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.

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.

Leishmaniavirus : a possible target against metastatic leishmaniasis

Leishmaniasis is widely spread disease found in bath tropical and temperate regions but limited to the habitat of its sand fly vector. lt affects over 12 million people with 2 million new cases each year. As cutaneous leishmaniasis patients show varying levels of immunity to the disease after recovery, the development of a vaccine has much promise as a prevention strategy. Unfortunately however, existing anti-leishmanial vaccines are plagued by safety issues and have only ever shown limited efficacy .So, despite much effort, no effective vaccine is currently available. Recent studies suggest a correlation between the presence of Leishmania RNA virus (LRV) and the development of mucocutaneous leishmaniasis (MCL), which is characterised by the presence of secondary lesions in nasal and buccal mucosa, causing destructive and disfiguring facial lesions. Moreover, recent research has associated the viral presence to treatment fa ilure in patients. ln the first part of this work, we propose that these viral particles may serve as promising vaccine candidates due to their powerful TLR-3 antigenicity, launching an early cell-mediated attack on stimulated cells and thus eliminating their virulent complications. The second part of this work discusses a preliminary study on the lymphocyte immune response against Leishmania guyanensis infection. The lymphocyte response (and in particular, the raie of CDS+ T cells) is controversial and varies greatly between Leishmania species. Here, we illustrate the importance of a small CDS+ T cell subpopulation, expressing the CDSaa+ receptor. These intraepithelial lymphocytes are mainly present in the skin, vagina and intestinal tissue and are best known for their raie in the early immune response against pathogens. Similarly to traditional CDS+ cells, they secrete the tissue-destructive enzymes, perforin and granzyme, which can result in a hyper-inflammatory cutaneous lesion, raising a possibility for their raie in Leishmania infection. lndeed, our initial results in a murine mode( of Leishmania guyanensis infection suggest a pathogenic raie for CDSaa+ T cells. Further research into species-specific immune responses against the various Leishmania parasites is critical to realising the clinical potential of immunotherapy in the treatment and prevention of this disfiguring disease . -- La Leishmaniose est une maladie infectieuse causée par le parasite Leishmania. Elle est localisée dans les régions où son vecteur se reproduit, c'est-à-dire dans des régions tropicales ou tempérées. Cette pathologie affecte 12 millions des personnes dans le monde et 2 millions de nouveaux cas sont recensés chaque année. D'autres facteurs, tels la déforestation, les conditions d'hygiène ou encore l'accès limité aux médicaments, aggravent la pathologie et renforcent sa propagation. Les patients affectés par la leishmaniose et qui arrivent à en guérir, présentent une protection contre une réinfection. Pour cette raison, le développement d'un vaccin reste la meilleure solution pour combattre ce fléau. Mais, à ce jour, et malgré beaucoup d'efforts, aucun vaccin efficace n'a encore été développé. Un autre facteur responsable de l'aggravation de la pathologie et de la résistance de ces parasites aux drogues est un virus qui peut infecter certaines souches de Leishmania. Ce virus, appelé Leishmania RNA virus, peut induire une réponse inflammatoire exagérée, ce qui a comme résultat l'aggravation de la pathologie, la survie et la dissémination de ce parasite au sein de l'hôte infecté. Vu l'absence d'un vaccin contre ce parasite, Leishmania, nous proposons de développer un vaccin non pas contre le parasite lui- même mais contre l'agent qui provoque l'exacerbation de la pathologie, c'est-à-dire le virus. Dans cette étude, nous décrivons le développement d'un vaccin contre LRV, qui empêche le parasite d'induire des inflammations exagérées dans les souris. En d'autres mots, nous essayons de prévenir toutes les complications générées par cet hyperpathogène qu'est le LRV, en utilisant sa capside comme cible pour le développement d'un vaccin. Dans la deuxième partie de ce manuscrit, nous avons aussi étudié plus en détail la réponse immunitaire, et en particulier la réponse des lymphocytes T COB suite à l'infection du parasite Leishmania guyanensis porteur du LRV.

Review and new therapeutic alternatives for the treatment of cutaneous Leishmaniasis

Leishmaniasis comprises a group of diseases caused by protozoa of the genus Leishmania and has two basic clinical forms, visceral Leishmaniasis and cutaneous Leishmaniasis. The clinical features of Leishmaniasis depend on the species of Leishmania, the interaction between host and parasite and the immune response. This work focuses on cutaneous leishmaniosis because although it is not a deadly disease it results in significant scars and facial disfigurements, thus being clinically important. Furthermore, the first-line treatment consists of intravenous or intramuscular administration of intralesional pentavalent antimonials, which are highly toxic, making hospitalization of patients compulsory during treatment, with the associated financial costs. Herein, we review studies on drugs and treatments with fewer side effects and easier routes of administration such as topical administration. Recent research shows that the topical route of administration holds promise for the future treatment of cutaneous leishmaniosis.

Review and new therapeutic alternatives for the treatment of cutaneous Leishmaniasis

Leishmaniasis comprises a group of diseases caused by protozoa of the genus Leishmania and has two basic clinical forms, visceral Leishmaniasis and cutaneous Leishmaniasis. The clinical features of Leishmaniasis depend on the species of Leishmania, the interaction between host and parasite and the immune response. This work focuses on cutaneous leishmaniosis because although it is not a deadly disease it results in significant scars and facial disfigurements, thus being clinically important. Furthermore, the first-line treatment consists of intravenous or intramuscular administration of intralesional pentavalent antimonials, which are highly toxic, making hospitalization of patients compulsory during treatment, with the associated financial costs. Herein, we review studies on drugs and treatments with fewer side effects and easier routes of administration such as topical administration. Recent research shows that the topical route of administration holds promise for the future treatment of cutaneous leishmaniosis.

Spatial distribution of canine Visceral Leishmaniasis in Ilha Solteira, São Paulo, Brazil

Visceral Leishmaniasis (VL) is caused by protozoan of genus Leishmania and transmitted by sand flies of genus Lutzomyia, which has been adapted to the peridomicile environment where dogs are their mainly food source, increasing the risk for human cases. In this study, techniques of geoprocessing and spatial statistics were utilized as a contribution to understanding the epidemiological dynamics of VL in the urban area of Ilha Solteira, SP.

Serological diagnosis of visceral leishmaniasis by an enzyme immunoassay using protein A in naturally infected dogs

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Leishmania chagasi using total antigen from lysed promastigotes. Fifty symptomatic mixed breed dogs from a region of high incidence of visceral leishmaniasis in Brazil were examined. The results showed that in the positive animals, diagnosed by cytological examination, the ELISA using protein A assay system (mean optical density ± SD / 2.078 ± 0.631) detected more antibodies than the anti-IgG assay (mean optical density ± SD / 1.008 ± 0.437), while in the negative animals, the results by both systems were similar. These results suggest that the ELISA assay using protein A peroxidase conjugated could be useful to detect early infected animals in endemic areas, and thus help to control the spread of the infection.

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in dogs with visceral leishmaniasis

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5%) were positive for N. caninum (IFAT>50) and 36 (32.7%) for T. gondii (IFAT>16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

Outbreak of autochthonous canine visceral leishmaniasis in Santa Catarina, Brazil

The present study reports the first outbreak of autochthonous canine visceral leishmaniasis in Florianópolis, Santa Catarina, southern Brazil. Following the report of two cases of CVL, the Control Center of Zoonotic Diseases conducted a serological survey by ELISA and IFAT assays in seven districts of the Santa Catarina Island. Eleven seropositive dogs of autochthonous transmission were used in the present study. Infection by Leishmania sp. was confirmed by parasitological examination of bone marrow, liver, spleen and lymph nodes, culture in Schneider's medium and PCR. Leishmania sp. isolates were characterized by PCR-RFLP and hybridization with specific probes, allowing for the identification of Leishmania infantum. Autochthonous transmission of this disease in an area with high tourist traffic presents a major public health concern and signifies the emergence of an important zoonosis in southern Brazil. Therefore, the implementation of surveillance and control measures is imperative to prevent the spread of the disease among the canine population as well as transmission to the human population.

Assessment of the electrocardiogram in dogs with visceral leishmaniasis

As myocarditis and arrhythmias have been shown to occur in both human beings and dogs with leishmaniasis, electrocardiograms of 105 dogs serologically positive for this disease were assessed for rhythm disturbances and changes in ECG waves. A few expressive alterations were seen, including sinus arrest, right bundle branch block, and atrial premature beats in 14.3%, 4.8%, and 4.8% of the studied subjects, respectively. Also, the analysis of ECG waves showed changes suggestive of left atrium and ventricle enlargements, and myocardial hypoxia in some animals. Although cardiac compromise has been previously reported in dogs with leishmaniasis, only a small subset of dogs showed any alteration in the electrocardiogram, which cannot support the occurrence of myocarditis in this investigation.

Immunologic patterns associated with cure in human American cutaneous leishmaniasis

Patients with American cutaneous leishmaniasis were studied before therapy (active lesion) and at the end of therapy (cured patients). Assays of lymphocyte proliferative responses of peripheral blood mononuclear cells induced in vitro by Leishmania braziliensis promastigote antigens (Lb) were performed. Antigen-stimulated cells were harvested for CD4 and CD8 phenotype analysis and the levels of gamma interferon (IFN-g) and interleukin 4 (IL-4) produced were also determined in the culture supernatants. Two different patterns of Lb-induced T cell responses were observed: a) predominance of responding CD4+ cells and mixed type 1 and type 2 cytokine production (IFN-g and IL-4) during the active disease, and b) similar proportions of responding CD4+ and CD8+ cells, and type 1 cytokine production (presence of IFN-g and very low IL-4) at the end of therapy (healed lesions). This last pattern is probably associated with a beneficial T cell response

Cytokine profile and pathology in human leishmaniasis

The clinical spectrum of leishmaniasis and control of the infection are influenced by the parasite-host relationship. The role of cellular immune responses of the Th1 type in the protection against disease in experimental and human leishmaniasis is well established. In humans, production of IFN-g is associated with the control of infection in children infected by Leishmania chagasi. In visceral leishmaniasis, an impairment in IFN-g production and high IL-4 and IL-10 levels (Th2 cytokines) are observed in antigen-stimulated peripheral blood mononuclear cells (PBMC). Moreover, IL-12 restores IFN-g production and enhances the cytotoxic response. IL-10 is the cytokine involved in down-regulation of IFN-g production, since anti-IL-10 monoclonal antibody (mAb) restores in vitro IFN-g production and lymphoproliferative responses, and IL-10 abrogates the effect of IL-12. In cutaneous and mucosal leishmaniasis, high levels of IFN-g are found in L. amazonensis-stimulated PBMC. However, low or absent IFN-g levels were observed in antigen-stimulated PBMC from 50% of subjects with less than 60 days of disease (24 ± 26 pg/ml). This response was restored by IL-12 (308 ± 342 pg/ml) and anti-IL-10 mAb (380 ± 245 pg/ml) (P<0.05). Later during the disease, high levels of IFN-g and TNF-a are produced both in cutaneous and mucosal leishmaniasis. After treatment there is a decrease in TNF-a levels (366 ± 224 pg/ml before treatment vs 142 ± 107 pg/ml after treatment, P = 0.02). Although production of IFN-g and TNF-a might be involved in the control of parasite multiplication in the early phases of Leishmania infection, these cytokines might also be involved in the tissue damage seen in tegumentary leishmaniasis

Electrocardiographic changes during low-dose, short-term therapy of cutaneous leishmaniasis with the pentavalent antimonial meglumine

The pentavalent antimonial (Sb5+) meglumine is the drug of choice for the treatment of cutaneous leishmaniasis (CL) in Brazil. Although the cardiotoxicity of high-dose, long-term Sb5+ therapy is well known, the use of low-dose, short-term meglumine has been considered to be safe and relatively free from significant cardiac effects. In order to investigate the cardiotoxicity of low-dose, short-term therapy with meglumine in cutaneous leishmaniasis, 62 CL patients treated with meglumine were studied. A standard ECG was obtained before and immediately after the first cycle of treatment (15 mg Sb5+ kg-1 day-1). The electrocardiographic interpretation was carried out blindly by two investigators using the Minnesota Code. There were no significant differences in qualitative ECG variables before and after meglumine treatment. However, the corrected QT interval was clearly prolonged after antimonial therapy (420.0 vs 429.3 ms, P<10-6). QTc augmentation exceeded 40 ms in 12 patients, 7 of whom developed marked QTc interval enlargement (500 ms) after meglumine therapy. This previously unrecognized cardiac toxicity induced by short-term, low-dose antimonial therapy has potentially important clinical implications. Since sudden death has been related to QTc prolongation over 500 ms induced by high-dose antimonial therapy, routine electrocardiographic monitoring is probably indicated even in CL patients treated with short-term, low-dose meglumine schedules. Until further studies are conducted to establish the interactions between pentavalent antimonials and other drugs, special care is recommended when using meglumine in combination with other medications, in particular with drugs that also increase the QTc interval.

The role of natural killer cells in the early period of infection in murine cutaneous leishmaniasis

In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 106 spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 106 spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.