A New Mouse Model That Spontaneously Develops Chronic Liver Inflammation and Fibrosis

Here we characterize a new animal model that spontaneously develops chronic inflammation and fibrosis in multiple organs, the non-obese diabetic inflammation and fibrosis (N-IF) mouse. In the liver, the N-IF mouse displays inflammation and fibrosis particularly evident around portal tracts and central veins and accompanied with evidence of abnormal intrahepatic bile ducts. The extensive cellular infiltration consists mainly of macrophages, granulocytes, particularly eosinophils, and mast cells. This inflammatory syndrome is mediated by a transgenic population of natural killer T cells (NKT) induced in an immunodeficient NOD genetic background. The disease is transferrable to immunodeficient recipients, while polyclonal T cells from unaffected syngeneic donors can inhibit the disease phenotype. Because of the fibrotic component, early on-set, spontaneous nature and reproducibility, this novel mouse model provides a unique tool to gain further insight into the underlying mechanisms mediating transformation of chronic inflammation into fibrosis and to evaluate intervention protocols for treating conditions of fibrotic disorders.

Chemokine Receptor Expression on Normal Blood CD56(+) NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population

Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56(+low) CD16(+) and CD56(+high)  CD16(-/+low) NK-cells. Conventional CD56(+low) and CD56(+high) NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56(+low) NK-cells are mainly CXCR1/CXCR2(+) and CXCR3/CCR5(-/+), whereas mostly CD56(+high) NK-cells are CXCR1/CXCR2(-) and CXCR3/CCR5(+). Both NK-cell subsets have variable CXCR4 expression and are CCR4(-) and CCR6(-). The CKR repertoire of the CD56(+low) NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56(+high) NK-cells mimics that of Th1(+) T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56(+int) NK-cells. These NK-cells are CXCR3/CCR5(+), they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57(-) and CD158a(-). In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56(+high) and CD56(+low) NK-cells populations.

Estudo funcional e fenotípico das células NK em esclerose sistémica

Introdução: A esclerose sistémica (ES) é uma doença reumática rara, onde se verifica uma reacção auto-imune, afectando o tecido conjuntivo. De etiologia desconhecida, esta patologia, dividide-se em dois subtipos: limitada e difusa, tendo como processos característicos e inter-relacionados: a lesão vascular, a reacção autoimune e a fibrose. As células NK modulam as doenças auto-imunes através das suas capacidades citotóxicas e de produção de citocinas. Deste modo procedeu-se ao estudo imunofenotípico e funcional de duas sub-populações de células Natural Killer (NK) (NK CD56dim e NK CD56bright). Métodos: Recorrendo à citometria de fluxo, caracterizamos fenotipicamente as células NK, com base na expressão de CD49e, CD29 e LAIR-1; funcionalmente, com base nas citocinas TNF-α e IFN-γ e citotoxicamente, com base na granzima B e na perforina. Resultados: Os nossos resultados sugerem uma diminuição da percentagem e valor absoluto de células NK totais e suas subpopulações, NK CD56dim e NK CD56bright, em doentes com ES, sobretudo na presença de úlceras digitais (UD) e fibrose pulmonar (FP). Observamos ainda uma diminuição da expressão de CD49e e um aumento da expressão de LAIR-1, nas células NK, em doentes com ES, principalmente nos que não apresentavam UD e FP. Num modo geral observou-se um aumento da frequência de células NK a expressar granzima B e perforina e um aumento da expressão destas duas proteínas. Conclusão: Concluímos que as células NK poderão ter um papel importante na fisiopatologia da ES, pelas alterações fenotípicas e funcionais, podendo, futuramente vir a ser utilizados na monitorização da severidade desta doença.

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.

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

International audience

Design and validation of a novel generic platform for the production of tetravalent IgG1-like bispecific antibodies

International audience

Role of chemerin and its receptors in mouse models of tumorigenesis

Dissertação de Mestrado, Oncobiologia: Mecanismos Moleculares do Cancro, Departamento de Ciências Biomédicas e Medicina, Universidade do Algarve, 2015

Fiber, a component for health gastrointestinal endocrine system

Dietary fiber was classified according to its solubility in an attempt to relate physiological effects to chemical types of fiber. Soluble fibers (B-glucans, gums, wheat dextrin, psyllium, pectin, inulin) were considered to have benefits on serum lipids, while insoluble fibers (cellulose, lignin, pectins, hemicelluloses) were linked with laxation benefits. More important characteristics of fiber in terms of physiological benefits are viscosity and fermentability. Viscous fibers (pectins, B-glucans, gums, psyllium) are those that have gel-forming properties in the intestinal tract, and fermentable fibers (wheat dextrin, pectins, B-glucans, gum, inulin) are those that can be metabolized by colonic bacteria. Objective: To summarize the beneficial effects of dietary fiber, as nutraceuticals, in order to maintain a healthy gastrointestinal system. Methods: Our study is a systematic review. Electronic databases, including PubMed, Medline, with supplement of relevant websites, were searched. We included randomized and non-randomized clinical trials, epidemiological studies (cohort and case-control). We excluded case series, case reports, in vitro and animal studies. Results: The WHO, the U.S. Food and Drug Administration (FDA), the Heart Foundation and the Romanian Dietary Guidelines recommends that adults should aim to consume approximately 25–30 g fiber daily. Dietary fiber is found in the indigestible parts of cereals, fruits and vegetables. There are countries where people don’t eat enough food fibers, these people need to take some kind of fiber supplement. Evidence has been found that dietary fiber from whole foods or supplements may (1) reduce the risk of cardiovascular disease by improving serum lipids and reducing serum total and low-density lipoprotein (LDL) cholesterol concentrations, (2) decreases the glycaemic index of foods, which leads to an improvement in glycemic response, positive impact on diabetes, (3) protect against development of obesity by increasing satiety hormone leptin concentrations, (4) reduced risk of developing colorectal cancer by normalizes bowel movements, improve the integrity of the epithelial layer of the intestines, increase the resistance against pathogenic colonization, have favorable effects on the gut microbiome, wich is the second genomes of the microorganisms, (5) have a positive impact on the endocrine system by gastrointestinal polypeptide hormonal regulation of digestion, (6) have prebiotic effect by short-chain fatty acids (SCFA) production; butyrate acid is the preferred energy source for colonic epithelial cells, promotes normal cell differentiation and proliferation, and also help regulate sodium and water absorption, and can enhance absorption of calcium and other minerals. Although all prebiotics are fiber, not all fiber is prebiotic. This generally refers to the ability of a fiber to increase the growth of bifidobacteria and lactobacilli, which are beneficial to human health, and (7) play a role in improving immune function via production of SCFAs by increases T helper cells, macrophages, neutrophils, and increased cytotoxic activity of natural killer cells. Conclusion: Fiber consumption is associated with high nutritional value and antioxidant status of the diet, enhancing the effects on human health. Fibers with prebiotic properties can also be recommended as part of fiber intake. Due to the variability of fiber’s effects in the body, it is important to consume fiber from a variety of sources. Increasing fiber consumption for health promotion and disease prevention is a critical public health goal.

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.

Infección por virus sincitial respiratorio y su relación con valores de IGG en niños críticos

Antecedente: La infección por el virus sincitial respiratorio (VSR) representa una elevada morbimortalidad, y en algunos casos necesidad de manejo en unidades de cuidado intensivo pediátrico (UCIP). La respuesta inmunológica influye de manera directa en la expresión de la severidad y pronóstico de los pacientes con infección respiratoria. Metodología: Estudio de una cohorte retrospectiva de pacientes con infección respiratoria grave secundaria a VSR, sin historia de inmunodeficiencia, atendidos en la UCIP del Hospital Universitario Clínica San Rafael. Se realizó análisis descriptivoglobaly de acuerdo a la categorización de las prueba de IgG. Resultados: De 188 pacientes que ingresaron a la UCIP, 13% presentaron infección por VSR (24), con una edad promedio de 7,3 (DE=3,6) meses. Pertenecían al sexo masculino79,83%. Se encontró que 12,5% tenían un valor de IgGbajo para su edad, 58,33% tenían valores en límite inferior y el 29,17% dentro de rangos normales para su edad. En los pacientes con IgG baja, fue mayor la presentación de choque séptico que no responde a líquidos (100 vs 92 vs 86%), la mediana de días de ventilación mecánica fue mayor (8 vs 6 vs 5 respectivamente), así como la mortalidad (67 vs 7,1 vs 0%). Conclusión: Nuestra serie encontró que aquellos pacientes con niveles bajos o valores en el límite inferior de IgG sérica tuvieron mayor compromiso sistémico, mayor duración de ventilación mecánica y mayor mortalidad. Se necesitan estudios prospectivos que relaciones niveles bajos de IgG con severidad y pronostico en estos pacientes con infección grave por VSR.

Impaired natural killing of MHC class I-deficient targets by NK cells expressing a catalytically inactive form of SHP-1.

NK cell function is negatively regulated by MHC class I-specific inhibitory receptors. Transduction of the inhibitory signal involves protein tyrosine phosphatases such as SHP-1 (SH2-containing protein tyrosine phosphatase-1). To investigate the role of SHP-1 for NK cell development and function, we generated mice expressing a catalytically inactive, dominant-negative mutant of SHP-1 (dnSHP-1). In this paper we show that expression of dnSHP-1 does not affect the generation of NK cells even though MHC receptor-mediated inhibition is partially impaired. Despite this defect, these NK cells do not kill syngeneic, normal target cells. In fact dnSHP-1-expressing NK cells are hyporesponsive toward MHC-deficient target cells, suggesting that non-MHC-specific NK cell activation is significantly reduced. In contrast, these NK cells mediate Ab-dependent cell-mediated cytotoxicity and prevent the engraftment with beta2-microglobulin-deficient bone marrow cells. A similar NK cell phenotype is observed in viable motheaten (mev) mice, which show reduced SHP-1 activity due to a mutation in the Shp-1 gene. In addition, NK cells in both mouse strains show a tendency to express more inhibitory MHC-specific Ly49 receptors. Our results demonstrate the importance of SHP-1 for the generation of functional NK cells, which are able to react efficiently to the absence of MHC class I molecules from normal target cells. Therefore, SHP-1 may play an as-yet-unrecognized role in some NK cell activation pathways. Alternatively, a reduced capacity to transduce SHP-1-dependent inhibitory signals during NK cell development may be compensated by the down-modulation of NK cell triggering pathways.

Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells.

Besides tumor cells, the tumor microenvironment harbors a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells. It is a complex and highly dynamic environment, providing very important cues to tumor development and progression. Tumor-associated endothelial cells play a key role in this process. On the one hand, they form tumor-associated (angiogenic) vessels through sprouting from locally preexisting vessels or recruitment of bone marrow-derived endothelial progenitor cells, to provide nutritional support to the growing tumor. On the other hand, they are the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby playing a central role in controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia is a critical parameter modulating the tumor microenvironment and endothelial/tumor cell interactions. Under hypoxic stress, tumor cells produce factors that promote tumor angiogenesis, tumor cell motility and metastasis. Among these factors, VEGF, a main angiogenesis modulator, can also play a critical role in the control of immune tolerance. This review discusses some aspects of the role of endothelial cells within tumor microenvironment and emphasizes their interaction with tumor cells, the extracellular matrix and with immune killer cells. We will also address the role played by circulating endothelial progenitor cells and illustrate their features and mechanism of recruitment to the tumor microenvironment and their role in tumor angiogenesis.

Síntese e estudos de sistemas de bioencapsulação associados a peptídeos antimicrobianos miméticos da toxina natural CcdB

Pós-graduação em Biotecnologia - IQ

Toxicity of synthetic and natural compounds on Tetranychus urticae and the predator Phytoseiulus macropilis

The objective of this work was to evaluate the toxicity of synthetic and natural compounds on Tetranychus urticae and the predator Phytoseiulus macropilis. Mortality and growth rates of T. urticae and its predator were evaluated after applications of: abamectin, clofentezine, fenpropathrin, fenpyroximate, propargite, sulfur and spiromesifen, at their recommended concentrations; neem oils (Natuneem and Sempre Verde Killer Neem at 1%); and aqueous extracts at 10% of Dieffenbachia brasiliensis, Annona squamosa, Ruta graveolens, Agave angustifolia, Melia azedarach, Sonchus oleraceus, Mentha spicata x M. suaveolens, Allium cepa, Laurus nobilis, and Eucalyptus saligna. The acute toxicity and the influence of the compounds on the instantaneous growth rate of the mites were carried out in laboratory. Extracts of A. cepa, A. angustifolia, neem oil-based products, spiromesifen, propargite, fenpyroximate, abamectin and fenpropathrin caused mortality higher than 83% on T. urticae. Extract of A. angustifolia, Natuneem and clofentezine did not cause significant mortality rates on P. macropilis. Agave angustifolia and Natuneem did not affect significantly the growth rate of this predator. Propargite, fenpyroximate, abamectin, fenpropathrin, spiromesifen and extract of L. nobilis severely affected P. macropilis population.