Immunological mediators are thought to be responsible for many of the pathophysiologic changes observed in human glomerulonephritis. Previous studies have shown the importance of immunological mediators such as immune-complexes, complement and neutrophils. Recently, a significant role for the monocyte/macrophage system in glomerular injury has been emphasized by several investigators. The present study was designed to investigate the effect of macrophage supernatant factor(s) on glomerular cell metabolism in an in vitro tissue culture system. Human glomerular cells are grown in vitro and were characterized by their phagocytic capacity and their morphologic characteristics. Light microscopy, scanning and transmission electron microscopy, and observation of their growth pattern revealed two basic cell types: an epithelial and a mesangial cell. Epithelial cells were represented by large (100-200 μ in length) and small (50-70 μ in length) flat polygonal cells. The larger epithelial cell was primarily seen in the initial outgrowth and was not easily maintained in culture. Therefore they were not used for the metabolic experiments. On the other hand, the smaller epithelial cell was maintained in culture for an average of 5 to 8 passages. The mesangial cells were medium-sized (75-120 μ in length), of variable morphology but mostly spindle-shaped and grew in a characteristic storiform pattern. Both cell types kept their morphologic appearance with subculturing and cryopreservation. Cultured glomerular cells were treated with dialyzed macrophage supernatants obtained from mouse or human peripheral blood monocytes. Undiluted or diluted macrophage supernatants were over-layed on glomerular cells cultured in 96-well flat-bottom microtiter plates. DNA, RNA and protein synthesis were evaluated by incorporation of radio-labelled precursors. Macrophage supernatants failed to stimulate DNA synthesis in epithelial cells as measured by incorporation of 3HTdR. The same macrophage supernatant did, however, significantly increase the uptake of 3HUdR and a 14-C amino acid mixture, indicating an increase in RNA and protein synthesis. The results with DNA metabolism are consistent with in vivo observations in that epithelial cells are not regarded as the intrinsic proliferating cell in the hypercellularity observed in glomerular injury. The stimulation of RNA and protein synthesis may be related to the in vivo thickening of the glomerular basement membrane. In addition, it may be related to the production of molecules which may directly or indirectly affect endothelial or mesangial cells and/or affect the local charges in the glomeruli which are known to be important in permeselectivity of the capillary wall. In the case of mesangial cells, exposure to macrophage supernatants led to a significant increase in DNA snythesis as measured by the increase in uptake of 3HTdR. No stimulation was seen in RNA and protein synthesis as measured by the radioactive label technique. The increase in DNA synthesis correlates with in vivo observations of mesangial cell proliferation in glomerular injury. The factor(s) in the macrophage supernatant which affect the metabolism of glomerular cells in vitro is non-dialyzable and denatured by freezing and thawing. In addition, preliminary results indicate that the activity stimulating RNA and protein synthesis (epithelial cells) is insensitive to heat treatment while the one affecting DNA synthesis (mesangial cells) seems to be sensitive to heat treatment. Neither was shown to be species specific since both human and mouse macrophage supernatant induced the same changes in glomerular cell metabolism. The results of this investigation suggest that both cell types are selectively affected by a factor(s) present in the macrophage supernatant. It is likely that more than one factor is responsible for the metabolic changes observed. The possibility that this factor(s) may be identical with macrophage factors previously described in other cell systems cannot be ruled out at this time. These in vitro observations further support an active role for the monocyte/macrophage system in glomerular injury in experimental and clinical glomerulonephritis.

published_or_final_version / Psychology / Doctoral / Doctor of Philosophy

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Typescript (photocopy) / xiv, 203 leaves, [2] leaves of col. plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1983

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2007. / Also available online.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-03-02Bitstream added on 2014-06-13T18:09:20Z : No. of bitstreams: 1 ramos_mm_me_botfm.pdf: 505331 bytes, checksum: c19ab9911d5a9bf7f687fb2f2987b64d (MD5) / Ministério da Saúde / Fundação para o Desenvolvimento Médico e Hospitalar (Famesp) / Secretaria do Estado da Saúde de São Paulo / O fluxograma de investigação laboratorial da hepatite C, proposto pelo Ministério da Saúde, envolve a utilização de metodologias que pesquisam anticorpos anti-VHC como testes de triagem para a infecção, sendo que os testes moleculares vêm sendo utilizados como confirmatórios mediante sorologia positiva. Neste fluxo, algumas amostras podem apresentar, nos testes de triagem, resultados reagentes próximos ao limiar de reatividade e estes resultados muitas vezes não se confirmam quando se utilizam outras metodologias. Estes resultados falso-positivos podem gerar descarte de bolsas de sangue e conseqüências psicológicas negativas para doadores e pacientes. Assim, o objetivo deste estudo foi estabelecer valores de corte entre resultados reagentes e fracamente reagentes no diagnóstico laboratorial da hepatite C pelo Imunoensaio Quimioluminescente de Micropartículas (CMIA), e avaliar estes resultados com outras metodologias. Foram analisados 2535 resultados anti- VHC por CMIA para a seleção das amostras reagentes independente do valor da razão S/CO. Destas, apenas as amostras com a razão S/CO entre 1,11 e 6,99 por CMIA foram testadas por MEIA, ELISA e RT-PCR. Das 2535 amostras, 100 (3,95%) foram reagentes pela CMIA e destas, a menor freqüência foi observada em amostras com a razão S/CO entre 6,00 e 6,99. No mesmo sentido, a detecção do RNA do VHC foi crescente a partir da razão S/CO 7,00 sugerindo uma maior ocorrência de resultados falso-positivos em amostras com valor de S/CO inferior a 7,00. No período do estudo, 56 amostras apresentaram-se reagentes por CMIA com razão S/CO entre 1,11 e 6,99 e 49 (87,5%) delas foram reagentes por MEIA, das quais 25 (44,6%) também foram reagentes por ELISA. Apenas uma amostra apresentou RNA do VHC detectável por RT-PCR. Esses resultados sugerem que os resultados reagentes pelos métodos de triagem com valores próximos ao limiar de... / Hepatitis C laboratorial investigation, proposed by Ministry of Health, includes anti-HCV tests for screening and molecular methodologies for confirmation of the positive results. Some time the samples can present reagents results near to the cut off and, these results, a lot of times, are not confirmed by other methodologies. False positives results can lead blood units discard and negative psychological consequences for donors and patients. The goal of this study was establish a threshold among resulted reagents and weakly reagents in the hepatitis C laboratorial diagnosis by CMIA, and to evaluate these results with other methodologies. In this study were analyzed 2535 resulted anti-VHC by CMIA for the selection of the reagents samples independent of the S/CO obtained. The samples with S/CO between 1.11 and 6.99 by CMIA were tested by MEIA, ELISA and RT-PCR. From 2535 samples, 100 (3.95%) were reagents for CMIA and the lower frequency it was observed in samples with S/CO between 6.00 and 6.99. In the same sense, the HCV RNA detection was confirmed in samples with S/CO upper than 7.00; suggesting an occurrence of false-positive results in samples with S/CO lower to 7.00. In study period 56 samples were reagents by CMIA with S/CO between 1.11 and 6.99 and 49 (87.5%) were reagents by MEIA, which 25 (44.6%) were also reagents by ELISA. One sample presented HCV RNA detected by RTPCR. Those results suggest that the reagents results by the screening tests, with S/CO near to cut off should be analyzed with precaution and different of the reagents results with upper S/CO

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