6 research outputs found
Genetic diversity of Leishmania amazonensis strains isolated in northeastern Brazil as revealed by DNA sequencing, PCR-based analyses and molecular karyotyping
Abstract\ud
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Background\ud
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Leishmania (Leishmania) amazonensis infection in man results in a clinical spectrum of disease manifestations ranging from cutaneous to mucosal or visceral involvement. In the present study, we have investigated the genetic variability of 18 L. amazonensis strains isolated in northeastern Brazil from patients with different clinical manifestations of leishmaniasis. Parasite DNA was analyzed by sequencing of the ITS flanking the 5.8 S subunit of the ribosomal RNA genes, by RAPD and SSR-PCR and by PFGE followed by hybridization with gene-specific probes.\ud
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Results\ud
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ITS sequencing and PCR-based methods revealed genetic heterogeneity among the L. amazonensis isolates examined and molecular karyotyping also showed variation in the chromosome size of different isolates. Unrooted genetic trees separated strains into different groups.\ud
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Conclusion\ud
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These results indicate that L. amazonensis strains isolated from leishmaniasis patients from northeastern Brazil are genetically diverse, however, no correlation between genetic polymorphism and phenotype were found.We thank Lucile FloeterWinter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii).We thank Lucile Floeter-Winter for critical reading of the manuscript and Artur T.L. de Queiroz for initial help with phylogenetic analysis. This work is supported by grants from CNPq, FAPESB and PAPES/FIOCRUZ. J.P.C. de Oliveira was supported by a CNPq fellowship; C.I.O. and F.M.C.F were supported by a FAPESB fellowship. AAC, AB, and CIO are senior investigators from CNPq. AB is a senior investigator for Instituto de Investigação em Imunologia (iii)
Keratinocytes Determine Th1 Immunity during Early Experimental Leishmaniasis
Experimental leishmaniasis is an excellent model system for analyzing Th1/Th2 differentiation. Resistance to Leishmania (L.) major depends on the development of a L. major specific Th1 response, while Th2 differentiation results in susceptibility. There is growing evidence that the microenvironment of the early affected tissue delivers the initial triggers for Th-cell differentiation. To analyze this we studied differential gene expression in infected skin of resistant and susceptible mice 16h after parasite inoculation. Employing microarray technology, bioinformatics, laser-microdissection and in-situ-hybridization we found that the epidermis was the major source of immunomodulatory mediators. This epidermal gene induction was significantly stronger in resistant mice especially for several genes known to promote Th1 differentiation (IL-12, IL-1β, osteopontin, IL-4) and for IL-6. Expression of these cytokines was temporally restricted to the crucial time of Th1/2 differentiation. Moreover, we revealed a stronger epidermal up-regulation of IL-6 in the epidermis of resistant mice. Accordingly, early local neutralization of IL-4 in resistant mice resulted in a Th2 switch and mice with a selective IL-6 deficiency in non-hematopoietic cells showed a Th2 switch and dramatic deterioration of disease. Thus, our data indicate for the first time that epidermal cytokine expression is a decisive factor in the generation of protective Th1 immunity and contributes to the outcome of infection with this important human pathogen
Utilization of human papillomavirus testing for cervical cancer prevention in a university hospital
Location of the β-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022
During the growth of Kluyveromyces marxianus var. marxianus ATCC 10022 on lactose, peaks of glucose, but not β-galactosidase activity, were detected in culture medium. Harvested and washed whole cells produced glucose and galactose from lactose, or ortho-nitro-phenol from the chromogenic substrate ortho-nitro-phenyl-β-D-galactopyranoside (ONPG), indicating that β-galactosidase is physically associated with cells. ONPG hydrolysis by whole cells presented a monophasic kinetics (Km 36.6 mM) in lactose exponential growth phase cells, but a biphasic kinetics (Km 0.2 and 36.6 mM) in stationary growth phase cells. Permeabilization with digitonin or disruption of cells from both growth phases led to monosite ONPG hydrolysis (Km 2.2 to 2.5 mM), indicating that β-galactosidase is not located in the periplasm. In addition, the energy inhibitors fluoride or arsenate, as well as the uncouplercarbonyl cyanide m-chlorophenylhydrazone (CCCP) prevented ONPG hydrolysis by whole cells. These findings indicate that energy coupled transmembrane transport is the rate-limiting step for intracellular ONPG cleavage. The taxonomic and physiologic implications of the exclusive intracellular location of β-galactosidase of K. marxianus var. marxianus ATCC 10022 are discussed. © 1996 Kluwer Academic Publishers
<i>Toll-like</i> receptors 2, 4, and 9 expressions over the entire clinical and immunopathological spectrum of American cutaneous leishmaniasis due to <i>Leishmania</i> <i>(V.) braziliensis</i> and <i>Leishmania (L.) amazonensis</i>
<div><p><i>Leishmania (V</i>.<i>) braziliensis</i> and <i>Leishmania(L</i>.<i>) amazonensis</i> are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCL<sup>DTH+/++</sup>), borderline disseminated cutaneous leishmaniasis (BDCL<sup>DTH±</sup>), anergic diffuse cutaneous leishmaniasis (ADCL<sup>DTH-</sup>), and mucosal leishmaniasis (ML<sup>DTH++++</sup>). It has recently been demonstrated, however, that while <i>L</i>. (<i>V</i>.) <i>braziliensis</i> shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), <i>L</i>. (<i>L</i>.) <i>amazonensis</i> drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of <i>Toll-like</i> receptors (<i>TLRs</i>) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused by<i>L</i>. (<i>L</i>.) <i>amazonensis</i>; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused by<i>L</i>. (<i>V</i>.) <i>braziliensis</i>. CD4<sup>+</sup> T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8<sup>+</sup> T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α<sup>+</sup>showed increased expression from ADCL to ML, while IL-10<sup>+</sup>and TGF-β<sup>+</sup> showed increased expression in the opposite direction, from ML to ADCL. With regards to <i>TLR</i>2, 4, and 9 expressions, strong interactions of <i>TLR</i>2 and 4 with clinical forms associated with <i>L</i>. (<i>V</i>.) <i>braziliensis</i> were observed, while <i>TLR</i>9, in contrast, showed a strong interaction with clinical forms linked to <i>L</i>. (<i>L</i>.) <i>amazonensis</i>. These findings strongly suggest the ability of <i>L</i>. (<i>V</i>.) <i>braziliensis</i> and <i>L</i>. (<i>L</i>.) <i>amazonensis</i> to interact with those <i>TLRs</i> to promote a dichotomous T-cell immune response in ACL.</p></div