Th.--Pharm.--Paris 5, 1985. N°: 112.

Texte remanié de: Thèse de doctorat--Médecine--Paris--Faculté de médecine, [1846]. Titre de soutenance : L'évolution du foetus. / Thèse présentée dans le cadre d'un : "Concours pour une chaire d'anatomie"

During normal pregnancy, the fetus is not rejected by the maternal immune system despite bearing paternal MHC antigen. It has been proposed that this is due to the local production of immunoregulatory factors by the maternal decidua. This study was designed to investigate immunosuppressive factors derived from early human decidua. In the first part of the study, assays were standardised in order to monitor the immunoregulatory activity of factors derived from human decidua. These included mitogen induced lymphocyte proliferation, mixed lymphocyte culture, and the proliferation of human monocytic and T lymphoid cell lines. Using these assays, two fractions from the FPLC separation of the 10-100 KDa crude decidual supernatant with immunosuppressive activity have been identified and partially characterised. One contained molecules of 183 KDa under non-reducing SDS-PAGE analysis, and 70 and 29 KDa under reducing conditions (large molecular weight fraction; LMWF). Another had molecules of 7-14 KDa under non-reducing conditions (small molecular weight fraction; SMWF). Both of them inhibited mitogen-induced lymphoproliferation and the mixed lymphocyte reaction. In addition they inhibited the growth of a monocytic cell line (Mono Mac 6), but not that of a T lymphoblastic cell line (Jurkat E6.1). The suppression of cellular growth by the LMWF, but not the SMWF, was due to the arrest of the cells in the G0/G1 phase of the cell cycle. Neither fraction affected the expression of MHC class II on Mono Mac 6 cells. The decidua-derived suppressive factors showed some similarity to human transforming growth factor type beta (hTGFbeta), but their activity could not be eliminated by neutralising antibodies to hTGFbeta1 and (32. In contrast to the decidua factors, hTGFbeta1 was able to significantly suppress the expression of MHC class II molecules. In addition, acidification of LMWF decreased their suppressive activity, whereas others have shown that similar treatment of TGFbeta isolated from decidua enhanced its activity. Although some similarity to human decidua-associated protein hDP200 could be demonstrated with LMWF, lgG-like molecules could not be demonstrated in the latter. A low molecular weight protein with immunosuppressive activity-PP14- has been demonstrated in human decidua. Despite similarity, the SMWF clearly showed a distinct M.w distribution pattern to PP14 when analysed by Western blot. This suggests that SMWF was not PP14. In the final part of the study, the supernatants of decidual mononuclear cell cultures (non-fractionated or fractionated) were used to investigate the cells which may produce the factors. The preliminary results suggest that non-leukocytic decidua cells are unlikely to be responsible for the activity. Either T lymphocytes or NK cells are the more likely source, as the suppressive activity was largely increased in populations enriched for these cells, and concanavalin A enhanced their suppressive activity. However, the precise cellular source of these factors needs to be elucidated further.

Inductively coupled plasma mass spectrometry (ICP-MS), a comparatively new multi-element analytical technique was employed for elemental determination in human hair, placenta and brain samples. The performance criteria of ICP-MS such as precision, accuracy and detection limits were assessed. For most elements sub ng ml-1 limits of detection were achieved. Amino acid analysis of human foetal brain extracts was performed using reverse phase high performance liquid chromatography. Prior to separation, samples were derivatised using o-phthaldialdehyde derivatising reagent. Precision and limits of detection were assessed. For most amino acids detection limits of sub nmoles ml-1 were achieved. Hair is commonly used as a diagnostic and monitoring tool to observe the effects of mineral supplementation on an individuals bodily status, especially prior and after pregnancy. Some of the factors influencing the elemental content of hair were investigated. Analysing scalp hair of thirteen female individuals showed that longitudinal variations exist in elemental composition of human scalp hair. Radial and longitudinal diffusion of Co, Zn and Cd were investigated in scalp hair, using tresses of "virgin" hair. Both radial and longitudinal diffusions occurred for all three elements. Analysis of scalp hair of thirty-seven females and twenty male subjects from United Kingdom showed that there were significant differences in concentrations of Mg, K, Ca, Mn and Fe between males and females. Effects of six months mineral supplementation (18.6 mg Mg, 31.0 mg Ca, 1.2 mg Cr, 4.0 mg Mn and 7.5 mg Zn daily) on elemental content of hair was investigated in six female subjects and no significant changes were observed in the levels of supplemented elements (Mg, Ca, Cr, Mn and Zn). For elemental analysis of placenta, the importance of defining the location of the sampling site was demonstrated by investigating levels of Mg, Ca, Mn, Fe, Cu, Zn and Pb in different regions of a placental disc. Significant variations were found in the elemental content of the various regions. The peripheral region of the placental discs from fifty black females from Soweto, South Africa, were analysed for their elemental content in relation to birthweight and gestational age of their neonates. Concentrations of Mg, Al, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Sr, Mo, Cd and Pb were determined. Positive correlation was found for Mg, K, Fe, Cu and As with both medical parameters and Zn with gestational age. Lower concentrations of Mg (P < 0.10), Cu (P < 0.05), Zn (P < 0.10) and As (P < 0.001) were found in low birthweight cases compared to high birthweight groups. Comparison with an English population showed that significant differences existed in the placental elemental levels of the two communities. The contributing factors are fully discussed. Analysis of Mg, P, Ca, Fe, Cu, Zn, Rb, Sr, Mo and Pb in foetal brain tissues of eleven stillbirths and ten socially terminated neonates showed that stillbirth brains contained significantly (P < 0.001) higher levels of Pb compared to the controls. Analysis of four different regions of the brains; hippocampus, cerebrum, cerebellum and basal ganglia, also showed higher concentrations of Pb in all the four regions in comparison with the controls. Most elements showed significant differences in their regional concentrations within each study group and the patterns were different in stillbirths compared to the social terminations. However, concentration of Zn was predominantly higher in hippocampus compared to the other regions, in both study groups. The concentrations of fifteen amino acids were measured in the same foetal brain samples used for elemental analysis. Stillbirth brain tissues had lower levels of alanine (P < 0.05), phenylalanine (P < 0.05) and Isoleucine (P < 0.05) compared to social terminations. Regional brain analysis of amino acids revealed higher concentrations of glutamic acid, aspartic acid and serine in the hippocampal region. The significance of these results and any relationship found between the amino acid and elemental content of the analysed foetal brain samples are discussed in detail. Overall this work demonstrates and supports the importance of an adequate balance of elements and amino acids for a successful outcome of pregnancy.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2014-11-17T19:30:12Z No. of bitstreams: 1 Alene Vanessa Azevedo dos Santos. Reacoes de oxido reducao como alvo de quimioterapia triconomicida.pdf: 13103565 bytes, checksum: ad5cc355a7ef3d05b87bd59e85b6f9e0 (MD5) / Made available in DSpace on 2014-11-17T19:30:12Z (GMT). No. of bitstreams: 1 Alene Vanessa Azevedo dos Santos. Reacoes de oxido reducao como alvo de quimioterapia triconomicida.pdf: 13103565 bytes, checksum: ad5cc355a7ef3d05b87bd59e85b6f9e0 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Centro de Pesquisa Gonçalo Moniz. Salvador, BA, Brasil / Trichomonas vaginalis e Tritrichomonas foetus são os agentes etiológicos da tricomoníase humana e bovina, respectivamente. A primeira é uma parasitose de grande importância em saúde pública, sendo a principal doença sexualmente transmissível não viral e a segunda ocasiona extensos prejuízos econômicos na pecuária. A tricomoníase humana pode promover a incidência de câncer de colo de útero e a transmissão do HIV. Na infestação humana a droga de escolha é o metronidazol, mas não há um composto eficiente para o tratamento de bovinos. Como este fármaco é usado para outras parasitoses e infecções bacterianas desde a década de 60, tem sido documentado o surgimento de casos refratários e cepas resistentes. Além disso, o fármaco pode ter efeitos mutagênicos e carcinogênicos. Assim sendo a busca por novos compostos tricomonicidas constitui uma demanda premente. No presente estudo foram avaliados os efeitos do dietilditiocarbamato de sódio (DETC) sobre os protozoários parasitas T. vaginalis e T. foetus. Observamos que a adição de DETC inibiu significativamente a proliferação de T. vaginalis, produzindo uma IC50 de 269,7nM, enquanto o metronidazol teve uma IC50 de 523,1nM. Em T. foetus os valores de IC50 foram semelhantes, sendo 497,8 e 459,7nM para o DETC e para o metronidazol, respectivamente. Estes compostos não afetaram significativamente a incorporação de [3H]timidina por esplenócitos murinos em concentrações de até 1000M. A atividade mitocondrial de macrófagos peritoneais murinos também não foi afetada por DETC, mesmo em 200μM, sugerindo seletividade no modo de ação. Objetivando determinar a existência de sinergismo entre DETC e metronidazol, foram determinados os valores de FIC (concentração inibitória fracionada) para os dois protozoários. Os valores de FIC foram 0,3 e 0,7 para T. vaginalis e T. foetus, respectivamente. Esta observação indica que ocorre a interação sinergística no protozoário parasita de humanos. A fim de determinar se a incubação com DETC poderia afetar a expressão de grupos sulfidrila dos parasitos empregamos a reação de Ellman para dosar os grupamentos tiol totais de T. vaginalis antes e após a adição do composto. Observamos que o tratamento com DETC por 24h significativamente (p<0,01) reduziu a concentração de grupos tióis do parasito. A detecção de tióis livres pela sonda fluorescente orto-phthalaldeído (OPA) em T. foetus sugere a participação de sulfidrilas no mecanismo de ação do DETC, uma vez que este reduz marcadamente a marcação dos parasitos pelo OPA, mas este efeito foi revertido pela pré-incubação com cisteína. Vale salientar que o aminoácido reverte, ainda, os efeitos do composto na proliferação parasitária. A mensuração de radicais livres por quimioluminescência amplificada pelo luminol indicou que a geração de espécies reativas de oxigênio foi significativamente (p <0,05) aumentada por DETC em T. vaginalis, mas não em T. foetus. O estresse oxidativo sobre os parasitas foi avaliado pela medida de substâncias reativas ao ácido tiobarbitúrico (TBARs). Observou-se que a combinação metronidazol-DETC significativamente aumenta a peroxidação lipídica em T. vaginalis (p <0,01) e T. foetus (p <0,05). A análise ultraestrutural por microscopia eletrônica de transmissão revelou que tanto DETC quanto o metronidazol produziram danos hidrogenossomais e desencadearam autofagia e esses efeitos foram mais acentuadas no parasitas incubados com a combinação de drogas. Tomados em conjunto, estes dados sugerem que a combinação metronidazol-DETC pode fornecer novas ferramentas para a efetiva quimioterapia da tricomoníase / Trichomonas vaginalis e Tritrichomonas foetus are the etiologic agents of the human and bovine trichomoniasis, respectively. The former is a parasitic disease of great relevance in public health – the major non-viral sexually-transmitted disease, whereas the latter causes remarkable losses in livestock productivity. Human trichomoniasis can promote the incidence of cervical cancer and HIV transmission. In human infestation the drug of choice is metronidazole, but there is no efficient compound for treating the cattle. Since the medication is also used for other parasitic and bacterial diseases, since the 60s, refractory cases and resistant strains have been documented. Besides, the drug may be mutagenic and carcinogenic. Therefore the search for new trichomonicidal compounds is required. In the present study we investigated the effects of sodium N,N-diethylthiolcarbamate (DETC) alone or combined with metronidazole upon the parasitic protozoa T. vaginalis and T. foetus. We notice that DETC significantly inhibited the T. vaginalis proliferation, producing an IC50 of 269.7nM, whereas metronidazole produced an IC50 of 523.1. In T. foetus the IC50 values were similar, being 497.8 and 459.7nM for DETC and metronidazole, respectively. These compounds did not significantly affect the incorporação de [3H]timidine incorporation by murine splenocytes in concentrations up to 1000μM. The mitochondrial activity of murine peritoneal macrophages was not affected by 200μM DETC, suggesting a selective mode of action. In order to determine whether there is synergism between DETC and metronidazole, we determined the fractional inhibitory concentrations (FIC) of these compounds upon both protozoa. The FIC values for T.vaginalis and T. foetus were 0.3 and 0.7, respectively. This observation indicates that synergistic interaction takes place only on the human pathogen. To determine whether the incubation with DETC could affect the expression of sulphydril groups of parasites we employed the Ellman’s reaction to measure the total thiol groups of T. vaginalis before and after the addition of the compound. We observed that treatment with DETC for 24h significantly (p <0.01) reduced the concentration of thiol groups of the parasite. The detection of free thiols by the fluorescent probe ortho- phthaldialdehyde (OPA) in T. foetus suggests the involvement of sulphydrils in the DETC mechanism of action, since it markedly reduces the OPA labeling of parasites, but this effect was reversed by cysteine preincubation with. It is noteworthy that the amino acid addition also reverts, the effects of the compound on the parasite proliferation. The measurement of free radicals by luminol-enhanced chemiluminescence indicated that reactive oxygen species generation was significantly (p <0.05) enhanced by DETC in T. vaginalis, but not T. foetus. The oxidative stress on the parasites was evaluated by measurement of the thiobarbituric acid-reactive substances (TBARs). We observed that the metronidazole-DETC combination significantly enhanced lipid peroxidation in T. vaginalis (p <0.01) and T. foetus (p <0.05). The ultrastructural analysis by transmission electron microscopy revealed that both DETC and metronidazole produced hydrogenosomal damage and triggered autophagy and these effects were more pronounced on the parasites incubated with the combined drugs. Taken together these data suggest that the metronidazole-DETC combination may furnish new tools in the effective chemotherapy of trichomoniasis.

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Reproduction de : Thèse d'exercice : Médecine. Gynécologie-Obstétrique : Nantes : 2008. / Bibliogr.

Reproduction de : Mémoire de Sage-femme : Médecine : Nantes : 2009. / Bibliogr.