Modulation of immune cells and Th1/Th2 cytokines in insulin-treated type 2 diabetes mellitus.

Background: The role of the immune system in insulin resistance associated with type 2 diabetes has been suggested. Objectives: We assessed the profile of Th1/Th2 cytokines along with the frequencies of immune cells in insulin-treated type 2 diabetic patients (T2DP). Methods: 45 T2D patients and 43 age-matched healthy subjects were selected. Serum concentrations of T-helper type 1 (Th1) and Th2 cytokines and the frequencies of innate and adaptive immunity cells were assessed. Results: T2DP were hyperglycemic and showed high level of insulin, normal levels of triglycerides and total-cholesterol and without any change in HDL-cholesterol.Compared to healthy subjects, T2DP exhibited significant decreased frequencies of neutrophils, without any change in monocytes, eosinophils and natural killer cells. The percentages of total lymphocytes (CD3+) and CD8+-T-cells decreased whereas those of regulatory T-cells increased without any change in CD4+ T-cells in T2DP. Interestingly, the frequencies of effector CD4+-T and B-cells increased in T2DP. Serum concentrations of IL-2, IFN-γ and IL-4 decreased while IL-10 significantly enhanced in T2DP, suggesting a differentiation of CD4+T helper cells towards IL-10-producing- Teff-cells in these patients. Conclusion: Insulin-treated type 2 diabetes is associated with anti-inflammatory profile consistent with differentiation of CD4+-Th-cells towards IL-10-producing-Teff-cells, concomitant with increased frequencies of Treg and B-cells, and this may probably offer prevention against certain infections or autoimmune/inflammatory diseases.

Linfócitos T : uma análise do perfil diferencial da expressão gênica na imunorregulação

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Pós-Graduação em Patologia Molecular, 2016.

Long-term consequences of Hodgkin lymphoma therapy on T-cell lymphopoiesis

International audience

Inmunoterapia autóloga con células dendríticas en modelos caninos con tumor venéreo transmisible

La inmunoterapia autóloga utiliza células del mismo cuerpo para estimular y restaurar las defensas naturales del sistema inmunológico. Algunas de las células que han sido utilizado en años recientes son: linfocitos infiltrantes de tumor, linfocitos T citotóxicos, células asesinas activadas por linfocinas y células dendríticas (CD). Las CD son células especializadas y su función es el capturar, procesar y presentar antígenos a los linfocitos para iniciar una respuesta inmune. El tumor venéreo transmisible (TVT), también conocido como sarcoma infeccioso o tumor de Sticker, es un cáncer transmisible en perros que afecta mayormente los genitales externos y se transmite durante el coito. En este trabajo se implementó por primera vez un modelo de TVT en el órgano genital (vagina) de los pacientes y se les administró la inmunoterapia autóloga con CD específicas contra el tumor. Para estudiar esta terapia se utilizaron tres grupos experimentales: el tumor sin tratamiento, el tumor tratado con sangre completa autóloga, y el tumor tratado con CD autólogas específicas para el TVT. Por último se evaluó la capacidad inmunológica del extracto tumoral total (ETT) del tumor como método de prevención in vivo. Para el tratamiento autólogo con las CD, se esperó que el tumor midiese 3cm, se realizó un cultivo primario de las células de TVT y se les extrajo el 4% de su peso corporal de sangre a los pacientes para realizar una diferenciación de los monocitos a CD. Para evaluar el efecto de la inmunoterapia autóloga con CD se observaron los efectos secundarios, el tamaño tumoral, las poblaciones de linfocitos, los niveles de IFN-γ en sangre y los linfocitos infiltrantes de tumor por histopatología. Los monocitos se diferenciaron a CD y se les realizó un análisis fenotípico mediante citometría de flujo. Los monocitos mostraron una expresión de CD14+ de 80.1%, CD80+ de 15.6%, CD83+ de 0.4% y un DLA II de1.8%. En las CD se obtuvo una expresión de 8.7% para CD14+, 80.3% para CD80+, 76.4% para CD83+ y 86.5% para DLA II y 62% en la prueba de fagocitosis. La terapia no mostró ningún efecto secundario en nuestro grupo experimental y hubo una regresión tumoral del 100% para la semana doce. Se encontró un aumento de expresión celular en sangre de CD4+ de 29%, de CD8+ de 34% y de IFN-γ de 120 pg/mL. Nuestros resultados demuestran que la inmunoterapia autóloga con CD específicas inducen una regresión del TVT en caninos.

Construção e produção de fragmentos de anticorpos anti-ssDNA em um contexto de desenvolvimento de linfócitos B

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2016.

Produção de novos anticorpos Anti-CD20 na forma de FvFc em células de mamíferos

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Pós-Graduação em Biologia Molecular, 2015.

Prevalencia del Helicobacter Pylori y factores asociados en escolares de la etnia Shuar del cantón Sucúa –Morona Santiago, 2014.

Introducción: Según la OMS, más del 50% de la población adulta está infectada con el Helicobacter Pylori, con prevalencias de hasta el 90%. La mayoría de contagios se produce antes de los 10 años de edad. Desde el descubrimiento del H. Pylori (1983), se lo ha relacionado con la úlcera péptica, gastritis, reflujo gastroesofágico e incluso, cáncer gástrico. Metodología: Se realizó un estudio transversal analítico. La muestra estuvo conformada por 250 niños escolares de la etnia Shuar del cantón Sucúa. La prueba utilizada para la detección del Helicobacter pylori es la identificación de antígenos en las heces por inmunocromatografía. Para establecer la significancia de asociación de variables se utilizó el OR con su intervalo de confianza al 95%. Resultados: El promedio de edad fue de 8.8 años (DS 2.0), con predominio de las mujeres (54.4%). El 56.4% consume agua potable, el 71.65% vive en hacinamiento, el 42% tiene servicios de letrinización y el 49.2% cuenta con servicios de alcantarillado. Se encontró asociación significativa con el nivel de instrucción OR 1.68, IC95% (1 – 2.84), p=0.049; letrinización OR 1.99, IC95% (1.17 – 3.36), p=0.01; deposiciones al aire libre OR 4.32, IC95% (2.13 – 8.77), p=0.000. Conclusiones: La prevalencia de Helicobacter pylori es alta en la población escolar de la etnia Shuar; está asociada a un nivel bajo de instrucción de los progenitores y una inadecuada infraestructura de servicios básicos.

Anomalies immunitaires chez les enfants exposés au VIH mais non infectés.

La thérapie antirétrovirale prévient la transmission mère-enfant du VIH dans plus de 98% des cas lorsqu’administrée pendant la grossesse, le travail et au nouveau-né. L’accessibilité à la thérapie antirétrovirale dans près de 70% des 1,5 millions cas de grossesses VIH+ dans le monde mène à la naissance de plus d’un million d’enfants exposés non infectés chaque année. Le nombre d’enfants exposés non infectés est à la hausse ainsi que les préoccupations concernant leur santé. En effet, plusieurs groupes ont signalé une augmentation de la morbidité et de la mortalité chez les enfants exposés non infectés. L’analyse des données rétrospectives de 705 enfants exposés non infectés de la cohorte mère-enfant du CMIS a révélé qu’à 2 mois d’âge, les enfants nés de mères ayant une charge virale supérieure à 1,000 copies d’ARN / ml avaient une fréquence de lymphocytes B significativement plus élevés par rapport aux enfants exposés non infectés nés de mères ayant une charge virale indétectable. L’objectif de cette étude est de caractériser ces anomalies. Les lymphocytes, provenant du sang de cordon ombilical et de sang veineux obtenu à 6 et 12 mois d’âge, ont été phénotypés par cytométrie en flux à l’aide des marqueurs CD3 / CD10 / CD14 / CD16 / CD19 / CD20 / CD21 / CD27 / IgM pour les lymphocytes B et CD4 / CD8 / CD3 / CCR7 / CD45RA pour les lymphocytes T. De plus, afin d’étudier les capacités fonctionnelles des lymphocytes B CD19+, la réponse antigène-spécifique au vaccin antitétanique a été mesurée par marquage avec des tétramères fluorescents de fragment C du toxoïde tétanique. Nos travaux ont mis en évidence des différences statistiquement significatives entre les enfants exposés non-infectés (ENI) nés de mères avec une charge virale détectable comparativement à ceux nés de mères avec une charge virale indétectable. À la naissance, les enfants ENI nés de mères avec une charge virale détectable avaient significativement moins de lymphocytes B totaux, plus de lymphocytes B mémoires classiques, activés, plasmablastes et lymphocytes T CD8+ mémoires centrales. À 6 mois, ils avaient significativement plus de lymphocytes B naïfs et significativement moins de lymphocytes T CD8+ effecteurs mémoires. À 12 mois d’âge, ils avaient significativement plus de lymphocytes B et T CD8+ totaux; significativement moins de lymphocytes T CD4+ totaux et leurs lymphocytes T affichaient un profil significativement plus activé (plus de cellules mémoires). L’analyse de la réponse antigène-spécifique a révélé une fréquence plus élevé de lymphocytes B mémoires IgM+ suggérant que les enfants nés de mères avec une virémie détectable ont plus de mal à établir une mémoire immunitaire efficace face au vaccin antitétanique. Nos données suggèrent qu’il y a exposition durant le premier trimestre de grossesse à la virémie maternelle et que cette exposition impacte le système immunitaire en développement du fœtus. Les mécanismes sous-jacents causant ces anomalies doivent encore être élucidés et l’épuisement du compartiment T à la naissance et à 6 mois reste à être investigué. Dans un pays industrialisé où l’accès aux soins est facilité, ces anomalies ont des conséquences modérées mais dans des pays à faible et moyen revenu, les conséquences peuvent être beaucoup plus tragiques voir fatales.

Anomalies immunitaires chez les enfants exposés au VIH mais non infectés

La thérapie antirétrovirale prévient la transmission mère-enfant du VIH dans plus de 98% des cas lorsqu’administrée pendant la grossesse, le travail et au nouveau-né. L’accessibilité à la thérapie antirétrovirale dans près de 70% des 1,5 millions cas de grossesses VIH+ dans le monde mène à la naissance de plus d’un million d’enfants exposés non infectés chaque année. Le nombre d’enfants exposés non infectés est à la hausse ainsi que les préoccupations concernant leur santé. En effet, plusieurs groupes ont signalé une augmentation de la morbidité et de la mortalité chez les enfants exposés non infectés. L’analyse des données rétrospectives de 705 enfants exposés non infectés de la cohorte mère-enfant du CMIS a révélé qu’à 2 mois d’âge, les enfants nés de mères ayant une charge virale supérieure à 1,000 copies d’ARN / ml avaient une fréquence de lymphocytes B significativement plus élevés par rapport aux enfants exposés non infectés nés de mères ayant une charge virale indétectable. L’objectif de cette étude est de caractériser ces anomalies. Les lymphocytes, provenant du sang de cordon ombilical et de sang veineux obtenu à 6 et 12 mois d’âge, ont été phénotypés par cytométrie en flux à l’aide des marqueurs CD3 / CD10 / CD14 / CD16 / CD19 / CD20 / CD21 / CD27 / IgM pour les lymphocytes B et CD4 / CD8 / CD3 / CCR7 / CD45RA pour les lymphocytes T. De plus, afin d’étudier les capacités fonctionnelles des lymphocytes B CD19+, la réponse antigène-spécifique au vaccin antitétanique a été mesurée par marquage avec des tétramères fluorescents de fragment C du toxoïde tétanique. Nos travaux ont mis en évidence des différences statistiquement significatives entre les enfants exposés non-infectés (ENI) nés de mères avec une charge virale détectable comparativement à ceux nés de mères avec une charge virale indétectable. À la naissance, les enfants ENI nés de mères avec une charge virale détectable avaient significativement moins de lymphocytes B totaux, plus de lymphocytes B mémoires classiques, activés, plasmablastes et lymphocytes T CD8+ mémoires centrales. À 6 mois, ils avaient significativement plus de lymphocytes B naïfs et significativement moins de lymphocytes T CD8+ effecteurs mémoires. À 12 mois d’âge, ils avaient significativement plus de lymphocytes B et T CD8+ totaux; significativement moins de lymphocytes T CD4+ totaux et leurs lymphocytes T affichaient un profil significativement plus activé (plus de cellules mémoires). L’analyse de la réponse antigène-spécifique a révélé une fréquence plus élevé de lymphocytes B mémoires IgM+ suggérant que les enfants nés de mères avec une virémie détectable ont plus de mal à établir une mémoire immunitaire efficace face au vaccin antitétanique. Nos données suggèrent qu’il y a exposition durant le premier trimestre de grossesse à la virémie maternelle et que cette exposition impacte le système immunitaire en développement du fœtus. Les mécanismes sous-jacents causant ces anomalies doivent encore être élucidés et l’épuisement du compartiment T à la naissance et à 6 mois reste à être investigué. Dans un pays industrialisé où l’accès aux soins est facilité, ces anomalies ont des conséquences modérées mais dans des pays à faible et moyen revenu, les conséquences peuvent être beaucoup plus tragiques voir fatales.

Fluoxetine for maintenance of remission and to improve quality of life in patients with Crohn's disease: a pilot randomized placebo-controlled trial.

BACKGROUND AND AIMS: Previous studies have shown that antidepressants reduce inflammation in animal models of colitis. The present trial aimed to examine whether fluoxetine added to standard therapy for Crohn's disease [CD] maintained remission, improved quality of life [QoL] and/or mental health in people with CD as compared to placebo. METHODS: A parallel randomized double-blind placebo controlled trial was conducted. Participants with clinically established CD, with quiescent or only mild disease, were randomly assigned to receive either fluoxetine 20 mg daily or placebo, and followed for 12 months. Participants provided blood and stool samples and completed mental health and QoL questionnaires. Immune functions were assessed by stimulated cytokine secretion [CD3/CD28 stimulation] and flow cytometry for cell type. Linear mixed-effects models were used to compare groups. RESULTS: Of the 26 participants, 14 were randomized to receive fluoxetine and 12 to placebo. Overall, 14 [54%] participants were male. The mean age was 37.4 [SD=13.2] years. Fluoxetine had no effect on inflammatory bowel disease activity measured using either the Crohn's Disease Activity Index [F(3, 27.5)=0.064, p=0.978] or faecal calprotectin [F(3, 32.5)=1.08, p=0.371], but did have modest effects on immune function. There was no effect of fluoxetine on physical, psychological, social or environmental QoL, anxiety or depressive symptoms as compared to placebo [all p>0.05]. CONCLUSIONS: In this small pilot clinical trial, fluoxetine was not superior to placebo in maintaining remission or improving QoL. [ID: ACTRN12612001067864.].

The role of inflammation in vascular function and pregnancy outcome in the pregnant stroke prone spontaneously hypertensive rat

During pregnancy, the maternal cardiovascular system undergoes major adaptation. One of these changes is a 40-50 % increase in circulating blood volume which requires a systemic remodelling of the vasculature in order to regulate maternal blood pressure and maximise blood supply to the developing placenta and fetus. These changes are broadly conserved between humans and rats making them an appropriate pre-clinical model in which to study the underlying mechanisms of pregnancy-dependent cardiovascular remodelling. Whilst women are normally protected against cardiovascular disease; pregnancy marks a period of time where women are susceptible to cardiovascular complications. Cardiovascular disease is the leading cause of maternal mortality in the United Kingdom; in particular hypertensive conditions are among the most common complications of pregnancy. One of the main underlying pathologies of these pregnancy complications is thought to be a failure of the maternal cardiovascular system to adapt. The remodelling of the uterine arteries, which directly supply the maternal-fetal interface, is paramount to a healthy pregnancy. Failure of the uterine arteries to remodel sufficiently can result in a number of obstetric complications such as preeclampsia, fetal growth restriction and spontaneous pregnancy loss. At present, it is poorly understood whether this deficient vascular response is due to a predisposition from existing maternal cardiovascular risk factors, the physiological changes that occur during pregnancy or a combination of both. Previous work in our group employed the stroke prone spontaneously hypertensive rat (SHRSP) as a model to investigate pregnancy-dependent remodelling of the uterine arteries. The SHRSP develops hypertension from 6 weeks of age and can be contrasted with the control strain, the Wistar Kyoto (WKY) rat. The phenotype of the SHRSP is therefore reflective of the clinical situation of maternal chronic hypertension during pregnancy. We showed that the SHRSP exhibited a deficient uterine artery remodelling response with respect to both structure and function accompanied by a reduction in litter size relative to the WKY at gestational day (GD) 18. A previous intervention study using nifedipine in the SHRSP achieved successful blood pressure reduction from 6 weeks of age and throughout pregnancy; however uterine artery remodelling and litter size at GD18 was not improved. We concluded that the abnormal uterine artery remodelling present in the SHRSP was independent of chronic hypertension. From these findings, we hypothesised that the SHRSP could be a novel model of spontaneously deficient uterine artery remodelling in response to pregnancy which was underpinned by other as yet unidentified cardiovascular risk factors. In Chapter 1 of this thesis, I have characterised the maternal, placental and fetal phenotype in pregnant (GD18) SHRSP and WKY. The pregnant SHRSP exhibit features of left ventricular hypertrophy in response to pregnancy and altered expression of maternal plasma biomarkers which have been previously associated with hypertension in human pregnancy. I developed a protocol for accurate dissection of the rat uteroplacental unit using qPCR probes specific for each layer. This allowed me to make an accurate and specific statement about gene expression in the SHRSP GD18 placenta; where oxidative stress related gene markers were increased in the vascular compartments. The majority of SHRSP placenta presented at GD18 with a blackened ring which encircled the tissue. Further investigation of the placenta using western blot for caspase 3 cleavage determined that this was likely due to increased cell death in the SHRSP placenta. The SHRSP also presented with a loss of one particular placental cell type at GD18: the glycogen cells. These cells could have been the target of cell death in the SHRSP placenta or were utilised early in pregnancy as a source of energy due to the deficient uterine artery blood supply. Blastocyst implantation was not altered but resorption rate was increased between SHRSP and WKY; indicating that the reduction in litter size in the SHRSP was primarily due to late (>GD14) pregnancy loss. Fetal growth was not restricted in SHRSP which led to the conclusion that SHRSP sacrifice part of their litter to deliver a smaller number of healthier pups. Activation of the immune system is a common pathway that has been implicated in the development of both hypertension and adverse pregnancy outcome. In Chapter 2, I proposed that this may be a mechanism of interest in SHRSP pregnancy and measured the pro-inflammatory cytokine, TNFα, as a marker of inflammation in pregnant SHRSP and WKY and in the placentas from these animals. TNFα was up-regulated in maternal plasma and urine from the GD18 SHRSP. In addition, TNFα release was increased from the GD18 SHRSP placenta as was the expression of the pro-inflammatory TNFα receptor 1 (Tnfr1). In order to investigate whether this excess TNFα was detrimental to SHRSP pregnancy, a vehicle-controlled intervention study using etanercept (a monoclonal antibody which works as a TNFα antagonist) was carried out. Etanercept treatment at GD0, 6, 12 and 18 resulted in an improvement in pregnancy outcome in the SHRSP with an increased litter size and reduced resorption rate. Furthermore, there was an improved uterine artery function in GD18 SHRSP treated with etanercept which was associated with an improved uterine artery blood flow over the course of gestation. In Chapter 3, I sought to identify the source of this detrimental excess of TNFα by designing a panel for maternal leukocytes in the blood and placenta at GD18. A population of CD3- CD161+ cells, which are defined as rat natural killer (NK) cells, were increased in number in the SHRSP. Intracellular flow cytometry also identified this cell type as a source of excess TNFα in blood and placenta from pregnant SHRSP. I then went on to evaluate the effects of etanercept treatment on these CD3- CD161+ cells and showed that etanercept reduced the expression of CD161 and the cytotoxic molecule, granzyme B, in the NK cells. Thus, etanercept limits the cytotoxicity and potential damaging effect of these NK cells in the SHRSP placenta. Analysing the urinary peptidome has clinical potential to identify novel pathways involved with disease and/or to develop biomarker panels to aid and stratify diagnosis. In Chapter 4, I utilised the SHRSP as a pre-clinical model to identify novel urinary peptides associated with hypertensive pregnancy. Firstly, a characterisation study was carried out in the kidney of the WKY and SHRSP. Urine samples from WKY and SHRSP taken at pre-pregnancy, mid-pregnancy (GD12) and late pregnancy (GD18) were used in the peptidomic screen. In order to capture peptides which were markers of hypertensive pregnancy from the urinary peptidomic data, I focussed on those that were only changed in a strain dependent manner at GD12 and 18 and not pre-pregnancy. Peptide fragments from the uromodulin protein were identified from this analysis to be increased in pregnant SHRSP relative to pregnant WKY. This increase in uromodulin was validated at the SHRSP kidney level using qPCR. Uromodulin has previously been identified to be a candidate molecule involved in systemic arterial hypertension but not in hypertensive pregnancy thus is a promising target for further study. In summary, we have characterised the SHRSP as the first model of maternal chronic hypertension during pregnancy and identified that inflammation mediated by TNFα and NK cells plays a key role in the pathology. The evidence presented in this thesis establishes the SHRSP as a pre-clinical model for pregnancy research and can be continued into clinical studies in pregnant women with chronic hypertension which remains an area of unmet research need.

Celiac disease in children from Madeira island and its prevalence in first degree relatives

Context - It is well recognized that celiac disease is an immune-mediated systemic disorder highly prevalent among relatives of celiac patients. Objectives - The aim of this study is to determine the prevalence of celiac disease in a group of first degree relatives of celiac children, and to access the frequency of human leukocyte antigen HLA-DQ2 and DQ8 in celiac disease patients and their affected relatives. Methods - A survey was conducted of 39 children with celiac disease with follow-up in the Pediatric outpatient’s clinic of Dr. Nélio Mendonça Hospital, in Madeira Island, Portugal. Were invited 110 first degree relatives to undergo serological screen for celiac disease with IgA antibody to human recombinant tissue transglutaminase (IgA-TGG) quantification. In all seropositive relatives, small intestinal biopsy and HLA typing was recommended. Results - HLA- typing was performed in 38 celiac patients, 28/74% DQ2 positive, 1/2% DQ8 positive and 9/24% incomplete DQ2. Positive IgA-TGG was found in five out of the 95 relatives, and CD was diagnosed in three of them. Three relatives had the presence of HLA-DQ2, two were DQ2 incomplete (DQB1*02). Conclusion - The prevalence of celiac disease among first degree celiac patients´ relatives was 3.1%, 4.5 times higher than the general Portuguese population (0,7%) witch reinforces the need of extensive diagnostic screening in this specific group. HLA-DQ2 typing may be a tool in the diagnostic approach.

Patrimonio arqueológico : valoración a través de una pedagogía virtual. Proyecto de Aplicación práctica (PAP)

educación y difusión que responde a la urgente necesidad de crear conciencia y sentido de pertenencia alrededor del patrimonio arqueológico de la nación, para generar su protección y respeto por parte de la comunidad mediante el desarrollo de un KIT PEDAGÓGICO INTERACTIVO compuesto por tres CD’s: CD1. Parque arqueológico virtual. CD2. Juegos virtuales relacionados con el parque arqueológico. CD3. Tutoriales para los docentes.

Establishment of antibody panels and histochemical techniques in routine tumor diagnosis in Veterinary Pathology

In Portugal, Veterinary Pathology is developing rapidly, and in recent years we assist to the emergence of private laboratories and the restructuring of universities,polytechnics and public laboratories.The Portuguese Society of Animal Pathology,through its actions and its associates has been keeping the discussion among its peers in order to standardizethe criteria of description,classification and evaluation of cases which are the subject of our daily work.One of the last challenges is associated with the use of routine histochemical techniques and immunohistochemistry, in an effort to establish standardized panels for tumour diagnosis, which could eventually reduce each analysis cost.For this purpose a simple survey was built, in which all collaborators answered questions about the markers used for carcinoma, sarcoma and round cell tumour diagnosis, as well as general questions related with the subject. We obtained twenty-one answered to the questions, from public and private laboratories.In general, in most cases immunohistochemical and histochemical methods are used for diagnosis.The wide spectrum cytokeratins are universally used to confirm carcinoma, and vimentin for sarcoma. The CD3 marker is used by all laboratories to identify T lymphocytes. For the diagnosis of B-cell lymphoma, the marker used is not consensual. In each laboratory there are different markers for more specific situations and only two labs perform PCR techniques for diagnosis. These data will be presented to promote extended discussion,namely to reach a consensus when different markers are used.