Orientador: Áureo Tatsumi Yamada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T23:37:33Z (GMT). No. of bitstreams: 1 Bianco_JuaresEdnaldoRomero_D.pdf: 17105644 bytes, checksum: 64a116dab9ebcd72e51d45d800243157 (MD5) Previous issue date: 2018-08-16T20:37:19Z / Resumo: As células Natural Killer uterinas (uNK) participam da interação materno fetal atuando ativamente como elemento imuno-modulador do micro-ambiente uterino na gestação bem sucedida. Apesar das uNK possuírem um potencial lítico, relacionado com a sua resposta imune inata, este não se manifesta em uma gestação normal. Por outro lado, diversos relatos sugerem o envolvimento destas células em abortos e interrupções da gestação. No presente trabalho, foi avaliado o possível envolvimento das células uNK em atividade citotóxica sob a influência "in loco" do líquido amniótico no útero pseudogestante de camundongos. Para tanto, camundongos fêmeas foram acasaladas com machos vasectomizados, e os cornos uterinos injetados com óleo mineral para indução do útero pseudogestante. O líquido aminótico coletado em condições assépticas dos sítios de desenvolvimento embrionário normal no 10° dia de gestação (dg), foi inoculado no volume constante de 3uL através de micro-injeção na parede uterina da região antímesometrial nosnódulos de decidualização dos animais no 9o dia de pseudogestação (dpg). Amostras dos sítios inoculados com líquido amniótico foram coletadas nos intervalos de 30 minutos, 1, 2 e 6 horas para análises morfológicas, citoquímicas, imunocitoquímicas, imunoquímica e expressões gênicas. Como controles, foram utilizados úteros gestantes normais no 8o dg e úteros pseudogestantes submetidos à introdução de agulha hipodérmica na parede do corno uterino e/ou inoculados com 3uL de solução fisiológica estéril.Pelas análises morfológicas realizadas em cortes histológicos avaliados pela coloração de hematoxilina-eosina, o endométrio decidualizado da região mesometrial e antímesometrial do útero pseudogestante inoculado com salina ou simples introdução da agulha hipodérmica não apresentaram variações. Igualmente, a morfologia das células uNK através da citoquímica com lectina DBA, não apresentou diferenças entre os grupos controles. Ainoculação de LA no útero pseudogestante promoveuhiperemia local após 30 minutos que, em cortes histológicos, foi constatada como estagnação do sangue e dilatação dos vasos venosos no endométrio. Nestes locais foram constatadas alterações degenerativas nas células uNK com perda de reatividade da superfície celular e no conteúdo dos grânulos através da lectina DBA. A análise immunocitoquímica, RT-PCR e ELISA para as citocinas IFN-y e TNF-a e, das proteínas perforina e granzima-A revelaram que, o líquido amniótico acentuava a expressão destas moléculas ou dos seus genes relacionados à resposta pró-inflamatória, enquanto não alteravam a expressão de moléculas anti-inflamatórias como a IL-10. A geração do óxido nítrico (NO), através da quantificação de nitrato e nitrito, demonstrou um aumento nas concentrações, principalmente do nitrito após a inoculação do líquido amniótico. O teste de TÚNEL e a imunocitoquímica para anti-caspase-3 revelaram aumento de marcações positivas nas células uNK e deciduais sob o efeito do líquido amniótico em relação aos grupo controles, enquanto se verificava uma relação inversa para a imunocitoquímica com o anti-PCNA aos 2 a 6 horas após líquido amniótico. O conjunto dos resultados obtidos comprovam que as células uNK são sensíveis à ação local do líquido amniótico, com padrão de resposta compatível à ativação da sua capacidade citotóxica e pró-inflamatória relacionada com a resposta imune inata. / Abstract: Uterine Natural Killer (uNK) cells participate on the maternal fetus interaction actively playing as a immunomodulator element of the uterine micro-enviroment during a well successful gestation. Despite uNK having a lytic potencial that is related to its innate immune responsive, this does not manifest in a normal gestation. Moreover, several reports suggest the involvement of these cells in abortions and interruptions of gestation. In this study, we investigated the possible involvement of uNK cells in cytotoxic activity under the influence "in loco" of amniotic fluid in the uterus of mice pseudopregnant. For this, female mice were mated with vasectomized males and the uterine horns injected with mineral oil for induction of the pseudopregnant uterus. The amniotic fluid collected aseptically from sites of normal embryonic development in the 10 th day of gestation (dg) was inoculated on the constant volume of 3ul_ through microinjection on the uterine wall in the antimesometrial region into uterine nodules from animals on the 9th day of pseudopregnancy (dpg). Samples of inoculated sites with amniotic fluid were collected at intervals of 30 minutes, 1, 2 and 6 hours for morphological, cytochemical, immunocytochemical, immunochemical and relative gene expressions analyses. As controls, normal pregnant uteri on the 8th dg, and uterus on 9th dpg were submitted to the introduction of a hypodermic needle into the wall of the uterus and/or inoculated with 3uL of sterile saline.For the morphological analysis performed on histological sections evaluated by hematoxylin-eosin, the decidualized endometrium of the mesometrial and antimesometrial regions of the pseudopregnant uterus were inoculated with saline or simple introduction of the hypodermic needle showed no variations. Also, the morphology of uNK cells by DBA lectin cytochemistry, evidenced no differences among control groups.The amniotic fluid inoculation on the pseudopregnant uterus promote Hyperaemia after 30 min, in histological slides that was observed as blood interruption flow and dilatation of the vessels on the endometrium. On these areas it was observed degenerative modifications on uNK cells with loss in the positive reaction to DBA lectin in the cellular membranes and in the granules.The immunocytochemical analysis, RT-PCR and ELISA for the cytokines IFN-y and TNF-a, and the perforin and granzyme-A protein revealed that the amniotic fluid enhanced the expression of these molecules or their genes related to pro-inflammatory response, while not altering the expression of anti-inflammatory molecules such as IL-10.The generation of nitric oxide (NO), was observed through the quantification of nitrate and nitrite that demonstrated an increase in concentrations, primarily of nitrite after inoculation of amniotic fluid.The TUNEL assay and immunocytochemistry for anti-caspase-3 revealed an increase on positive uNK and decidual cells under the effect of amniotic fluid in relation to the control group, while there was an inverse relationship to immunocytochemistry with anti-PCNA at 2 and 6 hours after the amniotic fluid. The overall results show that uNK cells are sensitive to local action of amniotic fluid, compatible with standard response to the activation of their cytotoxic and proinflammatory that are related to the innate immune response. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Se realizó un estudio descriptivo y transversal con el objetivo de describir las características de la oclusión decidua de niños de un Centro Poblado de Huancavelica alimentados con lactancia materna y con presencia o no de hábitos de succión no nutritiva. La muestra fue de 45 niños de 3 a 5 años de edad con dentición decidua completa que nunca se alimentaron con biberón, se realizó un examen clínico bucal al niño y un cuestionario a la madre. Se encontró que los niños de la muestra se alimentaron con lactancia materna en un promedio de 19 meses desde su nacimiento, siendo siempre exclusiva hasta los 6 meses, el 56% presentaron hábitos de succión no nutritiva de dedo, juguete o ambos y por un tiempo menor a 2 años. En el plano terminal predominó el escalón mesial (60%) y recto (36%) sin encontrarse presencia de escalón distal, el espacio primate en ambas arcadas se presentó en el 9% de la muestra sin tener relación estadística con los hábitos de succión no nutritivos. De los niños con hábitos de succión no nutritiva, solo el 4% (1) presentó mordida cruzada anterior relacionado al hábito de succión no nutritiva de un juguete; no hubo presencia de mordida abierta anterior ni mordida cruzada posterior. En la muestra los hábitos de succión no nutritiva no presentaron asociación con la oclusión decidua. Palabras claves: lactancia materna, hábitos de succión no nutritiva, oclusión decidua / This descriptive cross-sectional study was conducted with the aim of describing the characteristics of deciduous occlusion in children from a village in Huancavelica, breastfed with presence or absence of non-nutritive sucking habits. The sample consisted of forty-five 3 to 5 year olds with complete primary dentition who were never bottle fed. An oral examination and a questionnaire to the child's mother were performed. We found that children in the sample were breastfed for an average of 19 months, with exclusive breastfeeding during the first 6. 56% had non-nutritive sucking habits (finger, toy or both) for a less than a two-year period. In the flush terminal plane, the mesial step predominated (60%) and straight (36%) without any presence of distal step, the primate space in both arches appeared in 9% of the sample without statistical relationship with non-nutritive sucking habits. In the case of the children with non-nutritive sucking habits, only 4% (1) presented anterior crossbite related to the habit of non-nutritive sucking of a toy; there was no presence of anterior open bite or posterior cross bite. In the sample of non-nutritive sucking habits they presented no association with the deciduous occlusion. Keywords: breastfeeding, Non-nutritive sucking habits, occlusion decidua

<p>The tolerance towards the semi-allogenic foetus is obtained through both systemic and local changes in the maternal immune response. Locally, in the decidua, the cell composition differs from that found in the blood; natural killer (NK) cells and macrophages being the major cell types. Decidual macrophages (dMØ), which are alternatively activated, and trophoblasts, placental cells of foetal origin, are believed to participate in the foetal tolerance at the foetal-maternal interface. To test the recruitment ability of macrophages and trophoblasts, and to test if these cells are responsible for the special cell composition in the decidua, a migration assay was established. In this migration assay peripheral blood mononuclear cells (PBMC) were allowed to migrate through Matrigel-coated transwell inserts into lower wells containing a recruiting stimulus. After testing several conditions, a protocol was established for further use. The results showed that <em>in vitro</em> alternatively activated macrophages, which display many of the surface markers as dMØ, hold a recruiting ability and recruit monocytes. Further there was an indication that trophoblasts also hold a recruiting ability. Neither cell types were shown to recruit NK cells. In conclusion, this study presents a suitable protocol for assessing chemotactic factors and different cell type’s ability to recruit cells from blood. Although the experiments need to be repeated and extended and the recruitment ability of dMØ needs to be evaluated in detail before a final conclusion can be drawn, the preliminary data indicated that macrophages and trophoblasts can recruit monocytes.</p>

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

Neutrophils infiltrate myometrium and decidual tissue prior to parturition. Activated neutrophils release reactive oxygen species (ROS) and tumor necrosis factor α (TNFα), which might increase expression of pro-labor genes such as matrix metalloproteinase-1 (MMP-1), progesterone receptor (PR) A/B ratio, and cause demethylation of DNA. These changes might cause labor. Decidual tissue was obtained from consented, healthy women at term (37+ weeks of gestation) not in labor (no contractions, without cervical effacement), term labor and preterm labor (under 37 weeks of pregnancy). Decidual and myometrial cells in culture were treated with (1) ROS, (2) TNFα, or (3) 5-aza-2’-deoxycytidine. Total RNA was extracted, converted to cDNA and evaluated by qRT-PCR for MMP-1, PR-A+B and PR-B. TNFα increased MMP-1 by 17 fold in decidual cells and more than 12 fold in myometrial cells. PR-A/B was increased by 5.6 fold in decidua. ROS up-regulated MMP-1 by 6 fold and elevated the PR-A/B ratio by 4.5 fold in decidual tissue. DNA demethylation increased MMP-1 by about 4 and 11 fold in decidual and myometrium, respectively. The PR-A/B ratio was increased by 4 fold in decidua and the PR-B was decreased by 40% in the myometrium due to DNA demethylation. Decidual tissue in preterm labor showed a 7-fold increase in MMP-1 over term laboring and over a 15-fold increase over term not in labor tissue. In conclusion, MMP-1 expression and PR-A/B ratio was increased by neutrophil products possibly through a mechanism of DNA methylation in decidua and myometrium. Preterm decidua showed a dramatic increase in MMP-1 over normal labor tissue. TNFα and ROS increased expression of MMP-1 to possibly initiate parturition. These data might help explain mechanisms responsible for preterm labor unrelated to infection or premature rupture of membranes.

No presente estudo realizamos uma análise ultraestrutural e imuhistoquímica da organização das fibrilas de colágeno no endométrio durante a gestação de camundongos silvestres e deficientes em decorim. Os resultados mostraram que as fibrilas de colágeno em ambos os genótipos sofrem grande variação de forma e tamanho. Observou-se variação significante, na percentagem de distribuição dos diâmetros das fibrilas de colágeno existentes na região decidualizada em ambos os genótipos, porém foi maior nos animais Dcn-/-. Estes animais também apresentam maior percentagem de fibrilas finas quando comparados aos animais Dcn+/+. Observamos ainda que biglicam é expresso no endométrio não decidualizado dos animais Dcn-/-, no 3º dia de gestação. A expressão de lumicam mostrou-se nítida no estroma decidualizado e não decidualizado nos animais Dcn-/- no 7º dia de gestação e foi ausente nos animais Dcn-/-. Estes resultados mostraram que a ausência do decorim promove distúrbios no processo de agregação lateral das fibrilas espessas de colágeno. / The present study is an ultrastructural investigation into the organization of collagen fibrils in the pregnant endometrium of wild-type and decorin-deficient mice. Our results showed that collagen fibrils from both genotypes present a great variability of shape and size in cross section. Significant variation in the diameter of collagen fibrils exists in the decidualized endometrium from both groups of animals. In the decidualized endometrium, the diameter of collagen fibrils increases in both genotypes were higher in Dcn-/- than in Dcn+/+ animals. In the Dcn-/- animals the percentage of thin fibrils with diameter is also higher, when compared with Dcn+/+ animals. We also showed that Bgn is expressed in the non decidualized endometrium in the Dcn-/- animals, on day 3 of pregnancy. The expression of lumican showed a very sharp labeling in the decidualized stroma from day 7, and in the non decidualized estroma from Dcn-/- animals. These results suggest that the deficience of decorin may play a role on collagen fibrillogenesis in different stages of pregnancy.

Maternal age is a significant risk factor for adverse pregnancy outcomes, and is strongly associated with an increased risk of aneuploidy of the conceptus, as well as a significantly higher frequency of serious pregnancy complications known as the "Great Obstetrical Syndromes", including miscarriage, pre-eclampsia and fetal growth restriction. In the last 40 years average maternal age has increased considerably in many wealthy countries, and in the UK the number of babies born to women aged 35 and over is set to surpass those born to women under 25. The high incidence of aneuploidy in older mothers can be attributed to abnormalities in the oocyte and embryo, however the "Great Obstetrical Syndromes" do not appear to be related to the oocyte and may instead be linked to abnormal development of the placenta. In this thesis, I show that advanced maternal age in the mouse is associated with a drastically increased variability of developmental progression in utero, including developmental delays and growth restriction, severe embryonic abnormalities and higher resorption rates. I find that these embryonic defects are always accompanied by gross morphological and transcriptomic abnormalities in the placenta. Notably, I show that the increased risk of these complications can be rescued by transfer of embryos from aged females to a young surrogate mother, thus implicating the aged maternal uterus as the basis for embryonic and placental defects. Transcriptomic analysis of the decidua compartment in placentas from aged pregnancies revealed abnormal expression of genes involved in the decidualization process, which occurs during early pregnancy and facilitates implantation and development of the conceptus. I show that these defects are already obvious in the peri-implantation window, with endometrial stromal cells from aged females being unable to mount an adequate decidualization response due to a decline in their ability to respond to pregnancy hormones. This blunted decidualization reaction in turn may lead to abnormal development of the placenta. These age-associated decidualization defects are cell-intrinsic and can be recapitulated in vitro. The detected insufficient activation levels and abnormal intracellular distribution of phospho-STAT3, combined with highly variable progesterone receptor expression, may be possible causes of these defects. In addition, I examined the possible effects of ageing on the epigenome as a potential contributor to the decline in endometrial function. My results indicate that ageing of the uterus displays some of the common epigenetic hallmarks of tissue ageing. However, more importantly, decidual cells of aged females exhibit abnormal distributions of the histone modification H3K4me3, and are refractory to the profound DNA methylation remodeling that I find takes place during pregnancy. These age-related changes in the epigenome may underpin, or contribute to, the observed decline in uterine function during pregnancy. Understanding the mechanism underlying these epigenomic and functional changes in the ageing reproductive tract may pave the way for new therapeutic strategies to improve maternal and fetal outcomes of pregnancy in older mothers.

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Orientador: Aureo Tatsumi Yamada / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-06T17:18:35Z (GMT). No. of bitstreams: 1 Bianco_JuaresEdnaldoRomero_M.pdf: 23225852 bytes, checksum: 523781fa325c40e61fa0fe33c59406a1 (MD5) Previous issue date: 2006 / Resumo: Durante o período peri-implantacional na gestação de humanos e roedores, ocorre no estroma da mucosa uterina um conjunto de fenômenos que envolvem modificações dos componentes celulares e da matriz extracelular. Este conjunto de modificações é conhecido como decidualização ou reação decidual. Concomitante a este processo ocorre a migração de leucócitos provenientes de órgão hematopoéticos para este estroma. Dentre estes leucócitos predominam as subpopulações de linfócitos denominados de células ¿uterine Natural Killer¿ (uNK), que atuam na gestação reconhecendo o embrião alogeneico, modulando a reação decidual e o crescimento placentário. Porém, a sua participação nos casos de aborto e o possível desencadeamento do potencial lítico e citotóxico relacionado com a resposta imune inata, não foi ainda totalmente elucidado. O presente trabalho buscou desenvolver estratégias para o estudo in situ das células uNK, sem a influência de fatores oriundos do embrião, empregando o modelo da pseudogestação em camundongos. Foram utilizados fêmeas pseudogestantes obtidos através do acasalamento com machos vasectomizados e inoculação intraluminal do óleo vegetal (arachis) ou mineral (nujol) no útero de 4° dia de pseudogestação (dpg). Estes animais pseudogestantes foram subdivididos em grupos submetidos ou não a ovarectomia e com suplementação hormonal de estrógeno e/ou progesterona ou hormônio gonadotrófico coriônico (hCG). Foram coletados o útero e ovário e processados para análise em microscopia de luz e eletrônica de transmissão no 9°, 12° e 14° dpg. Fêmeas em gestação normal foram utilizados como grupo controle. As análises histológicas, citoquímicas e ultra-estruturais efetuadas demonstraram que a indução da pseudogestação com emprego tanto de óleo vegetal (arachis) quanto de óleo mineral resultam na reação decidual e presença de células uNK lectina DBA (Dolichos biflorus) positivas. A reação decidual resultante e mais exuberante com o óleo de arachis, porém, o período de manutenção do deciduoma é menor, mesmo com a suplementação hormonal de progesterona, se comparado ao grupo de animais induzidos com o óleo mineral e suplementada com HCG. As características morfológicas das células uNK foram melhor preservadas nos animais pseudogestantes induzidos com o óleo mineral, não ovarectomizados e suplementados com o HCG.Nestes, as células uNK foram encontradas aém do 12° dpg em meio ao endométrio que mantinha o deciduoma, enquanto nos demais grupos o deciduoma era destituído antes do 12º dpg. Porém, , à semelhança dos demais grupos experimentais, houve predominância de formas menos diferenciadas de células uNK (subtipos I e II), sendo raras as formas plenamente diferenciadas (subtipo III). Em nenhum dos grupos foi encontrado o subtipo IV, correspondentes à forma de degeneração destas células. Estes resultados demonstram que as características do endométrio e o comportamento das células uNK na pseudogestação, apresentam diferenças influenciadas pelos meios de indução utilizados e regimes hormonais adotados.Além disso, a ausência do concepto parece ser o fator limitante na continuidade da manutenção do utero seudogestante. Por conseguinte, a pseudogestação induzida com óleo mineral e mantida sob a regência ovariana supra-controlado pelo HCG poderia ser adotada como modelo experimental para estudo das células uNK sem a interferência do embrião. Este modelo deverá permitir novas estratégias para investigação dos diversos fatores de origem embrionário/fetal que possam afetar as atividades destas e/ou outras células de forma controlada in situ no ambiente uterino que simule a gestação / Abstract: During the period of pre-implantation in humans and rodents gestations, a set of phenomenon, involving, modifications of cellular components and extracellular matrix occur in the stroma of the uterus. These modifications, are known as decidualization or decidual reaction. During this processes a migration of leukocytes that came from the heamatopoetic organs occur into the uterine stroma. Among these leukocytes, there is a predominance of a subpopulation of leukocytes known as uterine Natural Killers (uNK) that play important roles during gestation by recognizing the allogeneic embryos and modulating the decidual reaction and the placental growth. However, the uNK participations in miss carriage and triggering of lytic and citotoxic potential related to innate immune response during pregnancy have not elucidated yet. The present work aimed to develop a strategy for uNK study without the influence of embryonaryfactors by using the pseudopregnancy model in mice. It was used the pseudopregnant females mice obtained by matting with vasectomized males and intra-luminal injection of arachis or mineral oil in the uterus on 4th day of pseudopregnancy (dpp). These animals were grouped according to treatment submitted of ovarectomy or not and with exogenous supplement of progesterone and/or estradiol or chorionic gonadotrophin hormone (HCG). The uteri and ovary were collected and processes for light and electron microscope on the 9th, 12thand 14thdpp. Normal pregnant females were used as controls. Histological analysis, cytochemistry and ultrastructure demonstrated that the induction of pseudopregnancy by using both arachis oil and mineral oil resulted in decidual reaction and presence of the DBA (Dolichos biflorus) lectin positive uNK cells. The decidual reactions induced by arachis oil were more intensive and exuberant, but the deciduomas were not sustained longer even under progesterone treatment, if compared to those, in the uterus induced with mineral oil, and supplemented with HCG. The morphological characteristics of the uNK cells were better preserved in the pseudopregnant animals induced with mineral oils, not ovarectomized and supplement with HCG. In these animals, the uNK cells were found over the 12th dpp in the endometrium preserving the deciduoma while it failed before 12th dpp in the other groups However, similarly to other experimental groups, there was predominance immature forms of uNK cells (subtypes I and II), being rare the fully differentiated forms (subtype III). No groups did not show the uNK sub type IV, corresponding to the degeneration form of this cell / Mestrado / Histologia / Mestre em Biologia Celular e Estrutural