[show abstract][hide abstract] ABSTRACT: Leishmania (Viannia) shawi parasite was first characterized in 1989. Recently the protective effects of soluble leishmanial antigen (SLA) from L. (V.) shawi promastigotes were demonstrated using BALB/c mice, the susceptibility model for this parasite. In order to identify protective fractions, SLA was fractionated by reverse phase HPLC and five antigenic fractions were obtained.
F1 fraction was purified from L. (V.) shawi parasite extract by reverse phase HPLC. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 μg of F1. After 1 and 16 weeks of last immunization, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 2 months, those same mice were sacrificed and parasite burden, cellular and humoral immune responses were evaluated.
The F1 fraction induced a high degree of protection associated with an increase in IFN-γ, a decrease in IL-4, increased cell proliferation and activation of CD8+T lymphocytes. Long-term protection was acquired in F1-immunized mice, associated with increased CD4+ central memory T lymphocytes and activation of both CD4+ and CD8+ T cells. In addition, F1-immunized groups showed an increase in IgG2a levels.
The inductor capability of antigens to generate memory lymphocytes that can proliferate and secrete beneficial cytokines upon infection could be an important factor in the development of vaccine candidates against American Tegumentary Leishmaniasis.
[show abstract][hide abstract] ABSTRACT: There were 49 patients studied, coming from The Liver Unit at the "Hospital das Clinicas da Faculdade de Medicina da USP (N=41) and from "Prof. Dr. Angelita Habr-Gama and Joaquim Gama-Rodrigues Surgery Institute", SP (N=8); all of which had hepatic metastasis of colorectal adenocarcinoma, with no evidence of concurrent metastasis in any other organs and were submitted to surgical treatment, during the period of 1992 to 2002, with the aim of analyzing the immunoexpression of the p53, ki-67, p16 and molecular markers in order to relate the disease-free period with the prognosis.
The patient's clinical data were analyzed retrospectively for verification of information such as age, gender, size of the hepatic metastasis and/or the largest lesion, number of satellite nodules resected and compromised, margin of resection free from neoplasia.
The immunoexpression of the p53 was associated with the shortest period of life free from disease (p = 0.04). The proliferation marker ki-67 was not associated with the reduction of the disease-free interval and survival; the immunoexpression of the proliferation marker p16 was not associated with the reduction of disease-free period and survival, however, it was associated with hepatic metastasis synchronism. In patients who received postoperative systemic chemotherapy with 5-FU and leucovorin, the immunoexpression on the hepatic metastasis was not associated with a longer disease-free interval.
Molcular markers may be useful to evaluate hepatic metastasis of colorectal Adenocarcinoma.
[show abstract][hide abstract] ABSTRACT: A novel l-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 microM and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 microg/ml.
[show abstract][hide abstract] ABSTRACT: Insulin-like growth factor (IGF)-I constitutively present in the skin is one of the first growth factors that Leishmania parasites encounter after transmission to the vertebrate host. We have previously shown that IGF-I is a potent growth-promoting factor for Leishmania parasites. IGF-I binds specifically to a single-site putative receptor at the parasite membrane, triggering a cascade of phosphorylation reactions. In the present article we characterize the receptor for IGF-I on Leishmania (Leishmania) mexicana promastigotes. The receptor is a monomeric glycoprotein with a molecular mass of 65 kDa and is antigenically related to the alpha chain of human type 1 IGF-I receptor. Upon IGF-I stimulation the receptor undergoes autophosphorylation on tyrosine residues with activation of its signaling pathway. Activation of the IGF-I receptor also leads to phosphorylation of an 185-kDa molecule that is homologous to the substrate of the insulin receptor present in human cells, the insulin receptor substrate 1 (IRS-1).
[show abstract][hide abstract] ABSTRACT: The mechanisms that control chronic infection in vivo and the immunologic mechanisms involved in the pathogenesis of chagasic megacolon are not completely characterized. Although autoimmunity may play a role in the pathogenesis of Chagas' disease, recent studies, both in mice and in humans, suggest a positive association of tissue parasitism, inflammation, and severity of lesions. The aim of this study was to evaluate the role of inflammatory cells and the subclasses of lymphocytes involved in neuropathic lesions in the colon of patients who underwent resection for advanced megacolon.
Specimens from 23 patients were selected based on histopathologic analysis. Paraffin-embedded tissue blocks were sectioned and evaluated by immunohistochemistry for cluster of differentiation 3, cluster of differentiation 8, cluster of differentiation 20, and natural killer cell antibodies by an avidin-biotin peroxidase method.
Almost all myenteric plexuses were damaged, characterized by degenerative changes, necrosis of ganglion cells, and inflammatory response. Mild lymphocytic infiltration around degenerated and normal ganglion cells was observed in all cases. Collagen fibers and mononuclear cells surrounded some ganglion cells. Most of the inflammatory cells were lymphocytes, identified as cluster of differentiation 3-positive cells. Cluster of differentiation 8-positive lymphocytes were associated with degenerated ganglion cells. Natural killer cell antibodies were detected in a lower proportion of cells and were distributed between muscle layers or in proximity to the myenteric plexus. All these findings were also observed in the submucosal plexus. Cluster of differentiation 20-positive lymphocytes were not present in muscle layers or in the vicinity of either plexus.
Pathogenesis of the megacolon is based on a continuous process of ganglion cell damage with participation of T lymphocytes expressing cluster of differentiation 8 and natural killer cell membrane antigens. B lymphocytes do not take part in the chronic inflammatory reaction.
Diseases of the Colon & Rectum 08/2001; 44(7):993-8. · 3.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: While the control or progression of leishmaniasis depends on host immune responses, the initial inflammatory process represents a key event. This process involves the participation of several cytokines and growth factors induced during inflammation as well as factors already present at the site of infection such as insulin-like growth factor (IGF)-I. We have previously demonstrated a potential role for IGF-I in experimental cutaneous leishmaniasis based on the significant increase in lesion size seen in mice injected with Leishmania promastigotes preactivated with IGF-I. In the present study we show that preactivation of Leishmania (Leishmania) amazonensis promastigotes with IGF-I induces an increase in the actual number of parasites at the lesion site from seven days postinfection, in addition to a more intense inflammatory infiltrate. There was a higher numerical density of polymorphonuclear neutrophils from 3 to 24 h, and of mononuclear cells from 48 h of infection onward. A higher density of polymorphonuclear neutrophils and mononuclear cells harboring parasites was also observed. The most important observation, however, was that more parasites per cell were present, revealing that IGF-I appears to favour parasite growth within the macrophages. These results strongly suggest an important role for IGF-I in the development of cutaneous leishmaniasis, where it influences both the inflammatory process and parasite growth.
International Journal of Experimental Pathology 09/2000; 81(4):249-55. · 2.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sera from patients with American visceral leishmaniasis (AVL) were studied before and after treatment based on their antibody isotypes and subtypes. The study was comprised of 33 Brazilian patients with well-defined diagnosis of AVL and 39 clinically healthy individuals. Antileishmanial antibody isotypes and subtypes were observed in almost all patients, except IgA that was detected in about 63% of them. The sensitivity and specificity of the immunofluorescence assay in the detection of antibody isotypes (IgG and IgM) and subtypes (IgG1, IgG2, IgG3, and IgG4) were high with no statistical difference, ranging from 0.937 to 1.000 and from 0.954 to 1.000, respectively. All IgG antibodies and its subtypes had their levels reduced after treatment. However, the IgG4 had an early decay and its conversion to negative was significantly high in children. Moreover, the profile of IgG4 before treatment corresponded to a unimodal curve that shifted to a patent bimodal curve after treatment, indicative of therapeutic success. Thus, the IgG4 shows to be a suitable immunological marker for the assessment of chemotherapy in AVL patients or communities. Our findings suggest that IgG4 correlates with IL-4 that also decreases after therapy.
[show abstract][hide abstract] ABSTRACT: In order to study the role of natural killer (NK) cells during the early period of Leishmania infection, BALB/c mice were selectively and permanently depleted of NK cells by injection with 90Sr and subsequently infected with Leishmania (Leishmania) amazonensis (HSJD-1 strain). 90Sr is known to selectively deplete NK cells, leaving an intact T- and B-cell compartment and preserving the ability to produce both interferon alpha and IL-2. This method of depletion has advantages when compared with depletion using anti-NK cell monoclonal antibodies because the effect is permanent and neither activates complement nor provokes massive cell death. In the present study, after one month of treatment with 90Sr, the depletion of NK cells was shown by a more than ten-fold reduction in the cytotoxic activity of these cells: 2 x 10(6) spleen cells from NK-depleted animals were required to reach the same specific lysis of target cells effected by 0.15 x 10(6) spleen cells from normal control animals. The histopathology of the skin lesion at 7 days after Leishmania infection showed more parasites in the NK cell-depleted group. This observation further strengthens a direct role of NK cells during the early period of Leishmania infection.
Brazilian Journal of Medical and Biological Research 04/1999; 32(3):323-5. · 1.14 Impact Factor
[show abstract][hide abstract] ABSTRACT: Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania-host interplay in the early stage during the establishment of the infection and presumably also in the later stages.
Proceedings of the National Academy of Sciences 11/1998; 95(22):13211-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Protein phosphorylation controls major steps of proliferation and differentiation in eukaryotic cells. However there are few studies done in protozoa particularly when being triggered by external stimuli. In this paper we have examined the tyrosine- and serine/threonine-phosphorylated proteins in both promastigote and amastigote-like forms of Leishmania (Leishmania) mexicana stimulated with insulin-like growth factor (IGF)-I. Stimulation with IGF-I induces major tyrosine phosphorylation of a 185-kDa protein in promastigotes and 60- and 40-kDa proteins in amastigotes. Analysis of total phosphorylation revealed additional sets of phosphorylated proteins: a 110-kDa protein band in promastigotes and two other proteins of 120 and 95 kDa in the amastigote-like forms. To further analyze the IGF-I-mediated response we compared it with the phosphorylation pattern obtained with a known inducer of protein kinase C, phorbol myristate acetate. This analysis showed overlapping phosphorylation of most of the proteins but mainly of the 185- and 110-kDa proteins in the promastigotes and the 95-, 60- and 40-kDa proteins in the amastigote-like forms. We thus conclude that there are phosphorylation-dependent pathways in Leishmania parasites induced by IGF-I that are stage-specific.
Journal of Eukaryotic Microbiology 01/1998; 45(3):352-5. · 2.16 Impact Factor
[show abstract][hide abstract] ABSTRACT: Tecidual reaction at the inoculation site of L. (L.)chagasi promastigotes in hamsters depleted and non-depleted of complement was studied within 2, 6, 12, 24, 48 and 72 hours of infection. The inflammatory reaction was characterized by early predominance of polymorphonuclear cells (PMN) at 2, 6 and 12 hours of infection, mixed infiltrate of PMN and mononuclear cells (MN) at 24 hours, followed by predominance of MN at 48 and 72 hours after infection. The group depleted of complement showed a higher number of PMN at 2 hours and lower numbers of MN at 72 hours after infection (P < 0.0001). In the depleted group the phagocytosis by PMN was lower at 2 and 24 hours and by MN was lower at 24, 48 and 72 hours after infection. Electron microscopy showed extracellular intact and degenerated parasites, and lysed intracellular parasites, in PMN; and, rarely, preserved intracellular parasites in MN at 2, 6 and 12 hours after infection. The groups examined at 24, 48 and 72 hours of infection showed only cellular and parasite debris in mononuclear inflammatory cells. C3b deposits were detected by immunofluorescence in the interstitium and in the cytoplasm of inflammatory cells in non-depleted group at 2, 6 and 12 hours of infection. No immunoglobulin was detected in either group. Visceralization was detected 240 days after infection. The complement system has an important role in the inflammatory reaction and phagocytosis. The ultrastructural findings showed that the escape of the parasite probably occurs soon after inoculation.
International Journal of Experimental Pathology 03/1996; 77(1):15-24. · 2.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Histopathological and ultrastructural studies of 23 patients who died with clinical diagnosis of measles were carried out. In 12 cases viral nucleocapsids were searched by electron microscopy and detected in 100% of the cases in the lungs and in 50% of the cases in the central nervous system. They were mostly intranuclear. Histopathological changes associated to neurological alterations and the detection of virion are discussed in relation to acute and delayed clinical manifestations.
Revista do Instituto de Medicina Tropical de São Paulo 01/1995; 37(2):137-43. · 0.96 Impact Factor
[show abstract][hide abstract] ABSTRACT: Diffuse intralobular fibrosis of the liver is rare in cases of New World visceral leishmaniasis. A patient with this disease from a newly endemic area in the northernmost area of the Brazilian Amazon region was studied. Hypertrophy and hyperplasia of the phagocytic mononuclear cell system were observed, with parasitism and Disse's space fibrosis diffusely involving the liver. This description is based on a biopsy carried out after seven days of treatment with Glucantime. Another biopsy performed almost two years later showed no fibrosis, no Kupffer cell hypertrophy and hyperplasia, and no parasitism. The mechanism of fibrosis and regression is discussed.
The American journal of tropical medicine and hygiene 12/1993; 49(5):616-24. · 2.53 Impact Factor