Elucidating in vitro and in vivo phenotypic behaviour of L. infantum/L. major natural hybrids.

The clinical manifestation and course of Leishmania infections depend on factors such as species, virulence and host-immunity. Although trypanosomatids are considered to have clonal propagation, genetic hybridization has produced successful natural hybrid lineages. Hybrids displaying strong selective advantages may have an impact on pathogenesis and the eco-epidemiology of leishmaniasis. Thus, characterization of phenotypic properties of Leishmania hybrids could bring significant insight into the biology, infectivity, pathogenicity and transmission dynamics of these atypical strains. The present study focuses on phenotypic features and survival capacity of Leishmania infantum/Leishmania major hybrid isolates as compared with representative putative parental species, L. infantum and L. major. In vitro assays (growth kinetics, susceptibility to different conditions) and in vivo infection (parasite detection and histopathological alterations) showed that hybrids present higher growth capacity and decreased susceptibility to reactive oxygen species. Furthermore, evaluation of infected spleen tissue suggests that hybrids induce a stronger immune reaction than their putative parents, leading to the development of white pulp hyperplasia in B-lymphocyte compartments. Overall, these hybrids have shown high plasticity in terms of their general behaviour within the different phenotypic parameters, suggesting that they might have acquired genetic features conferring different mechanisms to evade host cells.

The scavenger receptor MARCO is involved in Leishmania major infection by CBA/J macrophages.

CBA/J mice are resistant to Leishmania major infection but are permissive to L. amazonensis infection. In addition, CBA/J macrophages control L. major but not L. amazonensis infection in vitro. Phagocytosis by macrophages is known to determine the outcome of Leishmania infection. Pattern recognition receptors (PRR) adorning antigen presenting cell surfaces are known to coordinate the link between innate and adaptive immunity. The macrophage receptor with collagenous structure (MARCO) is a PRR that is preferably expressed by macrophages and is capable of binding Gram-positive and Gram-negative bacteria. No research on the role of MARCO in Leishmania-macrophage interactions has been reported. Here, we demonstrate, for the first time, that MARCO expression by CBA/J macrophages is increased in response to both in vitro and in vivo L. major infections, but not to L. amazonensis infection. In addition, a specific anti-MARCO monoclonal antibody reduced L. major infection of macrophages by 30%-40% in vitro. The draining lymph nodes of anti-MARCO-treated mice displayed a reduced presence of immunolabelled parasite and parasite antigens, as well as a reduced inflammatory response. These results support the hypothesis that MARCO has a role in macrophage infection by L. major in vitro as well as in vivo.

Inflammation and structural changes of splenic lymphoid tissue in visceral leishmaniasis: a study on naturally infected dogs.

The aim of this study was to identify splenic immuno-inflammatory patterns associated with natural infection by Leishmania chagasi. Spleen samples were obtained from 72 stray dogs from an endemic area of visceral leishmaniasis. The animals were grouped into four categories as follows: (i) potentially resistant to visceral leishmaniasis, with a positive leishmanin skin test result, and negative splenic culture for Leishmania parasites (ii) potentially susceptible to visceral leishmaniasis, with a negative leishmanin skin test and positive splenic culture for Leishmania (iii) infected with undefined susceptibility status, with a positive leishmanin skin test and positive splenic culture for Leishmania, and (iv) noninfected, with a negative leishmanin skin test, negative splenic culture for Leishmania, and negative serology for anti-Leishmania antibodies. Histopathological analyses showed that there was a higher frequency of perisplenitis (18/25, P < 0.0001), granuloma (7/25, P = 0.0102), structural disorganization (14/25, P < 0.0001), and atrophy of the lymphoid follicles (20/25, P = 0.0036) and of the marginal zone (15/25, P = 0.0025) in the potentially susceptible group than in the other groups. The data presented here show changes in the white pulp of the spleen that are associated with naturally acquired visceral leishmaniasis.

Comparative study of hepatitis C virus genotypes 1 and 3 in Salvador, Bahia Brazil.

UNLABELLED: Hepatitis C virus displays a high degree of genetic mutation, with considerable heterogeneity, motivating clinical and biomolecular investigations. It is necessary to understand the effects of genotypes on the course of the disease, as well as their peculiarities at the regional level. OBJECTIVE: The study objective was to compare epidemiological, biochemical and histological aspects of hepatitis C virus genotypes 1 and 3 in Salvador, Bahia. STUDY DESIGN: Data were collected retrospectively from outpatient medical records. MATERIALS AND METHODS: 127 patients with positive anti-HCV results were selected, based on detectable RNA-HCV (RT-PCR) of genotypes 1a, 1b and 3a. RESULTS: Thirty-nine (30.7%) individuals were infected by subtype 1a, 45 (35.4%) by subtype 1b and 43 (33.9%) by subtype 3a. Most (73.2%) patients were male, with an average age of 47.8 years. The subtype 1b-infected patients had the highest average age (512 +/-11.17; P=0.09). The use of illicit injected drugs was more frequent among subtype 3a infected individuals when compared with genotype 1 (6/43; 14% and 3/84; 3.6%, respectively; P=0,06). No significant differences were found for other epidemiological characteristics. Average values for GT, AST, ALT and ferritin did not differ between the groups (64, 78, 109, 276, respectively). Thyroid dysfunction occurred in 7/30 (23.3%) of those infected by genotype 3 (P=0.05). Cryoglobulinemia was also more frequent in this group (5/13, 38%, P=0.02). Most patients presented limited necro-inflammatory activity, stages 2 and 3 by the METAVIR Classification. In some cases, dissociation was noticed between inflammatory activity and fibrosis. No significant differences were found in the histopathological findings of the various genotypes. Younger patients had a significantly smaller degree of necrosis in stomatocytosis (P=0.032) and fibrosis (P=0.012). Intense parenchymatous activity and lymphoid follicles were more frequent among alcohol consumers (P=0.06 and P=0.04, respectively). CONCLUSIONS: In Bahia, genotype 3 dissemination seems to be associated with illicit drug use. The disease evolution depends on a function of complex interactions between virus and host. Age and alcohol consumption stand out as important variables in the development of cirrhosis.

In vivo lymphocyte activation and apoptosis by lectins of the Diocleinae subtribe.

This paper reports the overall effects of three lectins, extracted from Canavalia brasiliensis, Dioclea violacea, and D. grandiflora, on BALB/c mice popliteal draining lymph nodes. These lectins have presented high stimulatory capacity on lymph node T cells. Additionally, they were able to induce apoptosis and inflammation (frequently associated with high endothelial venule necrosis). The data presented here suggest that the Diocleinae lectins studied can stimulate in vivo T cell activation and apoptosis, as well as present important side effects.

1,8-cineole protects against liver failure in an in-vivo murine model of endotoxemic shock.

The effects of 1,8-cineole on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced shock model of liver injury was investigated in mice. The co-administration of GalN (700 mg kg(-1), i.p.) and LPS (5 microg kg(-1), i.p.) greatly elevated serum concentrations of tumour necrosis factor-alpha (TNF-alpha), alanine aminotransferase and aspartate aminotransferase, and induced massive hepatic necrosis and lethality in 100% of control mice. Pretreatment with 1,8-cineole (400 mg kg(-1), p.o.) and dexamethasone (1 mg kg(-1), s.c.), 60 min before GalN/LPS, offered complete protection (100%) against the lethal shock and acute elevation in serum TNF-alpha and serum transaminases. Hepatic necrosis induced by GalN/LPS was also greatly reduced by both 1,8-cineole and dexamethasone treatment. The results indicate that 1,8-cineole protects mice against GalN/LPS-induced liver injury through the inhibition of TNF-alpha production, and suggest that 1,8-cineole may be a promising agent to combat septic-shock-associated pathologies.

Different Leishmania species determine distinct profiles of immune and histopathological responses in CBA mice.

Most experimental studies on leishmaniasis compare two different inbred strains of mice that are resistant or susceptible to one species of Leishmania. In the present study we characterized some cytokines and nitric oxide production as well as histological changes related to resistance and susceptibility in isogenic CBA mice infected with Leishmania major or Leishmania amazonensis. CBA mice are capable of controlling infection with L. major, but they succumb to infection with L. amazonensis. Cells from susceptible L. amazonensis-infected CBA mice produced interleukin (IL)-4 and IL-10 but no interferon (IFN)-gamma. On the other hand, resistant L. major-infected CBA mice produced IFN-gamma and IL-10, but IL-4 was detected only in the first week of infection. Histopathological studies showed patterns of tissue responses at the site of the infection and in the draining lymph nodes that correlated with resistance or susceptibility. Resistant mice showed a mixed inflammatory cell infiltration and granulomas in the lesions, whereas in susceptible mice only heavily parasitized macrophages were seen. Our results indicate an important role of the parasite species in determining the pattern of immune response. L. amazonensis induces a Th2-type immune response, whereas L. major induces a Th1-type response. These factors must be identified and taken into account in the strategies for the development of vaccines against leishmaniasis. The model presented here will be useful for the study of such factors.

Indomethacin treatment slows disease progression and enhances a Th1 response in susceptible BALB/c mice infected with Leishmania major.

Prostaglandins of the E series inhibit the development of Th1 responses. When infected with Leishmania major, BALB/c mice fail to develop a Th1 response, but instead mount a Th2 response and die of the disease. Therefore, we treated L. major-infected BALB/c mice with indomethacin, which inhibits prostaglandin production. Indomethacin lessened disease severity (parasite burden and pathology), and promoted a Th1 response, but the mice still succumbed to infection. The explanation for these observations may be two-fold: (1) the beneficial effects of indomethacin were predominantly observed later in infection (beyond two weeks), a time at which indomethacin was unable to sufficiently block the development of a Th2 response; (2) indomethacin was unable to induce a Th1 response in BALB/c mice that was of the same magnitude as the Th1 response observed in C57BL/6 mice infected with L. major.

Biological behavior of Leishmania amazonensis isolated from humans with cutaneous, mucosal, or visceral leishmaniasis in BALB/C mice.

Leishmania amazonensis causes a wide spectrum of disease in humans. In this study, we evaluated BALB/c mice infected with five strains of L. amazonensis isolated from patients with either cutaneous, mucosal, or visceral leishmaniasis. Mice infected with cutaneous and mucosal isolates developed ulcerating footpad lesions with parasite-loaded macrophages and extensive tissue destruction. Skin metastases, early dissemination of parasites to the spleen, and high anti-Leishmania antibody levels were also noted. Mice infected with L. amazonensis strains isolated from patients with visceral disease had a controlled infection, with small footpad lesions with mononuclear cell infiltration, few infected macrophages, and granuloma formation. They had no skin metastases, delayed dissemination of the parasite to the spleen, lower levels of IgG and higher levels of IgG2a against L. amazonensis. These findings demonstrate an unexpected resistance of BALB/c mice to the infection with L. amazonensis isolated from patients with visceral leishmaniasis. This resistance seems to be due to differences in these parasites that may be related to the altered course of the disease in humans and in isogenic BALB/c mice.

Development of a safe live Leishmania vaccine line by gene replacement.

Vaccination with live Leishmania major has been shown to yield effective immunization in humans; however, this has been discontinued because of problems associated with virulence of the available vaccine lines. To circumvent this, we tested the ability of a dhfr-ts- null mutant of L. major, obtained by gene targeting, to infect and then to vaccinate mice against challenge with virulent L. major. Survival and replication of dhfr-ts- in macrophages in vitro were dependent upon thymidine, with parasites differentiating into amastigotes prior to destruction. dhfr-ts- parasites persisted in BALB/c mice for up to 2 months, declining with a half-life of 2-3 days. Nonetheless, dhfr-ts- was incapable of causing disease in both susceptible and immunodeficient (nu/nu) BALB/c mice. Animal infectivity could be partially restored by thymidine supplementation. When inoculated by the i.v., s.c., or i.m. routes into mice, dhfr-ts- could elicit substantial resistance to a subsequent challenge with virulent L. major. Thus, Leishmania bearing auxotrophic gene knockouts can be safe and induce protective immunity. Potentially, dhfr-ts- could be used as a platform for delivery of immunogens relevant to other diseases.

Ki-1 large cell lymphoma with regressing lesions in a child.

An 8-year-old boy was seen with a cutaneous Ki-1 anaplastic, large cell lymphoma with multiple lesions. Some of the lesions showed spontaneous regression. During more than seven years of disease no systemic involvement was observed, but recurrent, self-healing lesions did appear. Histopathologic examination of five lesions revealed a variety of findings, from an inflammatory infiltrate to a highly anaplastic pattern. The neoplastic cells expressed Ki-1 and leukocyte common antigens. Ultrastructurally, those cells showed ruffled indentations. The differential diagnosis includes microvillous malignant lymphoma. The patient has had a four-year follow-up without relapses.

Morphological aspects of early and late collagen degradation in granulation tissue.

A sequential histologic, ultrastructural and immuno pathologic study was carried out in the Selye's inflammatory pouch model to observe extracellular matrix and cellular changes during granulation tissue formation. Besides changes involving different components of the connective tissue, it was observed that collagen resorption occurred under a biphasic process. At an early phase (3rd to 15th day), in which exudative inflammatory changes predominated, signs of collagen synthesis and degradation were seen simultaneously. Extracellular breakdown and internalization of collagen fragments within fibroblasts and myofibroblasts were observed. Later on (30th to 60th day), changes affecting collagen had a different ultrastructural appearance. Collagen fragmentation, focal "lytic" and "electron dense" changes occurred in the extracellular space specially at the periphery of fibroblasts, myofibroblasts and smooth muscle cells. Collagen degradation, thus seems to be a continuous process in granulation tissue, occurring with different morphologies at different times.

Primary pregnancy in the liver. A case report.

A case of intra-hepatic pregnancy is reported. The patient was 32 years old and presented with an acute intra-abdominal hemorrhage. At surgery a spongy 3 x 2 x 1.5 cm mass was removed from the right liver lobe. Microscopically, well developed chorionic villi appeared invading the liver tissue. The patient had a relatively uneventful recovery.

Distinct ultrastructural aspects in different biopsies of a single patient with diffuse cutaneous leishmaniasis.

The authors investigated the relation between parasites and host-cells in active and regressed lesions of a patient with diffuse cutaneous leishmaniasis, evaluating the frequency of different cell types, and the location and integrity of amastigotes. No correlation was found between parasite integrity and size of parasitophorous vacuoles. They observed ultrastructural findings characterizing a cell mediated immune response: macrophages lysis, parasitic destruction inside macrophages, close contact between parasitized macrophages and lymphocytes and between parasites and lymphocytes, lymphocytic infiltration and fibrosis. They suggest that in DCL there is a limited cellular immune response, although insufficient to control infection.

Histopathologic changes induced by vaccination in experimental cutaneous leishmaniasis of BALB/c mice.

Highly susceptible BALB/c mice became partially resistant to Leishmania mexicana amazonensis infection after intravenous immunization with solubilized homologous promastigote antigen. Immunized BALB/c mice exhibited mixed mononuclear cell reactions, with granulomatous inflammation, collagen deposition, and fibrinoid necrosis at the site of infection. In contrast, naive animals displayed a monomorphic picture composed of largely vacuolated and parasitized macrophages with areas of coagulative necrosis. Electron microscopy revealed an increased number of eosinophils, sometimes in close contact with parasitized macrophages, in immunized animals. These findings illustrate that histologic changes reflect host immune status in cutaneous leishmaniasis, and that susceptibility of BALB/c mice to L m amazonensis, although dependent on genetic background, can be artificially modified.

Human extrahepatic biliary atresia: portal connective tissue activation related to ductular proliferation.

Surgical bile flow restoration in extrahepatic biliary atresia (EHBA) does not prevent the development of ongoing hepatic fibrosis and cirrhosis. Portal connective matrix was studied on liver biopsies obtained from seven children submitted to portoenterostomy. Electron microscopy and immunohistochemical techniques (using specific antibodies directed against collagen isotypes and associated glycoproteins) were performed. The study of extracellular and cellular components of connective matrix demonstrated the existence of two distinct areas according to their situation with regard to ductular proliferation: loose connective matrix--mainly composed of fibronectin, type III collagen, type IV collagen and laminin--associated with microvessels and myofibroblasts proliferation characterized periportal zones adjacent to bile ductules; in areas distant from ductular proliferation, connective matrix appeared dense, composed of type I and type III collagen associated with fibroblasts. The connective matrix pattern observed in periductular areas can be compared to that described in cicatricial and hypertrophic processes where the myofibroblastic cell population is known to play an important role in fibrosis development. Although the connective matrix activation process remains unclear in EHBA, it may be suggested that activation of a connective tissue cellular clone might be responsible for this portal fibromatosis.

[Diffuse cutaneous leishmaniasis. Anatomo-pathologic aspects]. / Leishmaniose tugementar difusa. Aspectos anátomo-patológicos.

We present a study of 21 biopsies from three patients with diffuse cutaneous leishmaniasis with periods of observation of up to 9 years. The biopsies were taken before, during, and after specific treatment. We observed that a lymphocytic infiltrate may be present in the lesions and that the intensity of the infiltrate is variable from lesion o lesion, even among lesions biopsied at the same time. Tuberculoid granulomatous reactions were not found in the patients following treatment. We conclude that the histologic features of importance in the classification of cutaneous leishmaniasis are: the tuberculoid granulomatous reaction and the intensity of parasitism.

Pneumonitis in congenital Chagas' disease. A study of ten cases.

Ten cases of pneumonitis in congenital Chagas' disease are described. Amastigotes were found in the lungs in seven cases, and in two of these cases parasitized cells were seen in the alveolar lumen. In five cases parasites were found both in the lungs and in the amniotic epithelium of the extraplacental membranes and umbilical cord. The authors conclude that infection of the amniotic epithelium originated in the lungs and was carried by the amniotic fluid, and emphasize the epidemiological importance of infection of the amniotic fluid as a probable means of transmission of Chagas' disease among professionals working in the area of obstetrics.