Type I signal peptidase from Leishmania is a target of the immune response in human cutaneous and visceral leishmaniasis.

The gene encoding type I signal peptidase (Lmjsp) has been cloned from Leishmania major. Lmjsp encodes a protein of 180 amino residues with a predicted molecular mass of 20.5 kDa. Comparison of the protein sequence with those of known type I signal peptidases indicates homology in five conserved domains A-E which are known to be important, or essential, for catalytic activity. Southern blot hybridisation analysis indicates that there is a single copy of the Lmjsp gene. A recombinant SPase protein and a synthetic peptide of the L. major signal peptidase were used to examine the presence of specific antibodies in sera from either recovered or active individuals of both cutaneous and visceral leishmaniasis. This evaluation demonstrated that sera from cutaneous and visceral forms of leishmaniasis are highly reactive to both the recombinant and synthetic signal peptidase antigens. Therefore, the Leishmania signal peptidase, albeit localised intracellularly, is a significant target of the Leishmania specific immune response and highlights its potential use for serodiagnosis of cutaneous and visceral leishmaniasis.

Influence of apoptosis on the cutaneous and peripheral lymph node inflammatory response in dogs with visceral leishmaniasis

In canine visceral leishmaniasis (CVL), the abnormalities most commonly observed in clinical examination on the animals are lymphadenomegaly and skin lesions. Dogs are the main domestic reservoir for the protozoon Leishmania (L.) chagasi and the skin is the main site of contamination by the vector insect. Some protozoa use apoptosis as an immunological escape mechanism. The aim of this study was to correlate the presence of apoptosis with the parasite load and with the inflammatory response in the skin and lymph nodes of dogs naturally infected with Leishmania (L.) chagasi. Thirty-three dogs from the municipality of Araçatuba (São Paulo, Brazil) were used, an endemic area for CVL. Muzzle, ear and abdominal skin and the popliteal, subscapular, iliac and mesenteric lymph nodes of symptomatic (S), oligosymptomatic (O) and asymptomatic (A) dogs were analyzed histologically. The parasite load and percentage apoptosis were evaluated using an immunohistochemical technique. Microscopically, the lymph nodes presented chronic lymphadenitis and the skin presented plasmacytic infiltrate and granulomatous foci in the superficial dermis, especially in the ear and muzzle regions. The inflammation was most severe in group S. The parasite load and apoptotic cell density were also greatest in this group. The cause of the lymphoid atrophy in these dogs was correlated with T lymphocyte apoptosis, thus leaving the dogs more susceptible to CVL. The peripheral lymph nodes presented the greatest inflammatory response. Independent of the clinical picture, the predominant inflammatory response was granulomatous and plasmacytic, both in the skin and in the peripheral lymph nodes. The ear skin presented the greatest intensity of inflammation and parasite load, followed by the muzzle skin, in group S. The ear skin area presented a non-significant difference in cell profile, with predominance of macrophages, and a significant difference from group A to groups O and S. It was seen that in these areas, there were high densities of parasites and cells undergoing apoptosis, in group S. The association between apoptosis and parasite load was not significant in the lymph nodes, but in the muzzle regions and at the ear tips, a positive correlation was seen between the parasite load and the density of cells undergoing apoptosis. The dogs in group S had the highest parasite load and the greatest number of apoptotic cells, thus suggesting that the parasite had an immune evasion mechanism, which could be proven statistically in the skin. © 2012 Elsevier B.V.

Cutaneous and Mucocutaneous Leishmaniasis

Tegumentary leishmaniases are caused by approximately 15 species of protozoa of the genus Leishmania. They prevail in tropical and subtropical areas of the Old and New World but human mobility also makes them a medical problem in nonendemic areas. Clinical manifestations may comprise cutaneous and mucocutaneous forms that may be localized, disseminated, or diffuse in distribution and may differ in Old and New World leishmaniases. Diagnosis and treatment vary according to the clinical manifestations, geographic area, and Leishmania species involved. This article highlights the diversity and complexity of tegumentary leishmaniases, which are worsened by human immunodeficiency virus/Leishmania coinfection.

The finding of Lutzomyia almerioi and Lutzomyia longipalpis naturally infected by Leishmania spp. in a cutaneous and canine visceral leishmaniases focus in Serra da Bodoquena, Brazil

To identify natural infections by Leishmania spp. in insect vectors of cutaneous and visceral leishmaniasis, we performed field studies in natural and anthropic environments in the Guaicurus Settlement (Bodoquena Range) of the Bonito municipality, Mato Grosso do Sul state, Brazil. From October 2002 to October 2003, a total of 1395 sandfly females were captured with Shannon and light traps and dissected in search of flagellates. The sample is composed of a total of 13 species, with Lutzomyia almerioi (59.9%) and Lutzomyia longipalpis (31.4%) predominant. Infections by flagellates were directly observed in three of the dissected of Lu. almerioi females (0.36%). To increase the sensitivity of detection, DNA extracted from pools of the 1220 dissected females (Lu. almerioi 808, Lu. longipalpis 399 and Nyssomyia whitmani 13) was subjected to small subunit rRNA-based polymerase chain reactions (SSU-PCR). DNA from Leishmania (L.) infantum chagasi was detected in at least 0.37% of Lu. almerioi females and in 0.25% of Lu. longipalpis females. The DNA of the Leishmania (Viannia) sp. was detected in 0.12% of Lu. almerioi and in 0.70% of Lu. longipalpis. Leishmania (L.) amazonensis was found in 1.25% of Lu. longipalpis. Mixed infections of L. (Leishmania) sp. and L. (Viannia) sp. were found in 0.50% of Lu. longipalpis. When considering that each positive pool contained at least a single infected specimen, we found a 1.23% rate of Leishmania spp. infection among the total population of dissected female sand flies as determined by PCR. This is the first report of natural infection by L. (L.) infantum chagasi and L. (Viannia) sp. in Lu. almerioi. It is also the first report of infection by L. (Viannia) sp. in Lu. longipalpis. The observation that Lu. longipalpis and Lu. almerioi are naturally infected by agents of both cutaneous and visceral leishmaniases suggests that these two species play a role in the transmission (continua) (continuação) of these diseases within the study area. Furthermore, the finding that Lu. longipalpis has been naturally infected by L. (L.) amazonensis and L. (Viannia) sp., and Lu. almerioi by L. (L.) infantum chagasi and L. (Viannia), suggests their participation as permissive vectors

Mucosal leishmaniasis ("espundia") responsive to low dose of N-methyl glucamine (Glucantime ®) in Rio de Janeiro, Brazil

Response to treatment with antimonial drugs varies considerably depending on the parasite strain involved, immune status of the patient and clinical form of the disease. Therapeutic regimens with this first line drug have been frequently modified both, in dose and duration of therapy. A regimen of 20 mg/kg/day of pentavalent antimony (Sb5+) during four weeks without an upper limit on the daily dose is currently recommended for mucosal disease ("espundia"). Side-effects with this dose are more marked in elderly patients, more commonly affected by this form of leishmaniasis. According to our experience, leishmaniasis in Rio de Janeiro responds well to antimony and, in cutaneous disease, high cure rates are obtained with 5 mg/kg/day of Sb5+ during 30 to 45-days. In this study a high rate of cure (91.4%) employing this dose was achieved in 36 patients with mild disease in this same geographic region. Side-effects were reduced and no antimony refractoriness was noted with subsequent use of larger dose in patients that failed to respond to initial schedule.

L. (L.) chagasi in aids and visceral leishmaniasis (kala-azar) co-infection

Concomitant skin lesions in visceral leishmaniasis (VL) or kala-azar are rare, being more common the description of post-kala-azar dermal leishmaniasis occurring post treatment of kala-azar. Skin lesions caused by Leishmania donovani are frequently seen in the aids-VL co-infection. In Brazil cutaneous or mucosal forms of tegumentary leishmaniasis concomitant with aids are more commonly registered. Here we present a case of aids-VL co-infection, with unusual cutaneous and digestive compromising attributed to L. (L.) chagasi, with special attention to ecthymatous aspect of the lesion, allied to the absence of parasite on the histological skin biopsy.

IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

Efecto de la temperatura de la piel en la leishmaniasis cutanea experimental

The literature on the thermosensitive properties of strains or species of Leishmania and of other miercorganisms is revised. Cutaneous or mucocutaneous strains that infect animais in the coldest areas of the skin or mucosa in general can not grow in tissue culture at 37°C or higher temperatures and their respiratory metabolism decreases at these temperatures. These facts suggest a thermosensitive event in some important metabolism phase of the organisme. The strains or species that are able to produce visceral leishmaniasis were probably originated from cutaneous strains after genetioally determined physiological adaptation, to warmer temperatures. These strains can not only visceralize in animais and man but will also grow in tissue culture at 36-37°C and the respiratory metabolism will be higher at such temperatures. There are reasons to believe that intermediate strains, i. e., with properties of both groupsí do exist. A thermosensitive physiological event is a more general phenomenon and examples of it can also be found in the fields of virology, bacteriology and mycology. It has practical applications since some of the diseases produced by these agents can be cured by treatments with heat or artificial fever. Experiments along these line were performed on hamsters with a Costa Rican strain of L. braziliensis as an experimental model. Even after intraperitoneal inoculation lesions appear in the nose, ears, paws and tail with a subcutaneous temperature bellow 33°C at 22-24°C. Healing of the lesión is accomplished by increasing room temperature. A good lesión is produced in the rump of the animal if the area is depilated (comercial cream depilatory) previously and the naked skin cooled artificially. Elevated temperature, or the growing back of the hair will tend to diminish or cure the lesion.

Human mucocutaneous leishmaniasis in Três Braços, Bahia - Brazil: an area of Leishmania braziliensis braziliensis transmission. I. Laboratory diagnosis

Leishmanial parasites were detected in 71.2% of patients with cutaneous disease and 48% of patients with mucosal disease, using principally scanning of imprints mears and histological sections and hamster inoculation. Parasites were more frequent in early cutaneous lesions (p < 0.005) o fless than two month duration. Also they were more common in multiple than single mucosal lesions (p < 0.02) in spite of considerable prior glucan time therapy in the former group. 93% of cutaneous lesions had a positive leishmanin skin test and most of the negatives occurred in patients with lesions of less than one month duration. 97% of patients with single mucosal lesion and 79% with multiple mucosal lesions had a positive skin test. 86% of cutaneous disease and 90% of mucosal disease was associated with a positive indirect immunofluorescent antibody test at a ≥ 1/20 dilution. In both groups multiple lesions were associated with higher titres and titres were significantly higher in patients with mucosal disease compared with cutaneous disease (p < 0.01).

Parasitological diagnosis of mucocutaneous leishmaniasis due to Leishmania b. braziliensis in Bolivia

Parasitological diagnosis, using staned smears, culture and pathological examination of biopsy, was studied in 146 patients infected with mucocutaneous leishmaniasis, in Bolivia and Peru. The most efficient parasite detecting technique appeared to be the smear examination in cutaneous lesions (33 % positive) and the pathology in case of mucous lesions (28 % positive). In both, cutaneous and mucous lesions, the parasites were found most frequently in old lesions.

Experimental canine mucocutaneous leishmaniasis (Leishmania braziliensis braziliensis)

Four mongrel dogs were intradermically inoculated with 3 x 10**6 Leishmania braziliensis braziliensis promastigotes. Three out of the four animals developed cutaneous lesions respectively 4, 7, and 8 months after. The fourth dog did not develop lesion at the inoculation site, but a mucosal ulcer was seen 16 months after the inoculum. Clinical, histopathological, and serological findings were similar to what is found in natural canine infection as well as in the human disease. These results suggest that dogs may be an useful model for L. b. braziliensis infection.

Resistance to oxidative stress is associated with metastasis in mucocutaneous leishmaniasis.

Mucocutaneous leishmaniasis (MCL) in South and Central America is characterized by the dissemination (metastasis) of Leishmania Viannia subgenus parasites from a cutaneous lesion to nasopharyngeal tissues. Little is known about the pathogenesis of MCL, especially with regard to the virulence of the parasites and the process of metastatic dissemination. We previously examined the functional relationship between cytoplasmic peroxiredoxin and metastatic phenotype using highly, infrequently, and nonmetastatic clones isolated from an L. (V.) guyanensis strain previously shown to be highly metastatic in golden hamsters. Distinct forms of cytoplasmic peroxiredoxin were identified and found to be associated with the metastatic phenotype. We report here that peroxidase activity in the presence of hydrogen peroxide and infectivity differs between metastatic and nonmetastatic L. (V.) guyanensis clones. After hydrogen peroxide treatment or heat shock, peroxiredoxin was detected preferentially as dimers in metastatic L. (V.) guyanensis clones and in L. (V.) panamensis strains from patients with MCL, compared with nonmetastatic parasites. These data provide evidence that resistance to the first microbicidal response of the host cell by Leishmania promastigotes is linked to peroxiredoxin conformation and may be relevant to intracellular survival and persistence, which are prerequisites for the development of metastatic disease.

Leishmaniasis dissemineted by Leishmania braziliensis in a mare (Equus cabalus) immunotherapy and chemotherapy assays

Cutaneous disseminated lesions caused by Leishmania sp. were found in a pregnant mare (Equus cabalus) from a rural city in the State of rio de Janeiro, Brazil. Before delivering, treatment was undertaken by immunotherapy followed by chemotherapy. Histopatology and serology were performed during treatment, as well as the biochemical characterization of the parasite (L. braziliensis) that was isolated from one of the lesions.

Phase 1 Study of an Inactivated Vaccine against American Tegumentary Leishmaniasis in Normal Volunteers in Brazil

A Phase 1 double-blind placebo-controlled study was performed to evaluate a vaccine against American tegumentary leishmaniasis in 61 healthy male volunteers. Side effects and the immune response to the vaccine were evaluated, with 1- and 2- dose schemes, with intervals of 7 or 21 days, each dose containing 1440 mg of protein N antigen of a single strain of Leishmania amazonensis (PH 8) diluted in merthiolated saline (1:10,000). Merthiolated saline and an inert substance were used as placebos. No significant clinical alterations were found following the respective injections in the vaccinated individuals as compared to the placebos, except for local pain, which was associated significantly with injection of the vaccine. The laboratory alterations we observed bore no association with the clinical findings and were unimportant. We observed no differences between the groups with regard to seroconversion or the Montenegro skin test. However, the group that received a single dose of the vaccine and the one that received two doses with a 21-day interval displayed cutaneous induration significantly larger than in the control group, with 100%, 100%, and 66% conversion in the skin test, respectively. We concluded that the vaccine does not present any major side effect that would contraindicate its use in healthy individuals.

B-cell infiltration and frequency of cytokine producing cells differ between localized and disseminated human cutaneous leishmaniases

Biopsies from human localized cutaneous lesions (LCL n = 7) or disseminated lesions (DL n = 8) cases were characterized according to cellular infiltration,frequency of cytokine (IFN-g, TNF-alpha) or iNOS enzyme producing cells. LCL, the most usual form of the disease with usually one or two lesions, exhibits extensive tissue damage. DL is a rare form with widespread lesions throughout the body; exhibiting poor parasite containment but less tissue damage. We demonstrated that LCL lesions exhibit higher frequency of B lymphocytes and a higher intensity of IFN-gamma expression. In both forms of the disease CD8+ were found in higher frequency than CD4+ T cells. Frequency of TNF-alpha and iNOS producing cells, as well as the frequency of CD68+ macrophages, did not differ between LCL and DL. Our findings reinforce the link between an efficient control of parasite and tissue damage, implicating higher frequency of IFN-gamma producing cells, as well as its possible counteraction by infiltrated B cells and hence possible humoral immune response in situ.