Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Histopathological and immunohistochemical study of type 3 complement receptors (CD11b/CD18) in livers and spleens of asymptomatic and symptomatic dogs naturally infected with Leishmania (Leishmania) chagasi.

Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate.

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 microg/kg wet organ (4 days after the first dose) to 2.07 microg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Controlled field trials of a vaccine against New World cutaneous leishmaniasis.

Two controlled, double blind field trials of a non-living promastigote vaccine against New World Cutaneous Leishmaniasis (NWCL) were conducted in 1981 and 1983 in Brazil. Brazilian Army conscripts were randomly assigned to the vaccine or placebo groups and tested during their training in the Amazon jungle, a high risk area for NWCL. The results obtained showed: no significant differences between the vaccine and the placebo groups with respect to a number of characteristics (age, race, previous contact with the jungle, etc.); no significant differences between the participants who got and who did not get NWCL during the trial, with respect to length of exposure, contact with the jungle, etc. and a reduction of 67.3 and 85.7% in the annual incidence rate of NWCL, in 1981 and 1983 respectively (although the difference between incidence rates of the disease in vaccinated and control groups in the 1983 trial was not statistically significant), among those vaccinated who had converted to a positive leishmanin skin test as compared with the placebo groups.

A field trial of a vaccine against American dermal leishmaniasis.

A field trial was carried out in the eastern part of the State of Minas Gerais (Brazil) of a vaccine containing killed promastigotes of five stocks of Leishmania. Tests with Montenegro antigen showed that a high proportion of the vaccinated persons became positive within three months, but circulating antibodies were not detected. A proportion of those vaccinated continued to give positive Montenegro reactions for up to three years. Lymphocyte sensitivity tests carried out, on a small sample, three years after vaccination were positive and gave no evidence of immunological depression. No cases of cutaneous or mucocutaneous leishmaniasis occurred in the trial area during the three years of observations.

Mastomys natalensis as an experimental host for Leishmania mexicana mexicana.

Young multimammate rats (Mastomys natalensis) can be readily infected when inoculated intradermally with the amastigotes of Leishmania mexicana mexicana. Lesions appear at the site of inoculation in the second week of infection and continue to increase in size for the first month; amastigotes can be detected during this period. In the second month, lesions begin to decrease in size and it is more difficult to detect amastigotes. Three months after inoculation, the rats show no sign of infection. The results suggest that M. natalensis could be a useful laboratory host for immunological studies on dermatotropic Leishmania and for chemotherapeutic assays.

Novel methods for the encapsulation of meglumine antimoniate into liposomes.

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Distribution of liposome-encapsulated antimony in dogs.

The achievement of complete cure in dogs with visceral leishmaniasis is currently a great challenge, since dogs are the main reservoir for the transmission of visceral leishmaniasis to humans and they respond poorly to conventional treatment with pentavalent antimonials. In order to improve the efficacy of treatment, we developed a novel formulation for meglumine antimoniate based on the encapsulation of this drug in freeze-dried liposomes (LMA). The aim of the present study was to evaluate the biodistribution of antimony (Sb) in dogs following a single intravenous bolus injection of LMA. Four healthy male mongrel dogs received LMA at 3.8 mg Sb/kg body weight and were sacrificed 3, 48 and 96 h and 7 days later. Antimony was determined in the blood, liver, spleen and bone marrow. In the bone marrow, the highest Sb concentration was observed at 3 h (2.8 microg/g wet weight) whereas in the liver and spleen it was demonstrated at 48 h (43.6 and 102.4 microg/g, respectively). In these organs, Sb concentrations decreased gradually and reached levels of 19.1 microg/g (liver), 28.1 microg/g (spleen) and 0.2 microg/g (bone marrow) after 7 days. Our data suggest that the critical organ for the treatment with LMA could be the bone marrow, since it has low Sb levels and, presumably, high rates of Sb elimination. A multiple dose treatment with LMA seems to be necessary for complete elimination of parasites from bone marrow in dogs with visceral leishmaniasis.

Immunotherapy as a treatment of American cutaneous leishmaniasis: preliminary studies in Brazil.

A prophylactic vaccine composed of killed promastigotes of five stocks of Leishmania was tested as an immunotherapeutic agent against American cutaneous leishmaniasis (ACL). The agent was administered by deep intramuscular injection daily for 10 days, followed by a 10-day interval. Out of 62 patients so treated, 47 (76%) were considered clinically cured; 41 required 2-10 treatment courses and the other six 11-19 courses. None of the patients treated by immunotherapy displayed adverse side-effects. Immunotherapy proved to be effective in the treatment of single cutaneous lesions, multiple cutaneous lesions and in cases of mucocutaneous leishmaniasis. In comparison with chemotherapy (Glucantime), immunotherapy is less efficient and more prolonged but can be safely used when antimonials are contra-indicated or are found to be ineffective. Consideration is given to the treatment of victims of ACL living in rural areas remote from a medical centre.

Histopathology and immunocytochemical study of type 3 and type 4 complement receptors in the liver and spleen of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi.

The objective of this study was to compare the histopathological changes and expression of CR3 and CR4 in the liver and spleen of dogs naturally and experimentally infected with L. chagasi. The basic histopathological lesions observed mainly in naturally infected dogs were: epithelioid hepatic granulomas, hyperplasia and hypertrophy of Kupffer cells, Malpigui follicles and mononucleated cells of the red pulp of the spleen. Sections from the liver and spleen by immunocytochemistry technique showed the presence of CD11b, c/CD 18 antigens in the control and infected animals and no qualitative or quantitative differences in the liver. Nevertheless, CD18 was always increased in the spleen of naturally and experimentally infected dogs. These results indicate that there is a difference in the activation of CD18 in both experimental and natural cases of canine visceral leishmaniasis that should play an important role in the immunological response to Leishmania chagasi infection.

[Optical and electron microscopic study of the kidney of dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi]. / Estudo, ao microscópio óptico e eletrônico, do rim de cães natural e experimentalmente infectados com Leishmania (Leishmania) chagasi.

Two naturally infected dogs (male and female) from Teófilo Otoni (MG-Brazil) were maintained for 18 months in our laboratory. Two other dogs, two months old males were infected with 1 x 10(6) promastigotes of MHO/BR/70/BH46 Leishmania (Leishmania) chagasi strain, endovenous route, and autopsied after 10 months and two years. The main findings concerning the kidney were: (1) focal or diffuse mesangial glomerulonephritis with proliferative and enlargement of mesangial cells; (2) increase in thickness of basement membrane with electron-dense deposits; (3) chronic interstitial nephritis with intense exudation of plasmocytes; (4) cloud swelling of renal tubules. The authors discuss the probable pathogenetic mechanisms.

Immune responses induced by a Leishmania (Leishmania) amazonensis recombinant antigen in mice and lymphocytes from vaccinated subjects.

In the search for Leishmania recombinant antigens that can be used as a vaccine against American Cutaneous Leishmaniasis, we identified a Leishmania (Leishmania) amazonensis recombinant protein of 33 kD (Larp33) which is recognized by antibodies and peripheral blood leukocytes (PBL) from subjects vaccinated with Leishvacin, Larp33 was expressed in Escherichia coli after cloning of a 2.2 kb Sau3 digested genomic fragment of L. (L.) amazonensis into the pDS56-6 His vector. Immunoblotting analysis indicated that Larp33 corresponds to an approximately 40-kD native protein expressed in promastigotes of L. (L.) amazonensis and L. (Viannia) braziliensis. Northern blots of total RNA also demonstrated that the gene coding for this protein is expressed in promastigotes of the major lineages of Leishmania causing American Cutaneous Leishmaniasis. Larp33 induced partial protection in susceptible mouse strains (BALB/c and C57BL/10) against L. (L.) amazonensis after vaccination using Bacille Calmette-Guerin (BCG) as adjuvant. In vitro stimulation of splenocytes from BALB/c protected mice with Larp33 elicited the secretion of IL-2 and IFN-gamma, suggesting that a Th1 cell-mediated protective response is associated with the resistance observed in these mice. As revealed by its immunogenic and antigenic properties, this novel recombinant antigen is a suitable candidate to compose a vaccine against cutaneous leishmaniasis.

Immunocytochemical identification of Leishmania and Trypanosoma cruzi amastigotes in situ with homologous and heterologous polyclonal antibodies.

The unlabelled antibody peroxidase-antiperoxidase method was used to study the immunocytochemical properties of Leishmania and Trypanosoma cruzi amastigotes in situ after tissues had been submitted to different fixation procedures. Antisera were obtained from rabbits chronically infected with different strains of T. cruzi or immunized with L. mexicana amazonensis and L. braziliensis guyanensis, and were applied on 5 microns thick sections. T. cruzi antigens were well stained by the three anti-T. cruzi sera and the two anti-Leishmania sera at optimum dilution between 1:1,000 and 1:2,000, regardless the parasite strain. Differently, the leishmanial antigens were revealed by anti-Leishmania sera only at low dilutions (between 1:60-1:160), whereas the anti-T. cruzi sera, at these low dilutions, gave rather weak stainings. Although there is no clear explanation for this immunocytochemical "reverse-monodirectional" cross-reactivity between Leishmania and T. cruzi, the present results show that polyclonal antibodies agains Leishmania species, when used for immunocytochemical detection of these parasites in situ, react more strongly with T. cruzi amastigotes than with the homologous amastigotes.

[Canine visceral leishmaniasis: evaluation of the serologic method used in epidemiologic studies]. / Leishmaniose visceral canina: avaliação da metodologia sorológica utilizada em inquéritos epidemiológicos.

A comparative study was made of eluates of the blood of dogs experimentally infected with different trypanosomatids. Using antigens prepared from promastigotes of Leishmania mexicana, L. braziliensis and L. chagasi, assessments were made by the indirect immunofluorescence test. The results showed a sensitivity of 87.5% in the diagnosis of canine visceral leishmaniasis, independent of antigen used. Cross-reactions occurred in 75% of cases of cutaneous leishmaniasis and 83.3% of dogs with chagas' disease. An epidemiological survey in an area of leishmaniasis confirmed that immunofluorescence tests on eluates of dogs' blood give cross-reactions between L. braziliensis and L. chagasi. The results suggest that such testing could be useful in public health campaigns but attention is drawn to the fact that the level of positive reactions cannot be used as an indicator of the prevalence of canine kala-azar.

Humoral immunological profile and parasitological statuses of Leishmune vaccinated and visceral leishmaniasis infected dogs from an endemic area.

Dogs represent the major reservoir of Leishmaniao chagasi and vaccination against the canine disease is a potential control strategy. However, seroconversion occurs post-vaccine and hence, there is need to discriminate between the former group and naturally infected dogs. The present study represents a comparison of the humoral immunological profiles of both groups using Leishmania soluble antigen (LSA) and fucose-mannose ligand (FML). For both categories, ear skin samples were evaluated immunohistochemically and through PCR, that was also performed in blood specimens, as well as their ability to infect Lutzomyia (Lutzomyia) longipalpis. All these tests showed negative results for the vaccinated dogs. Differences between groups were observed regarding IgG, IgG2 and IgE absorbances as determined by FML-ELISA, and for IgG1 and IgE absorbances as measured by LSA-ELISA, showing that Leishmune-immunised animals and VL naturally infected dogs present different immunological profiles, even though these differences cannot be used to distinguish between these two groups of dogs.