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Orientador: Julieta Rodini Engracia de Moraes / Banca: Ana Maria Ferreira Roselino / Banca: Angela Cleusa de Fatima Banzatto de Carvalho / Banca: Carlos Noriyuki Kaneto / Banca: Gilson Pereira de Oliveira / Resumo: A leishmaniose é uma enfermidade provocada por protozoários do gênero Leishmania, que pode produzir manifestações cutâneas, mucocutâneas ou viscerais. Os objetivos deste ensaio foram os de identificar a espécie Leishmania (Leishmania) chagasi, utilizando o método PCR-RFLP em amostras de tecido de cães com leishmaniose visceral; seqüenciar o fragmento amplificado de DNA das amostras de linfonodos de diferentes animais, em busca de homologia e polimorfismos; comparar os métodos de imunoistoquímica e de PCR dos rins, na identificação da Leishmania e comparar as técnicas sorológicas em relação à PCR como auxílio diagnóstico da leishmaniose. Para tanto, foram utilizadas amostras de 48 cães com leishmaniose, diagnosticados por meio de testes IFI, ELISA e por PCR. Realizou-se PCR com amostras de linfonodo poplíteo, baço e rins, utilizando 'primers' específicos para o gênero Leishmania. A partir de amostras amplificadas, o DNA foi submetido à digestão enzimática (técnica PCR-RFLP) com as enzimas Hae III, Bsr I e Rsa I. Para o seqüenciamento de DNA, produtos de PCR foram amplificados com 'primer sense', precipitados e submetidos ao seqüenciamento automático. Com fragmentos histológicos renais, realizou-se a técnica imunoistoquímica utilizando anticorpo policlonal anti-Leishmania produzido em coelho. O método PCR-RFLP permitiu identificar a espécie Leishmania (Leishmania) chagasi em todas as amostras de DNA dos diferentes tecidos. No seqüenciamento de DNA, os fragmentos amplificados mostraram-se pertencentes às espécies causadoras de leishmaniose visceral, porém não foi possível diferenciá-las. A técnica de imunoistoquímica mostrou presença de formas amastigotas íntegras em meio ao infiltrado inflamatório intersticial em dois (4%) dos 48 animais avaliados, enquanto a PCR confirmou Leishmania sp em 77% dos cães. Conclui-se que a técnica... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Leishmaniasis is a disease caused by organisms belonging to the genus Leishmania. Clinical forms of leishmaniasis are found as being cutaneous, mucocutaneous or visceral. The objectives of the present study were to identify the Leishmania (Leishmania) chagasi species in tissue specimen from dogs presenting visceral disease diagnosed by PCR-RFLP assay; to determine the fragment sequence (120bp) from lymph node samples from different animals searching for homologies and polymorphism; and to compare immunohistochemistry method to PCR assay with renal tissue and Leishmania local identification. Forty eight samples from dogs clinically diagnosed positive to leishmaniasis by IFAT, ELISA and PCR assays were used in this study. The PCR were performed with samples of popliteo lymph node, spleen and kidneys, and specific oligonucleotides to genus Leishmania. PCR products were digested with enzymes Hae III, Bsr I and Rsa I. The nucleotide sequences were determined automatically. For immunohistochemical purposes the sections of kidneys and anti-Leishmania polyclonal antibody were used. PCR-RFLP assay allowed us to identify the Leishmania (Leishmania) chagasi specie in all DNA samples from different tissues samples. DNA sequencing revealed products amplified belonging to specie responsible to cause visceral leishmaniasis, but it was not possible to distinguish the species. The immunohistochemical study revealed the presence of amastigotes organisms on intersticial inflammatory infiltrate from two dogs (4%), while the PCR assay detected the Leishmania sp in 37 dogs (77%). Based on the PCR-RFLP results, we concluded that it characterized as being Leishmania (Leishmania) chagasi species, etiologic agents of visceral leishmaniasis, in this group of animals; DNA sequence presented homology of 55 bp among all Leishmania spp studied. Additionally, the PCR performed... (Complete abstract click electronic access below) / Doutor

Thesis (M.S.)--University of Pretoria, 2005. / Title from PDF t.p. (viewed on Apr. 14, 2007). Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 63-70).

published_or_final_version / abstract / toc / Diagnostic Radiology / Master / Master of Research in Medicine

Kidney transplants from both living-related (LRD) and living unrelated (LURD) donors have superior function and survival than transplants from cadaver donors. This may be unsurprising as kidneys from living donors are procured under optimal conditions, from healthy donors with minimal ischaemia times. In contrast, cadaver kidneys are obtained from traumatised donors and may experience extended periods of cold ischaemic storage before transplantation. An immunohistochemical analysis has been performed on biopsies obtained before, and immediately after transplantation, to investigate the potential causes of early inflammatory events associated with cadaver renal transplantation that may influence subsequent graft outcome. An immunohistochemical analysis of biopsies obtained before transplantation demonstrated upregulated expression of endothelial E-selectin and proximal tubular expression of ICAM-1, VCAM-1 and HLA Class II antigens in cadaver donor kidneys. Analysis of donor parameters demonstrated that traumatic physiological events experienced in intensive care around the time of brain death were significantly associated with the induction of proinflammatory antigens. Antigen induction in cadaver donor kidneys before transplantation was significantly associated with early acute rejection. Furthermore, in cadaveric kidneys with long cold ischaemia times, glomerular neutrophil infiltration and deposition of activated platelets expressing P-selectin on intertubular capillaries were detected following reperfusion, in association with impaired short and long term graft function. Expression of inflammatory mediators were absent in all LRD renal allografts before and after reperfusion. A clinical trial was performed to determine whether ischaemia/reperfusion injury may be ameliorated by reflushing cadaver kidneys after cold storage to remove harmful products that may have accumulated in the vessel lumen. Reflushing did not prevent the inflammatory events observed after reperfusion or improve graft function. Therefore, a novel, oxygen free radical scavenger (lec-SOD) was obtained to assess its potential efficacy in preventing ischaemia/reperfusion injury. Lec-SOD bound with high affinity to macro- and microvascular endothelial cells under cold hypoxic conditions following incorporation into Marshall's preservation solution, significantly inhibiting cold hypoxia induced cell death, adhesion molecule induction and neutrophil adhesion. Furthermore, preservation of kidneys with lec- SOD for 18 hr in an experimental model of chronic renal allograft rejection, significantly attenuated neutrophil infiltration and MHC Class I induction day 1 post-transplant, with improved long term renal function. The results presented in this Thesis demonstrate that donor factors and cold ischaemia/ reperfusion injury elicit an early inflammatory response that may influence graft outcome of cadaver kidneys. Refinements in donor management and organ preservation may limit the deleterious effects of ischaemia/reperfusion injury in cadaver renal allografts, increasing graft survival to that observed in living donor transplantation.