Activated cytotoxic T cells within zoonotic cutaneous leishmaniasis lesions.

INTRODUCTION: Zoonotic cutaneous leishmaniasis (ZCL), due to infection by Leishmania (L). major, is characterized by polymorphic clinical manifestations which could be attributed to the host's immune response. In this study we investigated the involvement of cytotoxic cells on the outcome of the disease. METHODS: Expression of granzyme B (GrB), granulysine (Grly), and interferon (IFN)-γ was evaluated within ZCL lesion specimens using the technique of real-time quantitative polymerase chain reaction (RT-qPCR). Immunohistochemical staining was performed using anti-CD3, CD4, CD8, CD56, GrB, and IFN-γ antibodies to identify the phenotype of GrB and IFN-γ-producing cells. RESULTS: GrB and Grly mRNA was detected within 75% and 80% of ZCL lesions, respectively. Statistical analysis demonstrated a significant correlation between levels of GrB and Grly. Interestingly, expression of these molecules correlates negatively with the lesion's age. The highest levels were measured in early lesions (E-ZCL) (lesion age ≤1 month) comparing to late lesions (L-ZCL) (lesion age >1 month). Otherwise, IFN-γ mRNA was detected only within 56% and a positive correlation was found between levels of this cytokine and those of GrB. Immunohistochemical analysis showed that GrB is produced essentially by CD8+ T cells whereas IFN-γ is produced by both CD4+ and CD8+ T cells. CONCLUSION: Together our results demonstrate the presence of cytotoxic cells producing GrB and Grly within leishmaniasis cutaneous lesions.

Dok proteins are recruited to the phagosome and degraded in a GP63-dependent manner during Leishmania major infection.

Three adaptor molecules of the Dok family, Dok-1, Dok-2 and Dok-3 are expressed in macrophages and are involved in the negative regulation of signaling in response to lipopolysaccharide and various cytokines and growth factors. We investigated the role and the fate of these proteins following infection with Leishmania major promastigotes in macrophages. The protozoan parasite L. major causes cutaneous leishmaniasis and is known for its capacity to alter host-cell signaling and function. Dok-1/Dok-2(-/-) bone marrow-derived macrophages displayed normal uptake of L. major promastigotes. Following Leishmania infection, Dok-1 was barely detectable by confocal microscopy. By contrast, phagocytosis of latex beads or zymosan led to the recruitment of Dok-1 to phagosomes. In the absence of the Leishmania pathogenesis-associated metalloprotease GP63, Dok-1 was also, partially, recruited to phagosomes containing L. major promastigotes. Further biochemical analyses revealed that similar to Dok-1, Dok-2 and Dok-3 were targets of GP63. Moreover, we showed that upon infection with wild-type or Δgp63 L. major promastigotes, production of nitric oxide and tumor necrosis factor by interferon-γ-primed Dok-1/Dok-2(-/-) macrophages was reduced compared to WT macrophages. These results suggest that Dok proteins may be important regulators of macrophage responses to Leishmania infection.

Diversity of sand flies (Diptera, Psychodidae) in southwest Iran with emphasis on synanthropy of Phlebotomus papatasi and Phlebotomus alexandri.

Zoonotic Cutaneous Leishmaniasis (ZCL) is still a serious health problem in Iran. The objective of the study was to determine the differences in sand fly biodiversity in Shush (plain) and Khorramshahr (littoral) Counties, Khuzestan Province, southwest Iran. Sand flies were collected using sticky paper traps from urban, semi urban, agricultural and natural ecotypes. Alpha and beta diversity were calculated using Shannon-Weiner index and Jaccard's and Sorensen's coefficients, respectively. Synanthropic index was determined for the first time for Phlebotomus papatasi and Phlebotomus alexandri in different land use categories in Iran. Totally 11213 specimens, 68.47% in Shush and 31.53% in Khorramshahr, were collected. Eleven species of sand flies including, 2 of genus Phlebotomus and 9 of genus Sergentomyia were identified. Sergentomyia christophersi was found as a new record. Dominant species were P. papatasi and Sergentomyia sintoni. Shannon-Weiner index, richness and evenness in semi urban area of Shush County were more than other habitats. The analysis of α biodiversity showed that agricultural ecosystem of Khorramshahr County had the highest diversity due to maximal richness and diversity and also relatively high evenness. Comparison of similarity of the sand flies population composition between Shush and Khorramshahr indicated the maximum similarity between the urban area of Shush and the semi urban area of Khorramshahr (Sj=75% and Ss=86%). Synanthropic index of P. papatasi and P. alexandri were calculated to be -83.34 and -91.18, respectively in Shush County. Estimated synanthropic indices for P. papatasi and P. alexandri in three habitats (natural, semi urban and urban) of Khorramshahr County were -69.84 and -85.89, in the same order. The factors for having high diversity of sand flies in the plain area studied may be due to higher annual precipitation, the related land use and land cover. The changes on the composition of sand flies are perhaps due to human intervention in their natural habitats.

Avaliação do papel do receptor marco na infecção de macrófagos murinos por Leishmania major / Evaluation of the role of the receptor in March infection of murine macrophages with Leishmania major

Camundongos CBA são resistentes à infecção por Leishmania major e permissivos à infecção por L. amazonensis. Adicionalmente, macrófagos de camundongos CBA controlam à infecção por L. major, mas não por L. mazonensis in vitro. Em estudo comparativo realizado por nosso grupo foi demonstrado que o receptor scavenger MARCO teve expressão aumentada em resposta à infecção por L. major, mas não na infecção por L. amazonensis. Ainda, o bloqueio do receptor com o anticorpo específico reduziu a infecção por L. major em 30%, indicando que esta proteína tem participação no reconhecimento de promastigotas de L. major em macrófagos de CBA. Assim, nossa hipótese é que o receptor MARCO participa do reconhecimento e fagocitose de L. major por macrófagos, direcionando o curso da infecção. O objetivo do presente estudo consistiu em evidenciar o papel do receptor MARCO na infecção de macrófagos por L. major. Inicialmente, células J774 foram transfectadas com os vetores pcDNA3.1-MARCO (J774-MARCO) ou pcDNA3.1 (J774-MOCK)...

CBA mice are resistant to Leishmania major yet permissive to L. amazonensis infection. In addition, CBA macrophages control L. major, but not L. amazonensis infection in vitro. In a comparative study performed by our group increase in expression of the scavenger receptor MARCO has been detected in response to L. major, but not to L. amazonensis infection. Moreover, ED31 monoclonal antibody against MARCO reduced by 30% L. major infection in CBA macrophages. These findings indicate that MARCO plays a role in L. major recognition by CBA macrophages. We hypothesized that MARCO receptor participates in the recognition and phagocytosis of L. major by macrophages, directing the outcome of infection. In the present study, we aimed to further disclose the role MARCO plays in L. major infection of murine macrophages. First J774 cells were transfected with pcDNA3.1-MARCO vector (MARCO-J774) or pcDNA3.1 vector (MOCK-J774)...

An outbreak of suspected cutaneous leishmaniasis in Ghana: lessons learnt and preparation for future outbreaks.

Human cutaneous leishmaniasis (CL) has previously been reported in West Africa, but more recently, sporadic reports of CL have increased. Leishmania major has been identified from Mauritania, Senegal, Mali, and Burkina Faso. Three zymodemes (MON-26, MON-117, and MON-74, the most frequent) have been found. The geographic range of leishmaniasis is limited by the sand fly vector, its feeding preferences, and its capacity to support internal development of specific species of Leishmania. The risk of acquiring CL has been reported to increase considerably with human activity and epidemics of CL have been associated with deforestation, road construction, wars, or other activities where humans intrude the habitat of the vector. In the Ho Municipality in the Volta Region of Ghana, a localised outbreak of skin ulcers, possibly CL, was noted in 2003 without any such documented activity. This outbreak was consistent with CL as evidenced using various methods including parasite identification, albeit, in a small number of patients with ulcers. This paper reports the outbreak in Ghana. The report does not address a single planned study but rather a compilation of data from a number of ad-hoc investigations in response to the outbreak plus observations and findings made by the authors. It acknowledges that a number of the observations need to be further clarified. What is the detailed epidemiology of the disease? What sparked the epidemic? Can it happen again? What was the causative agent of the disease, L. major or some other Leishmania spp.? What were the main vectors and animal reservoirs? What are the consequences for surveillance of the disease and the prevention of its reoccurrence when the communities see a self-healing disease and may not think it is important?

Uptake and antileishmanial activity of meglumine antimoniate-containing liposomes in Leishmania (Leishmania) major-infected macrophages.

Leishmaniasis is a parasitic disease caused by the intramacrophage protozoa Leishmania spp. and may be fatal if left untreated. Although pentavalent antimonials are toxic and their mechanism of action is unclear, they remain the first-line drugs for treatment of leishmaniasis. An effective therapy could be achieved by delivering antileishmanial drugs to the site of infection. Compared with free drugs, antileishmanial agent-containing liposomes are more effective, less toxic and have fewer adverse side effects. The aim of this study was to develop novel meglumine antimoniate (MA)-containing liposome formulations and to analyse their antileishmanial activity and uptake by macrophages. Determination of the 50% inhibitory concentration (IC(50)) values showed that MA-containing liposomes were ≥10-fold more effective than the free drug, with a 5-fold increase in selectivity index, higher activity and reduced macrophage toxicity. The concentration required to kill 100% of intracellular amastigotes was ≥40-fold lower when MA was encapsulated in liposomes containing phosphatidylserine compared with the free drug. Fluorescence microscopy analysis revealed increased uptake of fluorescent liposomes in infected macrophages after short incubation times compared with non-infected macrophages. In conclusion, these data suggest that MA encapsulated in liposome formulations is more effective against Leishmania-infected macrophages than the non-liposomal drug. Development of liposome formulations is a valuable approach to the treatment of infectious diseases involving the mononuclear phagocyte system.

Anti-Leishmanial activity of homo- and heteroleptic bismuth(III) carboxylates.

Bismuth(III) complexes of NSAIDs (Non-Steroidal Anti Inflammatory Drugs) and substituted benzoic acids (o-methoxybenzoic acid, m-methoxybenzoic acid, o-nitrobenzoic acid, 3,5-diacetamidobenzoic acid, and 5-[(R/S)-2,3-dihydroxypropyl carbamoyl]-2-pyridine carboxylic acid) have been synthesised and fully characterised. Two new bis-carboxylato bismuth complexes have been characterised by single crystal X-ray diffraction, namely [PhBi(o-MeOC(6)H(4)CO(2))(2)(bipy)]·0.5EtOH (bipy=2,2'-bipyridine) and [PhBi(C(9)H(11)N(2)O(3)CO(2))(2)(H(2)O)]·6H(2)O. All compounds were tested against the parasite Leishmania major promastigotes for their anti-Leishmanial activity and were further assessed for their toxicity to mammalian cells. The NSAID free acids and their bismuth derivatives show negligible anti-Leishmanial activity at concentrations 1.95 to 250 µg/mL against the promastigotes of L. major whereas in the case of mammalian cells only bismuth complexes of naproxen and mefenamic acid have significant effect at concentration≥250 µg/mL. The bismuth(III) complexes of substituted benzoic acids show significant anti-Leishmanial activity against the promastigotes of L. major V121 at very low concentrations while their respective free carboxylic acids show no effective activity. However, the bismuth compounds inhibit the growth of the mammalian cells at all concentrations studied (1.95 to 500 µg/mL) following 48 h incubation. The comparatively low toxicity of BiCl(3) and Bi(NO(3))(3), suggests that overall toxicity of bismuth complexes towards the parasite is both ligand and metal dependent.

Vectors and reservoirs of cutaneous leishmaniasis in Marvdasht district, southern Islamic Republic of Iran.

An epidemiological study was made of vectors and reservoirs of cutaneous leishmaniasis in rural regions of Marvdasht, Fars province, southern Islamic Republic of Iran during 2003-04. Using live traps, 126 rodents were collected: 75.4% were Meriones libycus, 14.3% Cricetulus migratorius and 10.3% Microtus arvalis. Eight out of 95 Meriones libycus (8.4%) were found to be infected with Leishmania major, identified by nested-PCR; none of the other rodents were positive. Female sandflies were collected from indoor locations: 75% were Phlebotomus papatasi and only 2.7% were found naturally infected with L. major. This is the first report of P. papatasi as a proven vector of zoonotic cutaneous leishmaniasis in this area.

Activities of naphthylisoquinoline alkaloids and synthetic analogs against Leishmania major.

The current treatments for leishmaniasis are unsatisfactory due to their toxic side effects, high costs, and increasing problems with drug resistance. Thus, there is an urgent need for alternative drugs against leishmaniasis. Different approaches have been used to identify novel pharmacophores against Leishmania sp. parasites, and one strategy has been the analysis of naturally occurring plant-derived compounds, including naphthylisoquinoline alkaloids. In the present study, we examined the abilities of these alkaloids to inhibit the growth of Leishmania major promastigotes and evaluated their effects on macrophages, dendritic cells, and fibroblasts. Furthermore, we determined the efficacy of selected compounds in decreasing the infection rate of macrophages and regulating their production of cytokines and nitric oxide. Our results demonstrate that the naphthylisoquinoline alkaloids ancistrocladiniums A and B (compounds 10 and 11) and the synthetic isoquinolinium salt (compound 14) were effective against intracellular amastigotes in the low submicromolar range, while toxicity against mammalian cells was observed at concentrations that were significantly higher than those needed to impair parasite replication. The activities of compounds 11 and 14 were mainly directed against the amastigote stage of L. major. This effect was not associated with the stimulation of host macrophages to produce nitric oxide or secrete cytokines relevant for the leishmanicidal function. In conclusion, our data suggest that ancistrocladiniums A and B (compounds 10 and 11) and the synthetically prepared isoquinolinium salt (compound 14) are promising candidates to be considered as lead compounds for leishmanicidal drugs.

Meriones libycus is the main reservoir of zoonotic cutaneous leishmaniasis in south Islamic Republic of Iran.

A study was made in rural regions around Neiriz city, Fars province in the south of the Islamic Republic of Iran during 2002-03 to further investigate a new focus of zoonotic cutaneous leishmaniasis. Meriones libycus was the dominant rodent (100% of 65 rodents collected) and 4.6% were naturally infected with the amastigote form of Leishmania spp. Promasigotes were identified as L. major. This confirms M. libycus as the main reservoir host of zoonotic cutaneous leishmaniasis in southern parts of the country.

Leishmania major amastigotes induce p50/c-Rel NF-kappa B transcription factor in human macrophages: involvement in cytokine synthesis.

Invasion of a host by pathogens is frequently associated with activation of nuclear factor kappa B (NF-kappaB), which is implicated in various aspects of immune function required for resistance to infection. However, pathogens may also subdue these mechanisms to secure their survival. Here we describe the effect of Leishmania major infection on NF-kappaB transcription factor activation in both promonocytic human cell line U937 and fresh human monocytes. Infection by L. major amastigotes blocked nuclear translocation of a phorbol-12 myristate-13 acetate (PMA)-induced p50/p65 NF-kappaB complex in PMA-treated differentiated U937 cells and triggered expression of p50- and c-Rel-containing complexes in both U937 cells and fresh human monocytes. These p50/c-Rel complexes, triggered by direct cell-parasite interactions, were detectable within 30 min after the interaction and were transcriptionally active. The NF-kappaB inhibitor caffeic acid phenethyl ester inhibited production of both tumor necrosis factor alpha and interleukin-10 (IL-10) induced by Leishmania amastigotes in differentiated U937 cells. Similar results for IL-10 induction were observed with amastigote-infected human monocytes. Our results indicate that L. major amastigotes activate NF-kappaB by specifically inducing p50- and c-Rel-containing complexes which are likely involved in the regulation of cytokine synthesis.

Epidemiological study in a new focus of cutaneous leishmaniasis in the Islamic Republic of Iran.

An outbreak of cutaneous leishmaniasis (CL) in Sabzevar county prompted this study of the epidemiology and the ecology of vectors and reservoirs. Examination of 541 schoolchildren showed rates of 9.4% for scars and 5.9% for ulcers. Among 807 inhabitants of 4 villages, 10.4% had scars and 3.0% had active lesions. The most highly infected age group was 0-4 years with a rate of 5.9%. A total of 12 849 sandflies representing 7 species were collected in the study area. Leptomonad infection was found in Phlebotomus papatasi, P. caucasicus and Sergentomyia sintoni. Parasites from man, P. papatasi and Rhombomys opimus, were isolated and characterized as Leishmania major. Based on this survey, this is an epidemic of zoonotic CL, with R. opimus the main reservoir host, and P. papatasi the main vector.

Caracterização dos leucócitos e da produção de quimiocinas envolvidas na resposta imuno-inflamatória in situ de camundongos CBA infectados com Leishmania major ou Leishmania amazonensis / Characterization of leukocytes and production of chemokines involved in immune-inflammatory response in situ of CBA mice infected with Leishmania major or Leishmania amazonensis

Camundongos CBA infectados com L. major ou L. amazonensis apresentam perfis morfológicos distintos de resposta tecidual, relacionados, respectivamente, com resistência ou susceptibilidade. O perfil Th1, de resistência, é caracterizado por um infiltrado mononuclear misto, com aumento do número de linfócitos, necrose fibrinóide e formação de granulomas que culmina com cura das lesões. O perfil Th2, de susceptibilidade, apresenta um infiltrado macrofágico difuso, monomórfico, com intenso parasitismo e áreas de necrose lítica que resulta na morte dos animais. Esses diferentes perfis histopatológicos são determinados pelas células efetoras da resposta imuno-inflamatória e por citocinas e quimiocinas. A caracterização da resposta imuno-inflamatória relacionada com infecção por diferentes espécies de Leishmania pode trazer informações importantes quanto a patogênese das mesmas e oferecer subsídios à sua modulação. Avaliamos comparativamente a infecção de camundongos CBA com L. major (Lm), L. amazonensis (La), em diferentes períodos, em relação à constituição celular nos linfonodos popliteos e a caracterização do infiltrado inflamatório das lesões através das técnicas de imunohistoquímica e RT-PCR. Nos linfonodos de drenagem das lesões foi observado aumento no número de células MAC-1+ nos grupos infectados. No grupo La houve aumento no número de células T CD4+ e S, enquanto no grupo Lm houve aumento crescente na quantidade de células T CD8+. Nos infiltrados inflamatórios das lesões, foi observada uma maior expressão de RNAm para as moléculas TNF-a, MIG e CRG-2 no grupo Lm. Somente a expressão de RNAm para IL-4, detectada na fase tardia, foi maior...

Chemokines, natural killer cells and granulocytes in the early course of Leishmania major infection in mice.

In the present study the early recruitment of leukocytes into the infected skin and into the draining lymph node (LN) was investigated after subcutaneous infection of mice with Leishmania major promastigotes. Flow cytometric analysis of cells recovered from the infected skin revealed that GR-1+ granulocytes were present as early as 10 h after infection, thus representing the first leukocyte population to be recruited to the site of infection. The migration of granulocytes was shown to be associated with a rapid mRNA expression of the neutrophil-attracting chemokine KC in the infected skin. Moreover, L. major promastigotes were found to produce factor(s) that are chemotactic for human neutrophils in vitro. Experiments with human neutrophils revealed that these cells are able to phagocytose the parasites. Natural killer (NK) cells appeared at the site of infection 24 h after infection. The migration of NK cells in resistant mice was found to correlate with the expression of the NK cell-activating chemokine IP-10. Treatment of susceptible BALB/c mice with recombinant mouse IP-10 resulted in a significantly increased NK cell cytotoxic activity in the draining LN. These data suggest that both the early chemokine gene expression and the production of chemotactic factors by the parasites themselves regulate the site-directed migration and activation of cells of the innate immune response, and suggest a role of chemotactic factors in the early defense against the parasites.

Development of a natural model of cutaneous leishmaniasis: powerful effects of vector saliva and saliva preexposure on the long-term outcome of Leishmania major infection in the mouse ear dermis.

We have developed a model of cutaneous leishmaniasis due to Leishmania major that seeks to mimic the natural conditions of infection. 1,000 metacyclic promastigotes were coinoculated with a salivary gland sonicate (SGS) obtained from a natural vector, Phlebotomus papatasii, into the ear dermis of naive mice or of mice preexposed to SGS. The studies reveal a dramatic exacerbating effect of SGS on lesion development in the dermal site, and a complete abrogation of this effect in mice preexposed to salivary components. In both BALB/c and C57Bl/6 (B/6) mice, the dermal lesions appeared earlier, were more destructive, and contained greater numbers of parasites after infection in the presence of SGS. Furthermore, coinoculation of SGS converted B/6 mice into a nonhealing phenotype. No effect of SGS was seen in either IL-4- deficient or in SCID mice. Disease exacerbation in both BALB/c and B/6 mice was associated with an early (6 h) increase in the frequency of epidermal cells producing type 2 cytokines. SGS did not elicit type 2 cytokines in the epidermis of mice previously injected with SGS. These mice made antisaliva antibodies that were able to neutralize the ability of SGS to enhance infection and to elicit IL-4 and IL-5 responses in the epidermis. These results are the first to suggest that for individuals at risk of vector-borne infections, history of exposure to vector saliva might influence the outcome of exposure to transmitted parasites.

Leishmania major infection in major histocompatibility complex class II-deficient mice: CD8+ T cells do not mediate a protective immune response.

In order to evaluate the role of CD8+ T cells in cutaneous leishmaniasis, major histocompatibility complex (MHC) class II-deficient mice of C57BL/6 background lacking functional CD4+ T cells were infected with Leishmania major. In contrast to C57BL/6 wild-type mice which are resistant to infection with L. major, these mice developed severe skin lesions that did not heal. In comparison to susceptible BALB/c mice, however, lesion development in MHC class II-deficient mice was very much retarded, even though the increase in the parasite load in lymphoid organs was only slightly delayed. Lymph node cells from L. major-infected MHC class II-deficient mice produced very low levels of interferon-gamma upon stimulation with L. major antigen, whereas the response to the mitogen concanavalin A was not impaired. Interestingly, they did not release lymphokines associated with disease exacerbation (interleukin 4 and interleukin 10) either, suggesting that the delayed lesion development is caused by the lack of disease-promoting CD4+ cells rather than by the presence of protective CD8+ cells. The lack of L. major-reactive immunoglobulins in the serum of infected MHC class II-deficient mice indicates that B cells also cannot respond to parasite antigens in the absence of MHC class II-mediated helper signals. The data demonstrate that MHC class II-deficient mice are unable to restrict the spreading of L. major, although they contain highly increased proportions of CD8+ T cells. Thus, MHC class II-restricted immune responses, most likely mediated by functional CD4+ T cells, are essential for the control of primary infections with L. major.

Cutaneous leishmaniasis in Kenya: Sergentomyia garnhami (Diptera Psychodidae), a possible vector of Leishmania major in Kitui District: a new focus of the disease.

Investigations on phlebotomine sandflies in Tseikuru, Kitui District, Kenya were carried out to determine the vectors of Leishmania major. Sandflies were trapped from animal burrows over a period of one year using castor oil-smeared sticky traps. Nineteen phlebotomine sandfly species were identified and their prevalence in this habitat determined. Dissections on the parous sandflies resulted in the isolation of Leishmania parasites from Phlebotomus martini and Sergentomyia garnhami. The isolated parasites were cultured in NNN medium and were consequently mass cultured in RPMI medium for biochemical characterisation. The parasites from S. garnhami were typed as Leishmania major.