A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.

Muco-cutaneous leishmaniasis in the New World: The ultimate subversion.

Infection by the human protozoan parasite Leishmania can lead, depending primarily on the parasite species, to either cutaneous or mucocutaneous lesions, or fatal generalized visceral infection. In the New World, Leishmania (Viannia) species can cause mucocutaneous leishmaniasis (MCL). Clinical MCL involves a strong hyper-inflammatory response and parasitic dissemination (metastasis) from a primary lesion to distant sites, leading to destructive metastatic secondary lesions especially in the nasopharyngal areas. Recently, we reported that metastasizing, but not non-metastatic strains of Leishmania (Viannia) guyanensis, have high burden of a non-segmented dsRNA virus, Leishmania RNA Virus (LRV). Viral dsRNA is sensed by the host Toll-like Receptor 3 (TLR3) thereby inducing a pro-inflammatory response and exacerbating the disease. The presence of LRV in Leishmania opens new perspectives not only in basic understanding of the intimate relation between the parasite and LRV, but also in understanding the importance of the inflammatory response in MCL patients.

The protective capacities of histone H1 against experimental murine cutaneous leishmaniasis.

In a murine model of experimental cutaneous leishmaniasis, we investigated the protection elicited by injection of histone H1 isolated from parasites by perchloric extraction, of a H1 recombinant protein produced in E. coli, and of H1 long and short synthetic peptides, against infection by L. major. Partial protection was achieved in most of the animals as shown by reduction in lesion size, upon immunization with histone H1 or its peptides, provided that the region 1-60 was present in the molecule. These observations argue in favor of a thorough examination of the possibility of including histone H1 described here in a cocktail vaccine against human leishmaniasis.

Parasitic diseases, with an emphasis on experimental cutaneous Leishmaniasis

Parasites have to survive in their vertebrate host during a sufficiently prolonged period of time to achieve their life cycle through successful transmission via insect vectors. In their vertebrate hosts, parasites are often confronted by vigorous effector immune responses that they have to subvert somehow to be able to outlast and be successfully transmitted.

Local increase of arginase activity in lesions of patients with cutaneous leishmaniasis in ethiopia.

BACKGROUND: Cutaneous leishmaniasis is a vector-borne disease that is in Ethiopia mainly caused by the parasite Leishmania aethiopica. This neglected tropical disease is common in rural areas and causes serious morbidity. Persistent nonhealing cutaneous leishmaniasis has been associated with poor T cell mediated responses; however, the underlying mechanisms are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We have recently shown in an experimental model of cutaneous leishmaniasis that arginase-induced L-arginine metabolism suppresses antigen-specific T cell responses at the site of pathology, but not in the periphery. To test whether these results translate to human disease, we recruited patients presenting with localized lesions of cutaneous leishmaniasis and assessed the levels of arginase activity in cells isolated from peripheral blood and from skin biopsies. Arginase activity was similar in peripheral blood mononuclear cells (PBMCs) from patients and healthy controls. In sharp contrast, arginase activity was significantly increased in lesion biopsies of patients with localized cutaneous leishmaniasis as compared with controls. Furthermore, we found that the expression levels of CD3ζ, CD4 and CD8 molecules were considerably lower at the site of pathology as compared to those observed in paired PBMCs. CONCLUSION: Our results suggest that increased arginase in lesions of patients with cutaneous leishmaniasis might play a role in the pathogenesis of the disease by impairing T cell effector functions.

Intralesional regulatory T-cell suppressive function during human acute and chronic cutaneous leishmaniasis due to Leishmania guyanensis.

The levels of regulatory T cells (Treg cells), analyzed by Foxp3 mRNA expression, were determined in lesions from patients with acute cutaneous leishmaniasis (ACL) and chronic cutaneous leishmaniasis (CCL). We demonstrated that Treg cells preferentially accumulate in lesions from ACL patients during the early phase of infection (lesion duration of less than 1 month). In addition, levels of Foxp3 mRNA transcripts were significantly higher in specimens from patients with CCL than in those from patients with ACL, suggesting a critical role of intralesional Treg cells in CCL. Intralesional Treg cells from both ACL and CCL patients were shown to have suppressive functions in vitro, since they inhibited the gamma interferon (IFN-gamma) produced by CD4(+) CD25(-) T cells purified from peripheral blood mononuclear cells from the same patient in response to Leishmania guyanensis stimulation. Intralesional 2,3-indoleamine dioxygenase (IDO) mRNA expression was associated with that of Foxp3, suggesting a role for IDO in the suppressive activity of intralesional Treg cells. In addition, a role, albeit minor, of interleukin-10 (IL-10) was also demonstrated, since neutralization of IL-10 produced by intralesional T cells increased IFN-gamma production by effector cells in an in vitro suppressive assay. These results confirm the role of intralesional Treg cells in the immunopathogenesis of human Leishmania infection, particularly in CCL patients.

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.

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.

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.

Severe Cutaneous Leishmaniasis in a Human Immunodeficiency Virus Patient Coinfected with Leishmania braziliensis and Its Endosymbiotic Virus.

Leishmaniaparasites cause a broad range of disease, with cutaneous afflictions being, by far, the most prevalent. Variations in disease severity and symptomatic spectrum are mostly associated to parasite species. One risk factor for the severity and emergence of leishmaniasis is immunosuppression, usually arising by coinfection of the patient with human immunodeficiency virus (HIV). Interestingly, several species ofLeishmaniahave been shown to bear an endogenous cytoplasmic dsRNA virus (LRV) of theTotiviridaefamily, and recently we correlated the presence of LRV1 withinLeishmaniaparasites to an exacerbation murine leishmaniasis and with an elevated frequency of drug treatment failures in humans. This raises the possibility of further exacerbation of leishmaniasis in the presence of both viruses, and here we report a case of cutaneous leishmaniasis caused byLeishmania braziliensisbearing LRV1 with aggressive pathogenesis in an HIV patient. LRV1 was isolated and partially sequenced from skin and nasal lesions. Genetic identity of both sequences reinforced the assumption that nasal parasites originate from primary skin lesions. Surprisingly, combined antiretroviral therapy did not impact the devolution ofLeishmaniainfection. TheLeishmaniainfection was successfully treated through administration of liposomal amphotericin B.

Arginase as a marker of disease severity in human leishmaniasis

The leishmaniases are a group of diseases transmitted by the bite of Leishmania infected female phlebotomine sand flies. The diseases occur in different forms: localized, diffuse and muco-cutaneous leishmaniasis, and visceral leishmaniasis (VL). Inside macrophages, the main host cells of the obligate intracellular Leishmania parasites, nitric oxide synthase and arginase can regulate parasite killing or growth. In experimental leishmaniasis, we previously reported that non-healing disease is associated with higher arginase activity at site of pathology, correlating with local suppression of T cell function. To test whether these data translate to human leishmaniasis, the following study was initiated: I first tested the hypothesis that local suppression of T cell responses observed in persistent CL is associated with arginase induced L-arginine depletion. The results showed that arginase activity is increased at site of pathology compared to peripheral blood mononuclear cells (PBMCs) of LCL patients and intact skin of healthy controls. The phenotype of arginase expressing cells was identified in both compartments as CD15+ CD14|0W low-density granulocytes (LDGs). Finally, high arginase activity at site of pathology observed in cutaneous lesions of patients coincides with downregulation of CD3Ç, CD4 and CD8 molecules in CD4+ and CD8+ T cells at site of pathology. We concluded that increased arginase levels in lesions of LCL patients might contribute to CL pathogenesis by impairing T cell effector function at site of pathology. Next, it was tested whether arginase, an enzyme associated with immunosuppression, is higher in patients with VL and contributes to impaired T cell function through depletion of L- arginine. The results showed that higher level of arginase activity in the PBMC coincides with active phase of VL. Cells expressing arginase in PBMCs were also found to be LDGs. Importantly, increased arginase activity and frequency of degranulated neutrophils coincided with lower plasma L-arginine levels. Furthermore, downregulation of CD3Ç, in T cells correlated with low plasma arginine levels. VL/HIV co-infection is a frequently reported leishmaniasis complication in Ethiopia associated with poor prognosis, with up to 40% mortality rate and high relapse rate. Arginase activity was significantly increased in PBMCs and plasma of VL patients co-infected with HIV than in those having VL alone. Similarly, cells expressing arginase in PBMCs were found to be LDGs. In summary, the results presented here show that increased arginase activity is a marker of disease severity in human leishmaniasis with and without HIV; further, these results suggest that arginase mediated L-arginine depletion may inhibit T cell function and contribute to impaired control of infection. - Les leishmanioses sont un groupe de maladies transmises par la piqûre de mouches des sables femelles, appelées phlébotomes, ayant été infectées par Leishmania. Les maladies se manifestent sous différentes formes: la leishmaniose cutanée localisée, la leishmaniose diffuse et mucocutanée et la leishmaniose viscérale (LV). A l'intérieur des macrophages, les principales cellules hôtes des parasites, l'oxyde nitrique synthase et l'arginase, peuvent contrôler, soit la mort du parasite, soit sa croissance. Pour la leishmaniose expérimentale, nous avons déjà rapporté que le développement de lesions qui ne guérissent pas est associé à une activité plus grande d'arginase au site d'infection, en corrélation avec la suppression locale de la fonction des cellules T. Pour vérifier si ces données pouvaient s'appliquer à la leishmaniose humaine, j'ai d'abord vérifié l'hypothèse selon laquelle la suppression locale des réponses des cellules T observée dans la CL persistante, est associée à la la diminution de L- arginine induite par l'arginase. Les résultats ont montré que l'activité arginase est augmentée au site d'infection, par rapport aux cellules mononucléées du sang périphérique (CMSP) de patients LCL et à la peau intacte des contrôles sains. Le phénotype de cellules exprimant l'arginase a été identifié dans les deux compartiments comme des granulocytes CD15+ et CD 14" de basse densité (LDG). Enfin, l'activité arginase élevée au site de la pathologie, observée dans les lésions cutanées de patients, coïncide avec la reduction dde l'expression des molécules CD3Ç, CD4 et CD8 dans les cellules T CD4+ et CD8+ au site de pathologie . Nous avons conclu que l'augmentation des niveaux d'arginase dans les lésions de patients LCL pourrait contribuer à la pathogenèse de la CL, en altérant la fonction effectrice des celllules T au site de la pathologie. Ensuite, nous avons vérifié si l'arginase, une enzyme associée à l'immunosuppression, était plus élevée chez les patients atteints de VL et si elle contribuait à la mauvaise fonction des cellules T par la depletion en L-arginine. Les résultats ont montré qu'un niveau plus élevé de l'activité arginase dans les PBMC correspond à la phase active de la VL. Les cellules exprimant l'arginase dans les CMSP se sont révélées à être de type LDG . Il est important de souligner que l'augmentation de l'activité arginase et la fréquence des neutrophiles dégranulés a coïncidé avec des niveaux inférieurs de L-arginine plasmatique. En outre, la suppression de CD3Ç dans les cellules T correlle avec de faibles niveaux d'arginine plasmatique . Il a été fréquement rapporté que la co-infection VL/VIH est une complication de la leishmaniose en Ethiopie, associée à un mauvais prognostic, un taux de mortalité pouvant atteindre 40% et un pourcentage élevé de rechutes. L'activité de l'arginase a beaucoup plus augmentée dans les CMSP et le plasma de patients atteints de VL et co-infectés par le VIH, que chez ceux seulement attaints de VL. De même, les cellules exprimant l'arginase dans les CMSP sont aussi des LDG. En résumé, les résultats présentés ici montrent que l'augmentation de l'activité de l'arginase est un marqueur de gravité de la la leishmaniose humaine, avec ou sans VIH ; en outre, ces résultats suggèrent que la déplétion de L-arginine par l'arginase pourrait inhiber la fonction des cellules T et contribuer à un contrôle réduit de l'infection. - Les Leishmanioses sont des maladies parasitaires transmises par la piqûre d'une mouche des sables femelle (phlébotome) infectée par Leishmania. La maladie se manifeste sous différentes formes cliniques : la leishmaniose viscérale, une maladie progressive mortelle en l'absence de traitement, la leishmaniose muco-cutanée (MCL), la leishmaniose cutanée diffuse (LCD ) maladie mutilante, qui peut être de longue durée et la leishmaniose cutanée localisée maladie dont on guérit mais laissant une cicatrice inesthétique à vie. La maladie est largement répandue, elle affecte les populations les plus pauvres dans 98 pays et 350 millions de personnes à risque. Globalement on estime à 500.000 les nouveaux cas de la forme viscérale et 1-1.5 million ceux de la leishmaniose cutanée. La leishmaniose est fortement endémique en Ethiopie et se manifeste dans les formes viscérale et cutanée. Le parasite Leishmania infecte et se multiplie dans les cellules du système immunitaire, principalement les macrophages. Les macrophages sont capables de tuer le parasite Leishmania s'ils reçoivent des instructions correctes de la part d'autres cellules du système immunitaire, les lymphocytes. Les macrophages expriment deux enzymes importants, appelés oxide nitrique synthase inductible (iNOS ) et l'arginase, qui sont respectivement associés à la promotion de la mort du parasite et la multiplication. L'enzyme iNOS présent dans les macrophages métabolise l'arginine afin de générer de l'oxyde d'azote (NO) , une molécule effectrice nécessaire pour tuer le parasite . Au contraire, lorsque les macrophages sont activés d'une certaine manière conduisant à l'augmention de la régulation de l'arginase, ils métabolisent l'arginine en polyamines qui favorisent la croissance du parasite. Au cours du développement de la leishmaniose, les lymphocytes ne parviennent pas à transmettre aux macrophages les signaux nécessaires pour tuer le parasite. Les mécanismes cellulaires qui sont la cause de ce défaut, ne sont pas bien compris. En utilisant des modèles animaux, nous avons montré la régulation à la hausse de l'arginase au site de la pathologie, qui s'est traduit par l'altération de la fonction effectrice des lymphoctes. Nous avons initié des études de leishmaniose humaine en Ethiopie afin d'identifier le rôle de l'arginase dans la sévérité de la maladie. Nos résultats montrent, que l'arginase est fortement augmentée dans la lésion des patients CL, et dans le sang des patients VL et ceux co-infectés par VL / VIH. Le niveau d' arginase régulée à la hausse coincide avec l'expression inférieure d'une molécule de signalisation dans les lymphocytes, qui est essentielle à leur bon fonctionnement. En VL actif, l'augmentation d'arginase se traduit par la diminution de l'arginine qui est indispensable à la synthèse de NO et au bon fonctionnement des lymphocytes. Ainsi, l'incapacité des lymphocytes à envoyer des signaux adéquats aux macrophages pourrait être due à la suppression de l'arginine.

The therapeutic potential of immune cross-talk in leishmaniasis.

Veterans of infection, Leishmania parasites have been plaguing mammals for centuries, causing a morbidity toll second only to that of malaria as the most devastating protozoan parasitic disease in the world. Cutaneous leishmaniasis (CL) is, by far, the most prevalent form of the disease, with symptoms ranging from a single self-healing lesion to chronic metastatic leishmaniasis (ML). In an increasingly immunocompromised population, complicated CL is becoming a more likely outcome, characterized by severely inflamed, destructive lesions that are often refractory to current treatment. This is perhaps because our ageing arsenal of variably effective antileishmanial drugs may be directly or indirectly immunomodulatory and may thus have variable effects in each type and stage of CL. Indeed, widely differing immune biases are created by the various species of Leishmania, and these immunological watersheds are further shifted by extrinsic disturbances in immune homeostasis. For example, we recently showed that a naturally occurring RNA virus (Leishmania RNA virus (LRV)) within some Leishmania parasites creates hyperinflammatory cross-talk, which can predispose to ML: a case of immunological misfire that may require a different approach to immunotherapy, whereby treatments are tailored to underlying immune biases. Understanding the intersecting immune pathways of leishmaniasis and its co-infections will enable us to identify new drug targets, and thereby design therapeutic strategies that work by untangling the immunological cross-wires of pathogenic cross-talk.

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.