Visceral leishmaniasis and hemophagocytic syndrome

A 11 months old female infant from Portugal, free of family history, consults for apathy, weight loss, tachycardia, tachypnea, petechiae, pallor without icterus and hepatoslenomegaly. Seven months earlier, while being in Portugal, she presented a persistent bluish pimple on her buttock. Laboratory results showed anemia (35 g/l), leucopenia (3.3 G/l), thrombocytopenia (13 G/l), impaired coagulation (INR 1.4, PTT 41 sec.), hyponatremia (124 mmol/l), elevated CRP (139 mg/l), high ferritin (34.775 μg/l) and high triglycerides (5.22 mmol/l). After correction of vital parameters, a bone marrow aspiration and biopsy (BMB) revealed both the etiological diagnosis, namely a visceral leishmaniasis (VL) as well as one of its potential complications, the hemophagocytic syndrome (HS). Transfusions of whole blood, platelets and fresh frozen plasma were immediately started. Dexamethasone (10 mg/m2) and amphotericin B (3 mg/kg/day) have also been administrated. Visceral leishmaniasis is caused by a protozoan (Leishmania donovani) transmitted by the female sandfly. It is endemic in the Mediterranean basin (including France, Italy, Spain and Portugal), South America, sub-Saharan Africa as well as in India and Bangladesh. The parasite infects macrophages and, after several weeks of incubation, the disease occurs by affection of bloodlines (anemia, leucopenia, thrombocytopenia), hepatosplenomegaly, cachexia, gastrointestinal damage. The complications of the disease may lead to death. Liposomal amphotericin B is the currently recommended treatment. HS is caused by the proliferation and activation of macrophages in the marrow in response to a cytokine storm. It may be of primary cause. When it is secondary, it may be related to infections such as leishmaniasis. Patients present with fever and laboratory diagnostic criteria include cytopenia, hypertriglyceridemia, high ferritin and hemophagocytosis in the BMB. The treatment consists among other in the administration of high doses corticosteroids and, in secondary cases, in the treatment of the underlying cause. In conclusion, the clinical and biological features of VL may mimic haematological disorders as leukemia, but an enlargement of the liver and especially of the spleen should remind in this parasitic infection and its potential fatal complication, the HS.

Safety and effectiveness of amphotericin B deoxycholate for the treatment of visceral leishmaniasis in Uganda

Between September 2003 and April 2004, the supply of antimonial drugs to Amudat Hospital, in north-eastern Uganda, was interrupted and all cases of visceral leishmaniasis presenting at the hospital could only be treated with amphotericin B deoxycholate (AmB). This allowed the safety and effectiveness of the AmB to be evaluated, in comparison with an historical cohort of patients treated, at the same hospital, with meglumine antimoniate (Sb-V). Demographic and clinical data were collected before and after treatment. Adverse effects were recorded passively in all the subjects, and actively, using a standardized questionnaire, in a sub-group of the patients given AmB. The in-hospital case-fatality 'rates' were 4.8% [95% confidence interval (CI) =2.4%-8.8%] among the 210 patients treated with AmB and 3.7% (CI=1.4%-7.9%) among the 161 patients treated with Sb-V (P>0.20). Adverse effects requiring treatment interruption were rare in both cohorts. Treatment failures (i.e. non-responses or relapses) were observed in 2.9% (CI= 1.2%-6.4%) of the patients treated with AmB and 1.2% (CI=0.1%-4.4%) of the patients treated with Sb-V (P>0.20). For the treatment of visceral leishmaniasis in Uganda, AmB therefore had a similar effectiveness and safety profile to that of meglumine antimoniate.

MyD88 and TLR9 dependent immune responses mediate resistance to Leishmania guyanensis infections, irrespective of Leishmania RNA virus burden.

Infections with Leishmania parasites of the Leishmania Viannia subgenus give rise to both localized cutaneous (CL), and metastatic leishmaniasis. Metastasizing disease forms including disseminated (DCL) and mutocutaneous (MCL) leishmaniasis result from parasitic dissemination and lesion formation at sites distal to infection and have increased inflammatory responses. The presence of Leishmania RNA virus (LRV) in L. guyanensis parasites contributes to the exacerbation of disease and impacts inflammatory responses via activation of TLR3 by the viral dsRNA. In this study we investigated other innate immune response adaptor protein modulators and demonstrated that both MyD88 and TLR9 played a crucial role in the development of Th1-dependent healing responses against L. guyanensis parasites regardless of their LRV status. The absence of MyD88- or TLR9-dependent signaling pathways resulted in increased Th2 associated cytokines (IL-4 and IL-13), which was correlated with low transcript levels of IL-12p40. The reliance of IL-12 was further confirmed in IL12AB-/- mice, which were completely susceptible to infection. Protection to L. guyanensis infection driven by MyD88- and TLR9-dependent immune responses arises independently to those induced due to high LRV burden within the parasites.

Cutaneous leishmaniasis: progress towards a vaccine.

Leishmaniases are vector-borne diseases due to the protozoan parasite Leishmania . Since no prevention method is available and as current therapy is costly, often poorly tolerated and not always efficacious, the development of alternative therapies, including vaccines, constitutes the priority in the fight of Leishmania infection. This review focuses on recent advances in the development of vaccines against leishmaniasis, with emphasis on the cutaneous form. Indeed, the fact that recovery from leishmaniasis is associated with immunity against new infection provides a rational basis for the development of vaccination strategy against infection with Leishmania . Evidence from animal studies demonstrate that protection can be achieved following infection with live-attenuated Leishmania as well as through immunization with purified proteins or DNA vaccines. In addition, recent results have shown that immunization against the saliva of the insect vector could have synergistic effects with conventional vaccination. Finally, vaccination using dendritic cells was recently demonstrated as a possible tool for Leishmania vaccination.

Arginase activity in the blood of patients with visceral leishmaniasis and HIV infection.

BACKGROUND: Visceral leishmaniasis is a parasitic disease associated with high mortality. The most important foci of visceral leishmaniasis in Ethiopia are in the Northwest and are predominantly associated with high rates of HIV co-infection. Co-infection of visceral leishmaniasis patients with HIV results in higher mortality, treatment failure and relapse. We have previously shown that arginase, an enzyme associated with immunosuppression, was increased in patients with visceral leishmaniasis and in HIV seropositive patients; further our results showed that high arginase activity is a marker of disease severity. Here, we tested the hypothesis that increased arginase activities associated with visceral leishmaniasis and HIV infections synergize in patients co-infected with both pathogens. METHODOLOGY/PRINCIPAL FINDINGS: We recruited a cohort of patients with visceral leishmaniasis and a cohort of patients with visceral leishmaniasis and HIV infection from Gondar, Northwest Ethiopia, and recorded and compared their clinical data. Further, we measured the levels of arginase activity in the blood of these patients and identified the phenotype of arginase-expressing cells. Our results show that CD4(+) T cell counts were significantly lower and the parasite load in the spleen was significantly higher in co-infected patients. Moreover, our results demonstrate that arginase activity was significantly higher in peripheral blood mononuclear cells and plasma of co-infected patients. Finally, we identified the cells-expressing arginase in the PBMCs as low-density granulocytes. CONCLUSION: Our results suggest that increased arginase might contribute to the poor disease outcome characteristic of patients with visceral leishmaniasis and HIV co-infection.

Expression of cysteine proteinase type I and II of Leishmania infantum and their recognition by sera during canine and human visceral leishmaniasis.

In this study, the mature domains of type I (CPB) and type II (CPA) cysteine proteinases (CPs) of Leishmania infantum were expressed and their immunogenic properties defined using sera from active and recovered cases of human visceral leishmaniasis and sera from infected dogs. Immunoblotting and ELISA analysis indicated that a freeze/thaw extract of parasite antigens showed similar and intensive recognition in both active cases of human and dog sera but lower recognition in recovered human individuals. The total IgG of actively infected human sera was higher than in recovered cases when rCPs were used as antigen. In contrast to dog sera, both active and recovered human cases have higher recognition toward rCPB than rCPA. Furthermore, the asymptomatic dogs in contrast to the symptomatic cases exhibited specific lymphocyte proliferation to both crude antigens and rCPs.

Protection against cutaneous leishmaniasis in outbred vervet monkeys using a recombinant histone H1 antigen.

Infection with Leishmania major parasites results in the development of cutaneous ulcerative lesions on the skin. We investigated the protective potential of a single, recombinant histone H1 antigen against cutaneous leishmaniasis in an outbred population of vervet monkeys, using Montanide adjuvant. Protection was assessed by challenging the animals with a mixture of vector sand fly salivary-gland lysate and a low dose of in vitro-derived parasites, thus more closely mimicking natural infection induced by L. major. The course of infection in immunized monkeys was compared with that of animals that had healed from a primary infection and were immune. The monkeys immunized with recombinant histone H1 showed a reduced development of lesion size, compared with controls. Our study therefore illustrates the potential use of histone H1 as a vaccine candidate against cutaneous leishmaniasis in humans.

Experimental cutaneous Leishmaniasis: a powerful model to study in vivo the mechanisms underlying genetic differences in Th subset differentiation.

The murine model of infection with Leishmania major has allowed the demonstration in vivo of the importance CD4+ T cell subsets, distinguishable by the pattern of cytokines they produce, on the outcome of infectious diseases. Genetically determined resistance and susceptibility to infection with this parasite are the result of the development of Th1 and Th2 response, respectively. In this short paper, we present some results obtained in our group pertaining to the analysis of the mechanisms, operational during the early phase of this infection, responsible for the maturation of these functionally distinct CD4+ responses.

The immunological, environmental, and phylogenetic perpetrators of metastatic leishmaniasis.

Cutaneous leishmaniases have persisted for centuries as chronically disfiguring parasitic infections affecting millions of people across the subtropics. Symptoms range from the more prevalent single, self-healing cutaneous lesion to a persistent, metastatic disease, where ulcerations and granulomatous nodules can affect multiple secondary sites of the skin and delicate facial mucosa, even sometimes diffusing throughout the cutaneous system as a papular rash. The basis for such diverse pathologies is multifactorial, ranging from parasite phylogeny to host immunocompetence and various environmental factors. Although complex, these pathologies often prey on weaknesses in the innate immune system and its pattern recognition receptors. This review explores the observed and potential associations among the multifactorial perpetrators of infectious metastasis and components of the innate immune system.

In leishmaniasis due to Leishmania guyanensis infection, distinct intralesional interleukin-10 and Foxp3 mRNA expression are associated with unresponsiveness to treatment.

The presence of intralesional natural regulatory T cells, characterized by the expression of Foxp3 mRNA, was analyzed in patients with localized leishmaniasis due to Leishmania guyanensis infection that was unresponsive to treatment with pentamidine isethionate. Foxp3 mRNA levels were associated with unresponsiveness to treatment among patients with a lesion duration of 1 month, but this association was not observed among patients with a lesion duration of <1 month. In conclusion, high intralesional expression of Foxp3 might be an indicator of poor response to treatment, depending on the duration of lesions.

Metastatic leishmaniasis. A chronic inflammatory response to Leishmania's viral endosymbiont.

Leishmania parasites have been plaguing humankind for centuries as a range of skin diseases named the cutaneous leishmaniases (CL). Carried in a hematophagous sand fly, Leishmania usually infests the skin surrounding the bite site, causing a destructive immune response that may persist for months or even years. The various symptomatic outcomes of CL range from a benevolent self- healing reddened bump to extensive open ulcerations, resistant to treatment and resulting in life- changing disfiguration. Many of these more aggressive outcomes are geographically isolated within the habitats of certain Neotropical Leishmania species; where about 15% of cases experience metastatic complications. However, despite this correlation, genetic analysis has revealed no major differences between species causing the various disease forms. We have recently identified a cytoplasmic dsRNA virus within metastatic L. guyanensis parasites that acts as a potent innate immunogen capable of worsening lesionai inflammation and prolonging parasite survival. The dsRNA genome of Leishmania RNA virus (LRV) binds and stimulates Toll-Like-Receptor-3 (TLR3), inducing this destructive inflammation, which we speculate as a factor contributing to the development of metastatic disease. This thesis establishes the first experimental model of LRV-mediated leishmanial metastasis and investigates the role of non-TLR3 viral recognition pathways in LRV-mediated pathology. Viral dsRNA can be detected by various non-TLR3 pattern recognition receptors (PRR); two such PRR groups are the RLRs (Retinoic acid-inducible gene 1 like receptors) and the NLRs (nucleotide- binding domain, leucine-rich repeat containing receptors). The RLRs are designed to detect viral dsRNA in the cytoplasm, while the NLRs react to molecular "danger" signals of cell damage, often oligomerizing into molecular scaffolds called "inflammasomes" that activate a potent inflammatory cascade. Interestingly, we found that neither RLR signalling nor the inflammasome pathway had an effect on LRV-mediated pathology. In contrast, we found a dramatic inflammasome independent effect for the NLR family member, NLRP10, where a knockout mouse model showed little evidence of disease. This phenotype was mimicked in an NLR knockout with which NLRP10 is known to interact: NLRC2. As this pathway induces the chronic inflammatory cell lineage TH17, we investigated the role of its key chronic inflammatory cytokine, IL-17A, in human patients infected by L. guyanensis. Indeed, patients infected with LRV+ parasites had a significantly increased level of IL-17A in lesionai biopsies. Interestingly, LRV presence was also associated with a significant decrease in the correlate of protection, IFN-y. This association was repeated in our murine model, where after we were able to establish the first experimental model of LRV-dependent leishmanial metastasis, which was mediated by IL-17A in the absence of IFN-y. Finally, we tested a new inhibitor of IL-17A secretion, SR1001, and reveal its potential as a Prophylactic immunomodulator and potent parasitotoxic drug. Taken together, these findings provide a basis for anti-IL-17A as a feasible therapeutic intervention to prevent and treat the metastatic complications of cutaneous leishmaniasis. -- Les parasites Leishmania infectent l'homme depuis des siècles causant des affections cutanées, appelées leishmanioses cutanées (LC). Le parasite est transmis par la mouche des sables et réside dans le derme à l'endroit de la piqûre. Au niveau de la peau, le parasite provoque une réponse immunitaire destructrice qui peut persister pendant des mois voire des années. Les symptômes de LC vont d'une simple enflure qui guérit spontanément jusqu' à de vastes ulcérations ouvertes, résistantes aux traitements. Des manifestations plus agressives sont déterminées par les habitats géographiques de certaines espèces de Leishmania. Dans ces cas, environ 15% des patients développent des lésions métastatiques. Aucun «facteur métastatique» n'a encore été trouvé à ce jour dans ces espèces. Récemment, nous avons pu identifier un virus résidant dans certains parasites métastatiques présents en Guyane française (appelé Leishmania-virus, ou LV) et qui confère un avantage de survie à son hôte parasitaire. Ce virus active fortement la réponse inflammatoire, aggravant l'inflammation et prolongeant l'infection parasitaire. Afin de diagnostiquer, prévenir et traiter ces lésions, nous nous sommes intéressés à identifier les composants de la voie de signalisation anti-virale, responsables de la persistance de cette inflammation. Cette étude décrit le premier modèle expérimental de métastases de la leishmaniose induites par LV, et identifie plusieurs composants de la voie inflammatoire anti-virale qui facilite la pathologie métastatique. Contrairement à l'homme, les souris de laboratoire infectées par des Leishmania métastatiques (contenant LV, LV+) ne développent pas de lésions métastatiques et guérissent après quelques semaines d'infection. Après avoir analysé un groupe de patients atteints de leishmaniose en Guyane française, nous avons constaté que les personnes infectées avec les parasites métastatiques LV+ avaient des niveaux significativement plus faibles d'un composant immunitaire protecteur important, appelé l'interféron (IFN)-y. En utilisant des souris génétiquement modifiées, incapables de produire de l'IFN-y, nous avons observé de telles métastases. Après inoculation dans le coussinet plantaire de souris IFN-y7" avec des parasites LV+ ou LV-, nous avons démontré que seules les souris infectées avec des leishmanies ayant LV développent de multiples lésions secondaires sur la queue. Comme nous l'avons observé chez l'homme, ces souris sécrètent une quantité significativement élevée d'un composant inflammatoire destructeur, l'interleukine (IL)-17. IL-17 a été incriminée pour son rôle dans de nombreuses maladies inflammatoires chroniques. On a ainsi trouvé un rôle destructif similaire pour l'IL-17 dans la leishmaniose métastatique. Nous avons confirmé ce rôle en abrogeant IL-17 dans des souris IFN-y7- ce qui ralentit l'apparition des métastases. Nous pouvons donc conclure que les métastases de la leishmaniose sont induites par l'IL-17 en absence d'IFN-v. En analysant plus en détails les voies de signalisation anti-virale induites par LV, nous avons pu exclure d'autres voies d'activation de la réponse inflammatoire. Nous avons ainsi démontré que la signalisation par LV est indépendante de la signalisation inflammatoire de type « inflammasome ». En revanche, nous avons pu y lier plusieurs autres molécules, telles que NLRP10 et NLRC2, connues pour leur synergie avec les réponses inflammatoires. Cette nouvelle voie pourrait être la cible pour des médicaments inhibant l'inflammation. En effet, un nouveau médicament qui bloque la production d'IL-17 chez la souris s'est montré prometteur dans notre modèle : il a réduit le gonflement des lésions ainsi que la charge parasitaire, indiquant que la voie anti-virale /inflammatoire est une approche thérapeutique possible pour prévenir et traiter cette infection négligée.

Detection of Leishmania RNA virus in Leishmania parasites.

BACKGROUND: Patients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence. METHODOLOGY/PRINCIPAL FINDINGS: This study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice. CONCLUSIONS/SIGNIFICANCE: We propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Cytokines in parasitic diseases: the example of cutaneous leishmaniasis.

The essential role of cytokines in parasitic diseases has been emphasised since the in vivo description of the importance of T helper 1 (Th1) and T helper 2 (Th2) CD4+ T cell responses in resistance and susceptibility to infection with L. major in mice. Th1 cells produced IL-2, IFN-gamma and Lymphotoxin T (LT) and Th2 cells produce IL-4, IL-5 and IL-13. In this model of infection the correlation between on the one hand resistance to infection and the development of a Th1 response and on the other hand susceptibility and Th2 cell development allowed the identification of the mechanisms directing the differentiation of CD4+ T cell precursors towards either Th1 type or Th2 type responses. Cytokines are the crucial inducer of functional CD4+ T cell subset differentiation during infection with L. major. IL-12 and IFN-gamma direct the differentiation of Th1 response and IL-4 of a Th2 response. In susceptible mice, careful analysis of IL-4 production during the first days of infection has shown that the IL-4 produced as a result of a very early burst of IL-4 mRNA expression (16 hours) plays a essential role in the maturation of a Th2 CD4+ T cell response by rendering the CD4+ T cell precursors unresponsive to IL-12. Activation of a restricted population of CD4+ T cells expressing the V beta 4 V alpha 8 TCR heterodimer after recognition of a single antigen, the LACK (Leishmania Activated c Kinase) antigen, resulted in this rapid production of IL-4 required for the subsequent CD4+ T cell differentiation. Thus, tolerization of these cells might contribute a strategy for preventing infection with L. major.

Role of neutrophils in the early shaping of the Leishmania major specific immune response in experimental murine cutaneous Leishmaniasis

The development of a protective immune response to microorganisms involves complex interactions between the host and the pathogen. The murine model of infection with Leishmania major (L. major) allows the study of the factors leading to the development of a protective immune response. Following infection with the protozoan parasite L. major, most strains of mice heal their lesions, while a few fail to control infection, both processes linked to the development of specific T helper subsets. The early events occurring during the first days following parasite inoculation are thought to be critical in the development of the Leishmania-specific immune response. Neutrophils are the first cells arriving massively to the site of infection, and recent evidence points to their role as organizers of the immune response, yet their specific role in this process remains elusive. Through interactions with cells present at the parasite inoculation site, and possibly within the draining lymph nodes, neutrophils could have an impact not only on the recruitment of inflammatory cells but also on the activation of local as well as newly migrated cells that will be crucial in shaping the Leishmania-specific immune response.

Arginase activity - a marker of disease status in patients with visceral leishmaniasis in ethiopia.

The underlying mechanisms resulting in the profound immune suppression characteristic of human visceral leishmaniasis (VL) are not fully understood. Here, we tested the hypothesis that arginase, an enzyme associated with immunosuppression, is higher in patients with VL and contributes to impaired T cell responses. We recruited patients with VL before and after treatment and healthy controls and measured the arginase metabolism in the blood of these individuals. Our results show that arginase activity is significantly higher in the blood of patients with active VL as compared to controls. These high levels of arginase decline considerably once the patients are successfully treated. We identified the phenotype of arginase-expressing cells among PBMCs as neutrophils and show that their frequency was increased in PBMCs of patients before treatment; this coincides with reduced levels of L-arginine in the plasma and decreased expression levels of CD3ζ in T cells.