a canine infection. Both strains displayed comparable behaviour in their capacity of infecting the different macrophage types. Human peripheral blood macrophages and DH82 cells were less infectable by both strains. U-937, mouse peritoneal macrophages and mouse bone marrow derived macrophages are the most active cells to phagocytose the parasites. However, U-937 cell line appears to be the most useful as Leishmania infection model providing an unlimited source of homogeneous host cells with reproducibility of the results, is less time consuming, less expensive and tolerate high doses of first line drugs for human and canine visceral leishmaniasis treatment.     

Response to chemotherapy in experimental visceral leishmaniasis: T cell-dependent but interferon-gamma- and interleukin-2-independent

The capacity of Leishmania donovani-infected BALB/c mice to respond to conventional chemotherapy with pentavalent antimony (Sb) is T cell dependent and, in nude mice, can be restored in part by treatment with the T cell lymphokines, interferon-gamma (IFN-gamma) or interleukin-2 (IL-2). To document the presumed role of endogenous IFN-gamma and IL-2 in responsiveness to antileishmanial chemotherapy in the T cell-intact host, L. donovani-infected euthymic BALB/c mice were treated with anti-IFN-gamma or anti-IL-2 monoclonal antibodies (MAbs) before and after Sb administration. Treatment with MAbs exacerbated visceral infection but did not inhibit the in vivo efficacy of Sb. Thus, while combination therapy of Sb plus IFN-gamma or IL-2 may prove beneficial in T cell-deficient hosts with visceral leishmaniasis, T cell activities other than or in addition to IFN-gamma or IL-2 production may mediate in vivo responsiveness to antileishmanial chemotherapy in the euthymic host.     

CD8(+) T cells with parasite-specific cytotoxic activity and a Tc1 profile of cytokine and chemokine secretion develop in experimental visceral leishmaniasis

Recently, a prominent role for CD8(+) T cells in immunity against pathogens has emerged. The mode of action of CD8(+) T cells in murine visceral leishmaniasis and their contribution to the clearance of the parasite has been addressed in the present study. We showed that during the course of experimental infection cytotoxic clones specific for Leishmania infantum antigens developed in the spleen of susceptible BALB/c mice, showed an activated phenotype and became susceptible to apoptotic cell death late in the course of the disease. CD8(+) T cells exhibited considerable cytotoxic activity against cells expressing Leishmania antigens. This activity was mediated by both the perforin and the Fas/FasL pathway, as judged from in vitro and in vivo assays. The CD8(+) T cells also up-regulated mRNAs for cytokines (IFN-gamma and TNF-alpha) and C-C chemokines (RANTES and MIP-1alpha), which have a major role in immunity against the pathogen. CD8(+) T-cells thus displayed a Tc1 pattern of differentiation. In conclusion, CD8(+) T cells appear to play multiple roles in an experimental model of visceral leishmaniasis comprising both cytotoxic activity and secretion of cytokines and chemokines.     

Pathophysiology of experimental leishmaniasis: the role of parasite physiology in the development of metastatic disease

This paper addresses the issue of how physiological properties of Leishmania determine the pattern of development of disseminated leishmaniasis in the mammalian host. It presents direct experimental evidence from in vivo studies that species of Leishmania differ in their capacity to multiply in cutaneous and visceral sites which results in differences in the pattern and rate of development of leishmaniasis. It was found that Leishmania mexicana amazonensis begins to multiply in the cutaneous site of inoculation within 7 days. Parasites, detected in the liver and spleen at 4 weeks, increased 100-fold during the next 4 months. However, the slow multiplication of L. mexicana amazonensis in the liver and spleen was more apparent than real. Parasites implanted in those organs of athymic nude mice by an intravenous injection were rapidly eliminated with a half-time of 16 hr. Thus, the parasites found in small numbers in the liver during the development of disseminated cutaneous disease in mice are most likely those which have been recently removed from the blood. Those few parasites that are not removed from the blood can establish metastatic foci in distant cutaneous sites, and replicate progressively once there.