Dog hepatocytes are key effector cells in the liver innate immune response to Leishmania infantum.

Hepatocytes constitute the majority of hepatic cells, and play a key role in controlling systemic innate immunity, via pattern-recognition receptors (PRRs) and by synthesizing complement and acute phase proteins. Leishmania infantum, a protozoan parasite that causes human and canine leishmaniasis, infects liver by establishing inside the Kupffer cells. The current study proposes the elucidation of the immune response generated by dog hepatocytes when exposed to L. infantum. Additionally, the impact of adding leishmanicidal compound, meglumine antimoniate (MgA), to parasite-exposed hepatocytes was also addressed. L. infantum presents a high tropism to hepatocytes, establishing strong membrane interactions. The possibility of L. infantum internalization by hepatocytes was raised, but not confirmed. Hepatocytes were able to recognize parasite presence, inducing PRRs [nucleotide oligomerization domain (NOD)1, NOD2 and Toll-like receptor (TLR)2] gene expression and generating a mix pro- and anti-inflammatory cytokine response. Reduction of cytochrome P 450s enzyme activity was also observed concomitant with the inflammatory response. Addition of MgA increased NOD2, TLR4 and interleukin 10 gene expression, indicating an immunomodulatory role for MgA. Hepatocytes seem to have a major role in coordinating liver's innate immune response against L. infantum infection, activating inflammatory mechanisms, but always balancing the inflammatory response in order to avoid cell damage.

Leishmania infantum exerts immunomodulation in canine Kupffer cells reverted by meglumine antimoniate.

Kupffer cells (KC) are the liver macrophage population that resides in the hepatic sinusoids and efficiently phagocyte pathogens by establishing an intimate contact with circulating blood. KC constitute the liver host cells in Leishmania infection, nevertheless little is described about their role, apart from their notable contribution in granulomatous inflammation. The present study aims to investigate how canine KC sense and react to the presence of Leishmania infantum promastigotes and amastigotes by evaluating the gene expression of specific innate immune cell receptors and cytokines, as well as the induction of nitric oxide and urea production. Complementarily, the impact of a leishmanicidal drug - meglumine antimoniate (MgA) - in infected KC was also explored. KC revealed to be susceptible to both parasite forms and no major differences were found in the immune response generated. L. infantum parasites seem to interact with KC innate immune receptors and induce an anergic state, promoting immune tolerance and parasite survival. The addition of MgA to infected KC breaks the parasite imposed silence and increased gene expression of Toll-like receptors (TLR) 2 and TLR4, possibly activating downstream pathways. Understanding how KC sense and react to parasite presence could bring new insights into the control or even elimination of canine leishmaniasis.

Leishmania infantum antigens modulate memory cell subsets of liver resident T lymphocyte.

In the recent years, the liver has been recognized as an important immune organ with major regulatory functions and immune memory, adding to the well-described vital metabolic functions. There are evidences from experimental infections performed with visceral Leishmania species that immune responses to parasite infection can be organ-specific. The liver is the compartment of acute resolving infection, with minimal tissue damage and resistance to reinfection, whereas the spleen is the compartment of parasite persistence. Control of hepatic infection in mice requires a coordinated immune response that involves the development of inflammatory granulomas. It is also described that the liver harbors populations of resident lymphocytes, which may exhibit memory characteristics. Therefore, the present study aims to address the role of the liver as an immune memory organ in the context of Leishmania infantum infection, by characterizing phenotypically resident liver T lymphocytes. The dynamics of memory T cells in L. infantum infected BALB/c mice and the effect of anti-leishmanial treatment in the differentiation of memory cell subsets were analyzed. The potential of recognition, differentiation and selection of memory lymphocytes by three L. infantum recombinant proteins were also explored. L. infantum infection generates effector and central memory T cells, but the cells did not expand when recalled, demonstrating a possible parasite silencing effect. The treatment with a leishmanicidal drug (antimoniate meglumine) increases the levels of memory and effector T cells, eliciting a more robust hepatic immune response. L. infantum parasites with a decreased sensitivity to the leishmanicidal drug favor the expansion of memory CD8+ T cell subset, but inhibit the proliferation of CD8+ T effector cells, possibly assuring their own survival. The recombinant proteins LirCyp1 and LirSOD are strongly recognized by memory cells of treated mice, indicating that these proteins might be used in a prophylactic or therapeutic vaccine formulation. Thus, L. infantum released antigens induce the development of immune memory subsets in the liver resident T cell population that specifically recognized parasite antigens, including recombinant proteins.

Immunization with the Leishmania infantum recombinant cyclophilin protein 1 confers partial protection to subsequent parasite infection and generates specific memory T cells.

Control of zoonotic visceral leishmaniosis can be achieved using several available drugs. These drugs present high toxicity and require longer treatment regimens which complicate compliance to the treatment. Other control measures directed to the vector or the reservoirs are useful tools to restrain the spreading of this disease but the effects are transitory. A safe, affordable and efficient vaccine conferring long lasting immunity should be the most cost effective way of controlling zoonotic visceral leishmaniosis. The present study aims at characterizing a cyclophilin protein 1 of Leishmania infantum (LiCyP1) and investigating whether recombinant LiCyP1 (LirCyP1) is able to confer protection against infection by evaluating viable parasite load and the generation of specific CD4(+) and CD8(+) effector and central memory T cells in rodent model. LiCyP1 is present in the cytoplasm of L. infantum amastigotes and promastigotes. Immunization of BALB/c mice with LirCyP1 confers high protection to L. infantum infection, causing a marked reduction in parasite replication in the liver and spleen. Furthermore, helper and cytotoxic memory T cell subsets able to specifically recognize parasite antigens expanded in immunized and in challenged mice. CD4(+) T cell subpopulation of intermediate phenotype (CD62L(high)CD127(low)) of challenging mice also presented an accentuated expansion after the recall. This study demonstrated that LirCyP1 confers partial protection to L. infantum infection, promoting the generation of a desired long lasting immunity. LirCyP1 can be considered a potential candidate for the design of a vaccine against zoonotic visceral leishmaniosis.

Cytokine gene expression in the tissues of dogs infected by Leishmania infantum.

Canine leishmaniosis (CanL) caused by the protozoan parasite Leishmania infantum is a chronic systemic disease that is endemic in certain parts of the world. The domestic dog is the most important reservoir of L. infantum and is the main source of infection for other animals and for the human population. The aim of this study was to evaluate and compare the level of expression of genes encoding particular cytokines (interleukin [IL]-12, interferon [IFN]-γ, IL-2 and IL-4) in different tissues and organs of 53 adult dogs with or without clinical signs of leishmaniosis and after treatment for the disease. Asymptomatic dogs showed high expression of genes encoding IL-4 in blood leucocytes and of genes encoding IL-12 and IL-2 in lymph nodes. Blood leucocytes from symptomatic dogs had a mixed Th1 and Th2 cytokine gene expression profile, but lymph nodes from these animals had dominant IL-2 and IFN-γ gene expression, while bone marrow appeared to be unresponsive. The predominance of IL-4 gene expression in the blood of asymptomatic dogs may favour parasite replication, while the balance between Th1 and Th2 cytokine gene expression in the blood of symptomatic dogs may be important in reducing parasite replication and delaying the dissemination of Leishmania to other organs. The drugs used to treat CanL do not completely eliminate the parasite, so the high expression of the gene encoding IL-4 in blood leucocytes and the high expression of IL-12 and IL-4 mRNA in lymph nodes may reflect the persistence of residual Leishmania amastigotes. L. infantum appears able to regulate the host immune response in order to ensure its survival, but also to prevent the host from succumbing to infection. This guarantees its transmission and the completion of its life cycle.

Differential expression of cytokine genes among sickle-cell-trait (HbAS) and normal (HbAA) children infected with Plasmodium falciparum.

The human immune response to Plasmodium falciparum infection involves the release of cytokines that may contribute to the control of the parasites' replication. These cytokines are also involved in the pathogenesis of the malaria caused by the infection, leading to the appearance of symptoms of varying severity. In a cross-sectional study, the expression of the genes that code for pro-inflammatory cytokines (tumour necrosis factor, interferon-gamma, interleukin-6 and interleukin-12) and anti-inflammatory cytokines (interleukin-10 and interleukin-4) among 80 children infected with P. falciparum (from a malaria-endemic area of Sudan) and five healthy controls (from a non-endemic area) was explored. The infected children were either non-sicklers, with severe malaria (18 children), mild malaria (30) or no symptoms of malaria (18), or asymptomatic sicklers (14). Interleukin-12 was found to be weakly expressed by all the groups of children. In general, compared with the other groups, the asymptomatic non-sicklers had lower expression of all the cytokines studied. The asymptomatic sicklers had significantly lower expression of tumour necrosis factor than the non-sicklers with severe malaria, but these two groups showed similar expression of interferon-gamma, interleukin-4 and interleukin-6. Gene expression of the regulatory cytokine, interleukin-10, by the asymptomatic sicklers was significantly lower than that by the non-sicklers with severe malaria but higher than that recorded in the non-sicklers with mild malaria. Their regulation of cytokine release appears to protect sicklers from clinical malaria.

Efficacy of the liposome trifluralin in the treatment of experimental canine leishmaniosis.

Liposomes are used as carriers to deliver drugs and to treat diseases where infection is localised in the mononuclear phagocyte system cells, as is the case of leishmaniosis. Trifluralin is a dinitroaniline with proved anti-Leishmania activity in vitro. The efficacy of liposomal trifluralin (LIP/TFL) was studied in the treatment of experimental canine leishmaniosis through quantification of parasite burden using the limiting dilution assay, follow-up of anti-Leishmania antibodies by indirect fluorescent immunoassay and cytokine expression by Reverse Transcriptase-PCR, in the bone marrow, lymph nodes, skin and peripheral blood mononuclear cells in 5 female beagle dogs. After treatment, dogs showed a general remission of clinical signs related to parasite burden reduction and Th1 cytokine mRNA expression, but there was no significant decrease in antibody levels. Alternative treatment schemes with LIP/TFL are necessary to achieve optimal results.

Canine leishmaniasis chemotherapy: dog's clinical condition and risk of Leishmania transmission.

The aim of this study was to investigate whether treatment against canine leishmaniasis reduced the presence of Leishmania in the healthy skin of dogs, affecting the capacity of parasite transmission. A total of 37 dogs from an endemic region of leishmaniasis were studied. Thirteen symptomatic animals revealed parasites in the bone marrow and eight had also in the skin. Five of the 22 dogs that had been treated with meglumine antimoniate alone, meglumine antimoniate or trifluralin followed by allopurinol or just with allopurinol had the parasite in bone marrow but none showed Leishmania in the skin. One dog that was treated only with aminosidine was polisymptomatic and had parasites in bone marrow and skin. The different treatments used in this study did not completely eliminate the parasite allowing relapses to occur when the treatment is discontinued, but the use of meglumine antimoniate or allopurinol, alone or combined may improve dogs clinical condition and reduce or eliminate the parasite from the skin decreasing the probability of Leishmania transmission.

Leishmaniasis in Portugal: enzyme polymorphism of Leishmania infantum based on the identification of 213 strains.

This study reports isoenzyme polymorphism of Leishmania strains isolated in different regions of Portugal between 1982 and 2005. A total of 213 strains were obtained from cases of visceral and cutaneous leishmaniasis isolated from immunocompetent patients (adults and children) and immunocompromised adults, as well as from dogs and sandflies. Four zymodemes were identified: MON-1, MON-24, MON-29 and MON-80. Zymodeme MON-1 was identified in 96.7% of the strains, predominating in both immunocompetent and immunocompromised human patients, and it was the only zymodeme isolated from dogs. Isoenzyme diversity in HIV-infected patients was higher than in the immunocompetent group, in which all the strains from visceral leishmaniasis were MON-1. The domestic dog was confirmed as the reservoir host of zoonotic leishmaniasis in Portugal and Phlebotomus perniciosus and Phlebotomus ariasi as vectors. The overall low enzyme polymorphism observed in the Portuguese foci contrasts with the neighbouring foci in Spain.

Leishmania infantum: soluble proteins released by the parasite exert differential effects on host immune response.

The objective of this study was to analyse the modulatory effect of proteins released by cultured Leishmania infantum promastigotes on the cellular immune response of infected susceptible (BALB/c) and more resistant (C57BL/6) mice strains after 30 and 45 days of infection. One month after parasite inoculation, L. infantum released protein fractions (High, Inter, and Low according to molecular weight) stimulated C57BL/6 mice spleen cells to proliferate and to express cytokines. Following the decrease of parasite load only the Low protein fraction induced a considerable release of IL-4. In BALB/c mice, specific immune response to protein fractions was only observed at the higher parasitic level, with the fraction Inter promoting the production of IL-4 and fractions High and Low inducing high levels of IL-12. These results point out to a role of these proteins fractions in the modulation of host immunity, that depending on the host genetic background and parasite magnitude, seem to be critical in the control of parasite replication levels, thus avoiding premature host death.

Immunological and histopathological studies in a rodent model infected with Leishmania infantum promastigotes or amastigotes.

Infection with Leishmania infantum promastigotes (group I) and amastigotes (group II) was evaluated over 32 weeks, using Syrian golden hamsters as an experimental model. Spleen cells strongly responded to the specific antigen at 12 (group I) and 16 weeks (group II) post-inoculation (p.i.) and lower stimulation index values coincided with the parasite burden peak. Western-blot analysis detected antibodies during the 1st week p.i. and the number of recognized proteins increased with the time of infection, reaching a maximum at the peak parasite burden. Histopathology revealed hypoplasia in spleen white pulp and the liver showed a periportal infiltration of inflammatory cells and small granulomas, becoming increasingly more severe as the infection developed. Both organs exhibited a secondary amyloid deposition at the end of the experiment, especially the spleen. In this study, progressive visceral disease was observed as in natural human and canine infections; however, the incubation period was longer in the promastigote than in the amastigote infection.

Cell mediated immunity and specific IgG1 and IgG2 antibody response in natural and experimental canine leishmaniosis.

In the present study, we have followed up Leishmania infantum infection in dogs: (1) naturally infected; (2) experimentally infected with amastigotes; and (3) experimentally infected with culture promastigotes. The main objective was to evaluate the differences of the humoral and cellular immune responses of each group. Sera from 12 beagle dogs were analysed for total anti-leishmanial antibodies and IgG1 and IgG2 subclasses by enzyme-linked immunosorbent assay (ELISA). Lymphoproliferation to L. infantum antigen was also performed. All naturally infected animals were symptomatic with a marked humoral response. Dogs inoculated with amastigotes were asymptomotic and presented lower antibody titres than naturally infected. Dogs inoculated with culture promastigotes were asymptomotic with no significant humoral response. Strong proliferative responses to Leishmania antigen was observed in dogs inoculated with promastigotes. In our experimental model, IgG1 antibody levels presented a similar pattern in all infected animals, and IgG2 reactivity was high in naturally infected dogs.

Infectivity of promastigotes and amastigotes of Leishmania infantum in a canine model for leishmaniosis.

Seven dogs experimentally infected with amastigotes or culture promastigotes of Leishmania infantum MON-1 were observed for a period of up to 38 months. The course of infection was monitored by clinical and parasitological examinations, haematological and serum protein analysis, and by anti-leishmania antibody levels. Two of the three amastigote-inoculated dogs developed a symptomatic infection with haematological and protein alterations, and a strong humoral immune response. The third dog was asymptomatic with no haematological or protein alterations and developed a steady humoral response. Four promastigote-inoculated dogs remained asymptomatic throughout the observation period, with only transient antibody responses to leishmanial antigen, and no haematological or protein alterations. The detection of the parasite in biological material obtained at necropsy showed that dogs with no clinical signs or other manifestations of disease may be infected. This indicates that asymptomatic carriers may be present in the canine population, but not identifiable by the usual serological tests, and suggests that epidemiological surveys based on serology may underestimate the prevalence of canine leishmaniosis and the parasite transmission risk.

Canine experimental infection: intradermal inoculation of Leishmania infantum promastigotes.

Five mixed breed dogs were inoculated intradermally (ID) with cultured virulent stationary phase promastigotes of Leishmania infantum Nicole, 1908 stocks recently isolated. Parasite transformations in the skin of ID infected dogs were monitored from the moment of inoculation and for 48 h, by skin biopsies. Anti-Leishmania antibody levels were measured by indirect immunofluorescence assay, counterimmunoelectrophoresis and direct agglutination test, and clinical conditions were examined. Thirty minutes after ID inoculation the first amastigotes were visualised and 3 to 4 h after inoculation the promastigotes were phagocytized by neutrophils and by a few macrophages. These cells parasitised by amastigotes progressively disappeared from the skin and 24 h after inoculation parasites were no longer observed. Local granulomes were not observed, however, serological conversion for antibodies anti-Leishmania was achieved in all dogs. Direct agglutination test was the only technique positive in all inoculated dogs. Amastigotes were found in the popliteal lymph node in one dog three months after inoculation. This work demonstrates that, with this inoculum, the promastigotes were transformed into amastigotes and were up taken by neutrophils and macrophages. The surviving parasites may have been disseminated in the canine organism, eliciting a humoral response in all cases.

Performance of immunoblotting in diagnosis of visceral Leishmaniasis in human immunodeficiency virus-Leishmania sp.-coinfected patients.

This study evaluated the performance of immunoblotting with Leishmania infantum soluble antigens for the diagnosis of visceral leishmaniasis in human immunodeficiency virus (HIV)-infected and immunocompetent patients and assessed the humoral responses of patients coinfected with HIV and Leishmania. In this work, the results of the immunoblot analysis were compared to those of parasitological examination (Giemsa-stained smears and/or parasite isolation in Novy, Nicolle, and MacNeal medium from bone marrow) and indirect immunofluorescence and counterimmunoelectrophoresis techniques. Patients were considered to be infected if one or more of the comparison techniques gave a positive result. Immunoblotting was considered to be positive if at least one band was present. For 198 HIV-positive patients with a clinical suspicion of visceral leishmaniasis, immunoblot analysis had a sensitivity of 70.6%, a specificity of 73.2%, and an accuracy of 72.7%. For a separate group of 40 immunocompetent patients not infected with Leishmania, the immunoblot analysis was negative for all patients (100% specificity), and for a third group of 32 immunocompetent patients with confirmed visceral leishmaniasis, the immunoblot analysis was positive for all patients (100% sensitivity). Sera of coinfected patients recognized few bands and recognized bands at lower intensities compared with sera from immunocompetent patients. The most frequently detected band was 63 to 66 kDa (55.9%), with the difference being statistically significant compared to frequency of detection of the other bands. This study confirms that the humoral response in patients coinfected with HIV and Leishmania is much lower than that in immunocompetent patients and that the immunoblot method is a sensitive, noninvasive, and specific serological technique for the diagnosis of visceral leishmaniasis in immunocompromised patients.

The effect of chloroquine on the production of interferon-gamma, interleukin (IL)-4, IL-6, and IL-10 in Plasmodium chabaudi chabaudi in infected C57BL6 mice.

The effect of chloroquine (CQ) on the production pattern of interferon (IFN)-gamma, interleukin (IL)-4, IL-6, and IL-10 in female C57BL6 mice infected with Plasmodium chabaudi chabaudi AS was evaluated during a period of 35 days. Our data confirm that there is a switch from a T helper cell (Th)1 to a Th2 response during malaria infection in this model. Proliferation assays showed a decreased stimulation index in infected mice that was further reduced in infected mice treated with CQ. Noninfected control mice treated with CQ showed an increase production of IFN-gamma. However, no detectable changes in IL-4, IL-6, and IL-10 production were observed in this group. CQ treatment of infected mice resulted in parasite clearance that was associated with an earlier production of IL-4, IL-6, and IL-10 when compared with nontreated infected mice. We suggest that this earlier switch to a Th2 response is a consequence of parasite killing rather than CQ interference with cytokine production.

[Canine leishmaniasis. New concepts of epidemiology and immunopathology: their impact in the control of human visceral leishmaniasis]. / Leishmaniose canina. Novos conceitos de epidemiologia e imunopatologia e seus reflexos no controlo da leishmaniose visceral humana.

Visceral leishmaniasis (VL) is a zoonosis in most regions where it occurs. Dogs are the most important reservoir of the disease and are mainly responsible for the persistence of VL in the Paleartic and Neotropical regions. Canine leishmaniasis (CaL) is a viscerocutaneous, chronic infection with a worse prognosis than human disease. We now know that, as in man, there are some cases of asymptomatic infection. Former studies indicated that dog cutaneous parasitism becomes infectious to the insect vector in later periods of the disease, but recent studies performed by xenodiagnosis have shown that it is possible that transmission might occur earlier. The infected animal reacts with a great production of antibodies and depression of cellular immunity. Antibodies are not protective and resistance is related with active cellular immunity. The presence of Th 1 response in asymptomatic animals, sometimes without humoral response, means that the prevalence of CaL, found in epidemiological surveys by searching for antibodies, may be underestimated.

Reliability of serological methods for detection of leishmaniasis in Portuguese domestic and wild reservoirs

A direst agglutination test (DAT) and an immunofluorescence (IFAT) were compared for detection of Leishmania infantum infection in 43 dogs and five foxes from Alto-Douro and Arrabida, two known endemic areas in Portugal. In four dogs with proved canine leishmaniasis, both DAT and IFAT showed positive readings (titres ò1:320 and ò1:128). Of 34 samples collected form apparently healthly dogs, ten were positive by both serological tests and eight were serologically positive by one test or the other. Three foxes out of five captured in this area, scored titres indicative of leishmaniasis in both DAT and IFAT. The concordance between DAT and IFAT in all collected samples (48) was 81.25 per cent. Considering these and previous studies in the adjancent Mediterranean areas, the seroprevalence of L. infantum infection in the canine and vulpine populations appear to be high magnitude.