La perturbation de la barrière hémo-encéphalique et la migration des lymphocytes de la périphérie vers le système nerveux central (SNC) sont des événements précoces dans la formation des lésions cérébrales de sclérose en plaques (SEP). Dans ce contexte, les lymphocytes passent au travers des barrières hémo-encéphalique ou hémo-méningée pour atteindre le SNC et sont des contributeurs importants dans l’inflammation et les dommages tissulaires. Pour migrer à travers les barrières du SNC, les lymphocytes pathogéniques expriment des molécules d’adhérence. Identifier les acteurs clés à la migration des lymphocytes pathogéniques en estimant la contribution des molécules d’adhérence dans ce processus est la prochaine étape pour le développement de thérapies pour traiter la SEP. L’objectif de ce projet est d’explorer le rôle de deux molécules d’adhérence que sont ALCAM (de l’anglais : activated leukocytes cell adhesion molecule) et DICAM (de l’anglais : dual immunoglobulin domain containing cell adhesion molecule) dans la migration des lymphocytes pathogéniques vers le SNC pendant la SEP. Notre objectif principal se subdivise en deux sous-objectifs. En premier, notre but est de caractériser le rôle d’ALCAM dans le passage des lymphocytes B à travers les barrières du SNC dans un contexte neuroinflammatoire. En second, nous explorons le rôle de DICAM dans la migration des lymphocytes T auxiliaires 17 (TH17) vers le SNC en neuroinflammation. Nous faisons l’hypothèse qu’ALCAM contribue à la migration des lymphocytes B vers le SNC et que DICAM est impliqué dans la migration des lymphocytes TH17 à travers la barrière hémo-encéphalique pendant la SEP. Ces molécules d’adhérence seraient alors impliquées dans la pathogenèse de la SEP et seraient de potentielles cibles thérapeutiques pour traiter cette maladie. Nous avons d’abord utilisé une combinaison de spectrométrie de masse, PCR quantitative, cytométrie de flux et microscopie afin d’explorer l’expression de chacune de ses deux molécules d’adhérence sur les lymphocytes d’intérêt périphériques ex vivo ou différenciés in vitro. Des analyses en cytométrie en flux et microscopie nous ont permis de caractériser leur expression dans le sang périphérique et dans les lésions cérébrales de personnes atteintes de SEP. Ensuite, les expériences d’adhérence en flux et de migration in vitro effectuées en déplétant la molécule d’adhérence d’intérêt ont permis de mettre en évidence leur rôle dans différentes étapes de la migration des lymphocytes à travers les cellules endothéliales des barrières du SNC. Pour finir, le traitement de plusieurs modèles murins de SEP, appelés EAE (de l’anglais : experimental autoimmune encephalomyelitis), avec des anticorps bloquant anti-ALCAM ou anti-DICAM ont permis d’explorer le potentiel effet de tels traitements sur la sévérité de la maladie. Dans la première étude, nos résultats montrent qu’ALCAM est préférentiellement exprimée par les lymphocytes B pro-inflammatoires, mémoires et effecteurs au potentiel pathogénique. En tant que molécule d’adhérence, ALCAM contribue à leur migration à travers les cellules endothéliales des barrières hémo-encéphalique et hémo-méningée chez la souris et l’humain. De plus, nos expériences ont permis de montrer que la fréquence de lymphocytes B ALCAM+ est augmentée dans le sang périphérique des personnes atteintes de SEP et ces cellules sont aussi présentes dans les lésions et les infiltrats méningées en SEP. Finalement, bloquer ALCAM in vivo réduit la sévérité de la maladie EAE en diminuant l’infiltration des lymphocytes B au SNC. Dans la seconde étude, nous avons montré que parmi les sous-types de lymphocytes TH, DICAM est préférentiellement exprimée par les lymphocytes TH17. Dans les lésions de SEP, DICAM et son ligand αVβ3 co-localisent avec des marqueurs de cellules endothéliales suggérant que ces deux molécules pourraient être présentées à la lumière des vaisseaux aux lymphocytes TH17 circulants. Dans le sang périphérique, la fréquence de lymphocytes T CD4+ exprimant DICAM est augmentée chez les personnes atteintes de SEP et cette augmentation corrèle avec l’activité de la maladie. Nos expériences ont montré que DICAM est impliquée dans l’adhérence, l’arrêt et la diapédèse des lymphocytes TH17 à travers les cellules endothéliales de la barrière hémo-encéphalique in vitro et in vivo. Finalement, le traitement de souris EAE avec un anticorps bloquant DICAM permet de réduire la sévérité de la maladie et diminue la migration des lymphocytes TH17 vers le SNC. Nos résultats indiquent un rôle d’ALCAM dans la migration des lymphocytes B et que DICAM, préférentiellement exprimé par les TH17, médie leur migration vers le SNC. Bloquer ALCAM ou DICAM sont deux stratégies permettant de réduire l’accès au SNC de différents sous-types de cellules pathogéniques pendant la neuroinflammation. Ainsi, elles sont toutes deux de potentielles cibles thérapeutiques pour réduire la sévérité et la progression de la SEP. / Disruption of the blood-brain barrier and migration of lymphocytes from the periphery to the central nervous system (CNS) are early events in lesion formation during multiple sclerosis (MS). Lymphocytes readily cross the blood-brain barrier (BBB) and the blood-meningeal barrier (BMB) to infiltrate the CNS and are important contributors to inflammation and tissue damage. To migrate through the brain barriers, pathogenic lymphocytes express adhesion molecules. Identifying key players in lymphocyte migration by understanding the role of adhesion molecules is the next step to develop novel therapies to treat MS. The objective of this project is to explore the role of two distinct adhesion molecules ALCAM (activated leukocytes cell adhesion molecule) and DICAM (dual immunoglobulin domain containing cell adhesion molecule) in pathogenic lymphocytes migration to the CNS during MS. This thesis subdivides in two main objectives. First, we aim to characterize ALCAM role in B lymphocyte migration to the CNS during neuroinflammation. Second, we aim to explore DICAM role in T helper 17 (TH17) lymphocytes migration to the CNS in neuroinflammation. We hypothesized that ALCAM plays a role in B lymphocytes migration to the CNS during MS and that DICAM is involved in TH17 lymphocytes migration through the blood-brain barrier during MS. Those adhesion molecules might be involved in MS pathogenesis and therefore could become new therapeutic targets to treat MS. We first used mass spectrometry, quantitative PCR, flow cytometry and confocal microscopy to explore expression profiles of ALCAM and DICAM by peripheral lymphocytes subpopulations ex vivo and differentiated in vitro. Flow cytometry and confocal microscopy analysis also revealed how those adhesion molecules are expressed by lymphocytes in peripheral blood and brain lesions of people living with MS. Then, we performed flow adhesion and migration assay of lymphocytes depleted for the adhesion molecule of interest allowing us to address their role in multitstep migration process through brain barriers endothelial cells. Finally, using five distinct murine experimental autoimmune encephalomyelitis models (EAE), we explored how blocking ALCAM or DICAM in vivo could affect lymphocytes migration to the SNC and disease severity. In the first manuscript, we described that ALCAM is preferentially expressed by B lymphocytes with memory, pro-inflammatory and effector phenotypes. Functionally, ALCAM is involved in B lymphocyte migration through both the BBB and the BMB in mouse and human. Interestingly, we showed that ALCAM expressing B lymphocytes are increased in peripheral blood of people living with MS and they are recovered in meningeal and parenchymal MS lesions. Last, blocking ALCAM in vivo alleviates EAE severity by reducing B lymphocyte infiltration to the CNS. In the second manuscript, we showed that TH17 lymphocytes preferentially express DICAM and can adhere both to DICAM and its ligand αVβ3. Moreover, DICAM and αVβ3 are both overexpressed by inflamed brain endothelial cells. In MS lesions, we described that both molecules colocalize with endothelial cell markers suggesting that it could be presented to the vessel lumen to the circulating TH17 lymphocytes. In peripheral blood, we showed that DICAM+ memory CD4+ T lymphocytes frequency is increased in people living with MS and it correlates with active form of the disease. Then, we described DICAM as a player in TH17 lymphocyte adhesion, arrest and migration through BBB endothelial cells in vitro and in vivo. Last, we showed that treating mice with a neutralizing DICAM antibody in several distinct models of EAE, reduced disease severity and TH17 cell migration to the SNC. Our data provide evidence of the role of ALCAM in memory B lymphocyte migration and that DICAM is preferentially expressed by TH17 cells and mediate their migration to the CNS during neuroinflammation. Collectively, our findings indicate that blocking ALCAM or DICAM are two ways to restrict different pathogenic cells access to the CNS during neuroinflammation and thus potentially to reduce the severity and worsening of a disease like MS.

Toll-like receptors (TLRs) are a major group of pattern recognition receptors expressed on the surface of immune cells that recognize molecular patterns associated with all classes of pathogenic microorganisms. TLR4 recognizes the lipopolysaccharide component of Gram-negative bacterial cell walls and is the only TLR known to induce signaling through both the MyD88 and TRIF pathways. Basigin, a ubiquitous cell adhesion molecule, is a member of the immunoglobulin superfamily that has the ability to influence cell signaling mediated by the MyD88 and TRIF pathways, the same signaling pathways induced by the TLR4 receptor protein. Analysis of the Basigin protein sequence indicates the presence of a hydrophilic glutamate residue within the hydrophobic transmembrane domain, but no consensus binding sites for MyD88 or TRIF. The purpose of this study was to determine if Basigin uses TLR4 for signal transduction. It is hypothesized that Basigin interacts with TLR4 and that the glutamate residue plays a role in the interaction. Enzyme-linked immunosorbent binding assays were performed using endogenous TLR4 and recombinant Basigin proteins. These analyses demonstrated that binding of Basigin to TLR4 was significantly greater than that of the control protein and that the glutamate residue in the Basigin transmembrane domain does play a role in the interaction between Basigin and TLR4 as well as many hydrophobic residues in the Basigin transmembrane domain. The data suggest that Basigin interacts with TLR4 to influence signaling cascades using MyD88 and TRIF.

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-κB, facilitated nuclear translocation of NF-κB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-κB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-κB translocation, which are the characteristics of pulmonary endothelial cell activation.

<p>UVOD: Gojaznost je hronično, multifaktorijalno i kompleksno oboljenje povezano sa povećanim rizikom od aterosklerotskih kardiovaskularnih bolesti (KVB). Disfunkcija vaskularnog endotela predstavlja rani događaj u patofiziolo&scaron;kom kontinuumu aterosklerotskog procesa, a produženo izlaganje vaskularnog endotela faktorima rizika za aterosklerozu udruženim sa gojaznosti (insulinska rezistencija, dislipidemija, proinflamatorno/protrombozno stanje), može doprineti procesima aktivacije/disfunkcije endotela i progresiji ateroskleroze u supkliničku, odnosno kliničku formu bolesti. CILJ: Uporediti koncentracije solubilne forme adhezionih molekula &ndash; intracelularnog adhezivnog molekula &ndash;1 (sICAM&ndash;1) i E selektina (sE&ndash;selektin), između ispitanika sa hiperalimentacionim tipom gojaznosti i normalno uhranjenih zdravih ispitanika, kao i utvrditi eventualno postojanje razlika u koncentraciji sICAM&ndash;1 i sE&ndash;selektina između ispitanika kod kojih je merenjem debljine kompleksa intima medija karotidne arterije (IMK) uočen supklinički stadijum ateroskleroze i ispitanika koji imaju normalnu debljinu IMK. Ispitati povezanost parametara telesne kompozicije (ukupne masne mase tela i masne mase abdominalnih depoa), cirkuli&scaron;ućih koncentracija biomarkera disfunkcije vaskularnog endotela (sICAM&ndash;1 i sE&ndash;selektina) i IMK kod ispitanika sa hiperalimentacionim tipom gojaznosti. MATERIJAL I METODE: U istraživanje je uključeno 60 ispitanika sa hiperalimentacionim tipom gojaznosti bez pridruženih komorbiditeta i 30 zdravih normalno uhranjenih učesnika usklađenih sa ispitanicima po godinama života i polu koji su činili kontrolnu grupu. Svim ispitanicima su urađena antropometrijska merenja, analiza komponenata telesne kompozicije (bioelektrična impedansna analiza, Tanita Body Composition Analyzer BC &ndash; 418 MA III), laboratorijska analiza uzoraka krvi na automatizovanim analizatorskim sistemima sa određivanjem parametara metabolizma glukoze (bazalno i 2 h u toku oralnog glukoza tolerans testa), lipida i lipoproteina, inflamacije i homocisteina. Određivanje serumske koncentracije sICAM&ndash;1 i sE&ndash;selektina je vr&scaron;eno ELISA tehnikom (R&amp;D Systems, Inc. Minneapolis, USA). Vrednosti IMK&ndash;a su određivane pomoću karotidnog dupleks ultrazvuka (Aloka SSD&ndash;650 US system, Tokyo), a na osnovu izmerenih (IMK) i normalno očekivanih vrednosti IMK za svakog ispitanika je izračunavan IMK Z&ndash;skor. Supklinički stadijum ateroskleroze je definisan kao vrednost IMK Z&ndash;skora veća od 1 (&scaron;to odgovara vrednosti IMK većoj od 95 percentila normalno očekivane vrednosti u odnosu na pol i godine života ispitanika). REZULTATI: Ispitanici sa hiperalimentacionim tipom gojaznosti su imali statistički značajno vi&scaron;e vrednosti medijane serumske koncentracije sE&ndash;selektina u poređenju sa medijanom serumske koncetracije sE&ndash;selektina učesnika u kontrolnoj grupi (36,2 (33,21&ndash;43.7) vs. 25,14 (23,1&ndash;29,48) ng/mL, P=0,00). Gojazni ispitanici III stepena gojaznosti su imali statistički značajno vi&scaron;u medijanu serumske koncenracije sE&ndash;selektina u odnosu na medijanu sE&ndash;selektina u ispitanika I stepena gojaznosti (41,5 (36,58&ndash;49,48) vs. 34,34 (22,49&ndash;36,62) ng/mL, P=0,00), odnosno medijanu sE&ndash;selektina u ispitanika II stepena gojaznosti (41,5 (36,58&ndash;49,48) vs. 32,1 (26,1&ndash;43,64) ng/mL, P=0,00). Nije uočena statistički značajna razlika u medijani serumske koncentracije sE&ndash;selektina između ispitanika I i II stepena gojaznosti (34,34 (22,49&ndash;36,62) vs. 32,1 (26,1&ndash;43,64) ng/mL, P=0,12). Gojazni ispitanici su imali statistički značajno vi&scaron;e vrednosti medijane serumske koncentracije sICAM&ndash;1 u poređenju sa medijanom serumske koncetracije sICAM&ndash;1 učesnika u kontrolnoj grupi (266,8 (245,8&ndash;326,73) vs.183,32 (167,9&ndash;208,57), P=0,00). U ispitivanoj grupi gojaznih uočena je statistički značajna razlika u medijani koncentracije sICAM&ndash;1 između ispitanika u I, II i III stepena gojaznosti (200,6 (190,26 - 264,4) vs. 278,5 (219,54 - 343,24) vs. 329,6 (259,2 - 350,34) ng/mL, P=0,00). Učestalost IMK Z&ndash;skor &gt; 1 je bila statistički značajno eća u gojaznih ispitanika u odnosu na kontrolnu grupu (36/60 vs. 7/30, P=0,00). Ispitanici sa IMK Z&ndash;skor &gt; 1 su imali statistički značajno vi&scaron;u medijanu koncentracije sICAM&ndash;1 u odnosu na ispitanike kod kojih je IMK Z&ndash;skor &le; 1 (295,4 (238,46&ndash;340,38) vs. 244,2 (227,35&ndash;260,38), P=0.00). Regresionom analizom (R2=0,71, korigovani R2=0,59) je utvrđeno da su parametri hsCRP (&beta;=0,45, P=0,00), HOMA&ndash;IR (&beta;=0,44, P=0,035) i ISI (&beta;=&ndash;0,36, P=0,028) nezavisno i statistički značajno povezani sa serumskom koncentracijom sE&ndash;selektina. Regresionom analizom (R2=0,65, korigovani R2=0,56) je utvrđeno da parametri ITM (&beta;=0,55, P=0,00), trigliceridi (&beta;=0,30, P=0,00), HDL holesterol (&beta;=&ndash;0,31, P=0,00), odnos TG/HDL&ndash;holesterol (&beta;=0,33, P=0,01), hsCRP (&beta;=0,31, P=0,00) i fibrinogen (&beta;=0,34, P=0,00) su nezavisno i statistički značajno povezani sa serumskom koncentracijom sICAM&ndash;1. U faktorskoj analizi je izdvojeno pet faktora &ldquo;gojaznost&rdquo;, &ldquo;insulinska rezistencija&rdquo;, &ldquo;aterogeni faktor&rdquo;, &ldquo;endotelna disfunkcija i vaskularna inflamacija&rdquo; i &ldquo;metabolički faktor&rdquo; koji obja&scaron;njavaju 69.72% ukupne varijanse ispitivanog uzorka. U multivarijabilnom modelu sa svim faktorima zajedno kojim je obja&scaron;njeno ukupno 75% varijanse, jedino je faktor gojaznost imao statički značajan i nezavistan uticaj na vrednost IMK Z&ndash;skor &gt; 1 (OR=2,74 (CI 1,18&ndash;6,33), P=0,019). U faktoru gojaznost su se izdvojili parametri: FAT trunk (%), FAT (%), OS (cm), ITM (kg/m2), LDL &ndash; holesterol (mmol/L), SP (mmHg), HOMA1&ndash;%B, fibrinogen (g/L), ApoB/apoA-I i hsCRP (mg/L). Univarijantom logističkom regresijom je uočeno da porast u koncentraciji LDL&ndash;H (OR=5,33 (CI 1,9&ndash;14,2), P=0,02) i koncentraciji hsCRP&ndash;a (OR=2,53 (CI 1,3&ndash;3,98),P=0,017) povećava rizik za postojanje vrednosti IMK Z&ndash;skor &gt; 1. ZAKLJUČAK: Cirkuli&scaron;uće serumske koncentracije biomarkera disfunkcije vaskularnog endotela, sE&ndash;selektina i sICAM&ndash;1, su značajno vi&scaron;e kod ispitanika sa hiperalimentacionim tipom gojaznosti u odnosu na njihove koncentracije u normalno uhranjenih ispitanika. U gojaznih ispitanika, koncentracija sE&ndash;selektina je povezana sa vrednostima indeksa insulinske rezistencije i biomarkera inflamacije, dok je koncentracija sICAM&ndash;1 značajno povezana sa udelom masne mase u ukupnoj telesnoj masi, vrednostima biomarkera inflamacije i proaterogenih lipidskih parametara. Ispitanici kod kojih postoji uvećanje abdominalnih masnih depoa i ukupnog udela masnog tkiva u telesnoj masi, vrednosti SKP, koncentracije LDL &ndash; holesterola, vrednosti lipoproteinskog indeksa ApoAI/apoB, bazalne insulinemije i biomarkera inflamacije, imaju trostruko povećan rizik od supkliničkog stadijuma ateroskleroze. U gojaznih osoba prilikom procene rizika od aterosklerotskih KVB, potrebno je utvrditi fenotipske osobine vaskularnog endotela i eventualno postojanje supkliničkog stadijuma ateroskleroze, da bi se definisale adekvatne preventivne mere i sagledale potencijalne terapijske mogućnosti.</p> / <p>INTRODUCTION: Obesity is a chronic, multifactorial and complex disease associated with an increased risk of atherosclerotic cardiovascular diseases (CVD). Vascular endothelial dysfunction is an early event in the pathophysiological continuum of atherosclerotic process. The prolonged exposure of vascular endothelium to classical and obesity associated risk factors (insulin resistance, dyslipidemia, proinflammatory state) could further promote deterioration of endothelial function and progression of atherosclerosis to subclinical or clinical form of disease. OBJECTIVE: The aim of the study was to compare the concentration of soluble forms of adhesion molecules, intracellular adhesion molecule-1 (sICAM-1) and E-selectin (sE-selectin), between obese subjects and normal weight healthy subjects, as well as to determine the possible existence of differences in concentration of sICAM-1 and sE-selectin among subjects with subclinical stage of atherosclerosis (assessed by measuring the thickness of the intima media complex of the carotid artery (IMT)), and subjects who have a normal value of IMT. Also, the aim was to determine the association between the parameters of body composition (total body fat mass and fat mass intra-abdominal depots), circulating concentrations of sICAM-1 and sE-selectin, and value of IMT in obese subjects. MATERIALS AND METHODS: The study included 60 obese nondiabetic subjects, without preexisting CVD and other associated comorbidity, and 30 healthy normal weight age and sex matched participants. All subjects underwent anthropometric measurements, analysis of the components of body composition (bioelectrical impedance analysis, Tanita Body Composition Analyzer BC - 418 MA III), laboratory analysis of blood samples (automated analyzer systems) with determining the parameters of glucose metabolism (basal and 2 h during the oral glucose tolerance test), lipids and lipoproteins, inflammation and homocysteine. Serum concentrations of sICAM-1 and sE-selectin were determined by ELISA (R &amp; D Systems, Inc., Minneapolis, USA). The values of IMK were determined by carotid duplex ultrasound (Aloka &ndash; ProSound ALPHA 10). IMK Z-score was calculated using the measured and the normal expected values of IMT for each patient. Subclinical stage of atherosclerosis was defined as the value of IMT Z-score greater than 1 (corresponding to the 95th sex-age-specific percentile of IMT measurements). RESULTS: Obese subjects had significantly higher median sE-selectin serum concentrations compared to median serum concentrations of sE-selectin in the normal weight subjects (36.2 (33.21-43.7) vs 25.14 (23.1-29.48) ng/mL, P=0.00). Morbid obesity subjects had significantly higher sE-selectin median serum concentration compared to the median sE-selectin concentration in moderate obese subjects (41.5 (36.58-49.48) vs 34.34 (22.49-36.62) ng/mL, P=0.00), and compared to the median sE-selectin concentration in severely obese subjects (41.5 (36.58-49.48) vs. 32.1 (26.1-4364) ng / mL, P=0.00). Obese subjects had significantly higher median sICAM-1 serum concentration compared to median sICAM-1 serum concentration in the control group (266.8 (245.8-326.73) vs. 183.32 (167.9-208.57), P=0.00). In the obese group, we observed a statistically significant difference in median sICAM-1 serum concentrations between moderate, severely and morbid obese subjects (200.6 (190.26-264.4) vs. 278.5 (219.54-343.24) vs. 329.6 (259.2-350.34) ng/mL, P=0.00). The frequency of IMT Z-score&gt; 1 was significantly higher in the obese group compared to control group (36/60 vs. 7/30, P=0.00). Subjects with IMT Z-score&gt; 1 had significantly higher median concentrations of sICAM-1 compared to those in which the IMK Z-score &le; 1 (295.4 (238.46-340.38) vs. 244.2 ( 227.35-260.38), P=0.00). In regression analysis (R2=0.71, adjusted R2=0.59), hsCRP (&beta;=0.45, P=0.00), HOMA-IR (&beta;=0.44, P=0.035) and ISI (&beta;=-0.36, P=0.028) were independently and significantly associated with serum sE-selectin concentration. In regression analysis (R2=0.65, adjusted R2=0.56), BMI (&beta;=0.55, P=0.00), triglycerides (&beta;=0.30, P=0.00), HDL cholesterol (&beta;=-0.31, P=0.00), the ratio of TG/HDL-cholesterol ratio (&beta;=0.33, P=0.01), hsCRP (&beta;=0.31, P=0.00 ) and fibrinogen (&beta;=0.34, P=0.00) were independently and significantly associated with serum sICAM-1 concentration. In the Factor analysis, five factors &quot;obesity&quot;, &quot;insulin resistance&quot;, &quot;atherogenic factor,&quot; &quot;endothelial dysfunction and vascular inflammation&quot; and &quot;metabolic factor&quot; explained 69.72% of the total variance of the test sample. In a multivariate model with all the factors together (75% of the total variance), &quot;obesity&quot; factor was significantly and independently associated with IMT Z-score&gt; 1 (OR=2.74 (CI 1.18-6.33), P=0.019). The &quot;obesity&quot; factor consisted of parameters: trunk fat (%), fat (%), waist (cm), BMI (kg/m2), LDL &ndash; cholesterol (mmol/L), systolic blood presure (mmHg), HOMA1-% B, fibrinogen (g/L), Apo B/apoA-I and hsCRP (mg/L). Logistic regression analysis showed that independent predictors of IMT Z-score&gt; 1 were LDL-cholesterol (OR=5.33(CI 1.9-14.2), P=0.02) and hsCRP (OR=2.53 (CI 1.3-3.98), P=0.017). CONCLUSION: Circulating serum concentrations of endothelial dysfunction biomarkers, sE-selectin and sICAM-1, were significantly higher in obese subjects compared to concentration in the normal weight subjects. In obese subjects, the concentration of sE-selectin was associated with insulin resistance and biomarkers of inflammation, whereas sICAM-1 concentration was associated with fat mass, inflammation biomarkers and the proatherogenic lipid parametars. In individuals with increased abdominal fat depots and total proportion of fat mass in the body weight, values of SBP, LDL-C, ApoB/apoA-I, basal insulin levels and biomarkers of inflammation, there is threefold increased risk of subclinical stages of atherosclerosis. In order to define an adequate preventive measures and possible therapeutic options for atherosclerotic CVD in obese subjects, it is necessary to assess the phenotypic characteristics of vascular endothelium and possible presence of subclinical stage of atherosclerosis.</p>

INTRODUÇÃO: A obesidade é uma das causas de hipogonadismo (HG) secundário no homem. A terapia de reposição padrão de testosterona (TRT) é associada à melhora dos parâmetros metabólicos, mas pode levar à infertilidade. Apenas recentemente indicou-se que não há novas evidências nível 1 para apoiar uma conexão definitiva entre TRT e eventos cardiovasculares (CV). OBJETIVO: Avaliar os efeitos do Citrato de Clomifeno (CC) em homens jovens com hipogonadismo associado à obesidade diagnosticado por testosterona total (TT) <= 300 ng/dL em duas ocasiões, sintomas positivos no questionário ADAM, hormônio Luteinizante (LH) baixo ou inadequadamente normal (VR: 1,7 - 8,6 UI/L). MÉTODOS: Estudo randomizado, duplo cego, controlado por placebo (PLB), longitudinal em centro único. Setenta e oito pacientes com idade entre 36,5±7,8 anos, índice de massa corporal (IMC) 46,2±8,5 kg/m2 foram randomizados (1:1) para receber CC 50 mg ou PLB durante 12 semanas. Os pacientes foram avaliados através de: 1) Parâmetros clínicos: Questionário ADAM, número de intercursos sexuais, queixa de insatisfação com a vida sexual; 2) Parâmetros hormonais: dosagem sérica de TT, testosterona livre, Estradiol (E2), LH, hormônio folículo estimulante (FSH), SHBG, relação TT:E2; 3) Parâmetros de composição corporal: IMC, circunferência abdominal (CA) e análise de bioimpedanciometria; 4) Parâmetros metabólicos: pressão arterial sistólica e diastólica, glicemia em jejum (GJ), hemoglobina glicada (HbA1c), índice HOMA-IR, colesterol total e frações, triglicérides; 5) Parâmetros de resposta CV: dilatação fluxo mediada artéria braquial (FMDAB), níveis circulantes de sICAM-1, sVCAM-1, Selectina-sE e quantificação de células endoteliais progenitoras (CEPs) por citometria de fluxo; 6) Efeitos adversos: hematócrito, antígeno prostático específico sérico (PSA), questionário internacional de sintomas prostáticos (I-PSS), dosagem sérica de alanina aminotransferase (ALT), aspartato aminotransferase (AST), e efeitos adversos autorreferidos. RESULTADOS: Na randomização os dois grupos foram semelhantes em relação à idade (CC: 35,5±7,8 anos, PLB: 35,6±7,8; p= 0,951), IMC (CC: 45,5±11,3 kg/m2; PLB: 47,2±9,6; p= 0,470), CA (CC: 137,5±17,9 cm; PLB: 140,2±19,6; p= 0,526) e testosterona total (CC: 225,8±70,0 ng/dL; PLB: 216,0±72,1; p= 0,543). Não houve diferenças nos parâmetros de resposta clínica, exceto com relação à queixa de perda de vigor nas ereções (p < 0,001). Observou-se elevação significativa (p= < 0,001) de TT, Testosterona livre, E2, LH, FSH e SHBG no grupo CC em comparação com PLB. Houve um aumento significativo (p < 0,001) na massa magra e na massa muscular; e também na massa livre de gordura (p= 0,004). O CC reduziu HDL em comparação com PLB (p < 0,001) e não mostrou efeito em outros parâmetros metabólicos. Não houve significância estatística nos parâmetros CV, indicando efeito nulo do tratamento. CC reduziu ALT (p < 0,001) e aumentou o PSA (p= 0,023) dentro dos limites da normalidade. CONCLUSÕES: CC foi efetivo para melhorar os parâmetros de resposta hormonal e afetou positivamente um parâmetro de resposta clínica (perda de vigor nas ereções). Apesar das alterações na composição corporal, não se observou melhora do perfil metabólico. No entanto, o CC não ocasionou resposta adversa nos parâmetros CV. O tratamento CC para HG parece ser uma alternativa efetiva em jovens obesos que desejam preservar sua fertilidade, mas ensaios clínicos de seguimento em longo prazo e com maior número de participantes são necessários para melhor análise do perfil metabólico e de sintomas, além de impactos CV / INTRODUCTION: Obesity can cause secondary hypogonadism in man. The standard testosterone replacement therapy (TRT) improves metabolic parameters but can lead to infertility. Only recently TRT was not clearly associated with adverse cardiovascular (CV) events, but its impacts on endothelial function are still controversial. AIM: To evaluate the effects of Clomiphene Citrate (CC) in out clinic young man with obesity related hypogonadism: total testosterone (TT) <= 300 ng/dL on two occasions, positive symptoms in ADAM questionnaire, Luteinizing Hormone (LH) low or inappropriate normal (RV: 1.7-8.6 IU/liter). METHODS: This is a randomized, double blind, placebo-controlled, parallel group, single-center study. Seventy eight patients aged 36.5±7.8 years, Body mass index (BMI) 46.2±8.5 kg/m2 were randomized (1:1) to receive CC 50 mg or Placebo (PLB) during 12 weeks. MAIN OUTCOME MEASURES: 1) Clinical symptomology: ADAM Questionnaire, number of sexual intercourses and satisfaction with sexual life; 2) Hormonal monitoring: serum TT, Free testosterone, Estradiol (E2), LH and Follicle-stimulating hormone (FSH), SHBG, TT/E2 ratio; 3) Body composition and anthropometric measurements: BMI, waist circumference (WC) and Bioelectric Impedance analysis parameters; 4) Metabolic response parameters: systolic and diastolic blood pressure, fasting blood glucose (FBG), glycated hemoglobin (HbA1c), serum cholesterol and fractions, triglycerides; 5) CV assessment by endothelial function parameters: Flowmediated dilatation of the brachial artery (FMDAB), circulating levels of sICAM-1, sVCAM-1, E-selectin and flow cytometry endothelial progenitor cells (EPCs); 6) Adverse outcomes: Hematocrit, serum Prostate-Specific Antigen (PSA), International Prostate Symptom Score (I-PSS), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Selfreported Adverse Effects. RESULTS: Two groups were similar with regard to age (CC: 35.5±7.8 years; PLB: 35.6±7.8; P=0.951), BMI (CC: 45.5±11.3 kg/m2; PLB: 47.2±9.6; P=0.470), WC (CC: 137.5±17.9 cm; PLB: 140.2±19.6; P=0.526) and total testosterone (CC: 225.8±70.0 ng/dL; PLB: 216.0±72.1; P=0.543) in baseline data. There was an improvement in one sexual complaint (weaker erections) (P < 0.001) and there were significant improvements (P < 0.001) in TT, Free Testosterone, E2, LH, FSH and SHBG in CC group (vs. PLB). There was a gain in lean mass (P < 0.001), free fat mass (P=0.004) and muscle mass (P < 0.001). CC reduced HDL compared to PLB (P < 0.001) and showed no effect in other metabolic parameters. No statistical significance was seen in CV parameters. CC reduced ALT (P < 0.001) and increased PSA (P=0.023). CONCLUSIONS: CC was effective in increase hormonal response parametersand improved one sexual complaint (weaker erections). Despite body composition changes, CC did not improved metabolic profile and lowered LDL cholesterol. CC showed no adverse response in CV parameters. CC treatment for HG appears to be an effective alternative in young obese men wishing to preserve their fertility but long-term follow-up trials to better analyze the metabolic profile and CV outcomes are needed

INTRODUÇÃO: A obesidade é uma das causas de hipogonadismo (HG) secundário no homem. A terapia de reposição padrão de testosterona (TRT) é associada à melhora dos parâmetros metabólicos, mas pode levar à infertilidade. Apenas recentemente indicou-se que não há novas evidências nível 1 para apoiar uma conexão definitiva entre TRT e eventos cardiovasculares (CV). OBJETIVO: Avaliar os efeitos do Citrato de Clomifeno (CC) em homens jovens com hipogonadismo associado à obesidade diagnosticado por testosterona total (TT) <= 300 ng/dL em duas ocasiões, sintomas positivos no questionário ADAM, hormônio Luteinizante (LH) baixo ou inadequadamente normal (VR: 1,7 - 8,6 UI/L). MÉTODOS: Estudo randomizado, duplo cego, controlado por placebo (PLB), longitudinal em centro único. Setenta e oito pacientes com idade entre 36,5±7,8 anos, índice de massa corporal (IMC) 46,2±8,5 kg/m2 foram randomizados (1:1) para receber CC 50 mg ou PLB durante 12 semanas. Os pacientes foram avaliados através de: 1) Parâmetros clínicos: Questionário ADAM, número de intercursos sexuais, queixa de insatisfação com a vida sexual; 2) Parâmetros hormonais: dosagem sérica de TT, testosterona livre, Estradiol (E2), LH, hormônio folículo estimulante (FSH), SHBG, relação TT:E2; 3) Parâmetros de composição corporal: IMC, circunferência abdominal (CA) e análise de bioimpedanciometria; 4) Parâmetros metabólicos: pressão arterial sistólica e diastólica, glicemia em jejum (GJ), hemoglobina glicada (HbA1c), índice HOMA-IR, colesterol total e frações, triglicérides; 5) Parâmetros de resposta CV: dilatação fluxo mediada artéria braquial (FMDAB), níveis circulantes de sICAM-1, sVCAM-1, Selectina-sE e quantificação de células endoteliais progenitoras (CEPs) por citometria de fluxo; 6) Efeitos adversos: hematócrito, antígeno prostático específico sérico (PSA), questionário internacional de sintomas prostáticos (I-PSS), dosagem sérica de alanina aminotransferase (ALT), aspartato aminotransferase (AST), e efeitos adversos autorreferidos. RESULTADOS: Na randomização os dois grupos foram semelhantes em relação à idade (CC: 35,5±7,8 anos, PLB: 35,6±7,8; p= 0,951), IMC (CC: 45,5±11,3 kg/m2; PLB: 47,2±9,6; p= 0,470), CA (CC: 137,5±17,9 cm; PLB: 140,2±19,6; p= 0,526) e testosterona total (CC: 225,8±70,0 ng/dL; PLB: 216,0±72,1; p= 0,543). Não houve diferenças nos parâmetros de resposta clínica, exceto com relação à queixa de perda de vigor nas ereções (p < 0,001). Observou-se elevação significativa (p= < 0,001) de TT, Testosterona livre, E2, LH, FSH e SHBG no grupo CC em comparação com PLB. Houve um aumento significativo (p < 0,001) na massa magra e na massa muscular; e também na massa livre de gordura (p= 0,004). O CC reduziu HDL em comparação com PLB (p < 0,001) e não mostrou efeito em outros parâmetros metabólicos. Não houve significância estatística nos parâmetros CV, indicando efeito nulo do tratamento. CC reduziu ALT (p < 0,001) e aumentou o PSA (p= 0,023) dentro dos limites da normalidade. CONCLUSÕES: CC foi efetivo para melhorar os parâmetros de resposta hormonal e afetou positivamente um parâmetro de resposta clínica (perda de vigor nas ereções). Apesar das alterações na composição corporal, não se observou melhora do perfil metabólico. No entanto, o CC não ocasionou resposta adversa nos parâmetros CV. O tratamento CC para HG parece ser uma alternativa efetiva em jovens obesos que desejam preservar sua fertilidade, mas ensaios clínicos de seguimento em longo prazo e com maior número de participantes são necessários para melhor análise do perfil metabólico e de sintomas, além de impactos CV / INTRODUCTION: Obesity can cause secondary hypogonadism in man. The standard testosterone replacement therapy (TRT) improves metabolic parameters but can lead to infertility. Only recently TRT was not clearly associated with adverse cardiovascular (CV) events, but its impacts on endothelial function are still controversial. AIM: To evaluate the effects of Clomiphene Citrate (CC) in out clinic young man with obesity related hypogonadism: total testosterone (TT) <= 300 ng/dL on two occasions, positive symptoms in ADAM questionnaire, Luteinizing Hormone (LH) low or inappropriate normal (RV: 1.7-8.6 IU/liter). METHODS: This is a randomized, double blind, placebo-controlled, parallel group, single-center study. Seventy eight patients aged 36.5±7.8 years, Body mass index (BMI) 46.2±8.5 kg/m2 were randomized (1:1) to receive CC 50 mg or Placebo (PLB) during 12 weeks. MAIN OUTCOME MEASURES: 1) Clinical symptomology: ADAM Questionnaire, number of sexual intercourses and satisfaction with sexual life; 2) Hormonal monitoring: serum TT, Free testosterone, Estradiol (E2), LH and Follicle-stimulating hormone (FSH), SHBG, TT/E2 ratio; 3) Body composition and anthropometric measurements: BMI, waist circumference (WC) and Bioelectric Impedance analysis parameters; 4) Metabolic response parameters: systolic and diastolic blood pressure, fasting blood glucose (FBG), glycated hemoglobin (HbA1c), serum cholesterol and fractions, triglycerides; 5) CV assessment by endothelial function parameters: Flowmediated dilatation of the brachial artery (FMDAB), circulating levels of sICAM-1, sVCAM-1, E-selectin and flow cytometry endothelial progenitor cells (EPCs); 6) Adverse outcomes: Hematocrit, serum Prostate-Specific Antigen (PSA), International Prostate Symptom Score (I-PSS), Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST) and Selfreported Adverse Effects. RESULTS: Two groups were similar with regard to age (CC: 35.5±7.8 years; PLB: 35.6±7.8; P=0.951), BMI (CC: 45.5±11.3 kg/m2; PLB: 47.2±9.6; P=0.470), WC (CC: 137.5±17.9 cm; PLB: 140.2±19.6; P=0.526) and total testosterone (CC: 225.8±70.0 ng/dL; PLB: 216.0±72.1; P=0.543) in baseline data. There was an improvement in one sexual complaint (weaker erections) (P < 0.001) and there were significant improvements (P < 0.001) in TT, Free Testosterone, E2, LH, FSH and SHBG in CC group (vs. PLB). There was a gain in lean mass (P < 0.001), free fat mass (P=0.004) and muscle mass (P < 0.001). CC reduced HDL compared to PLB (P < 0.001) and showed no effect in other metabolic parameters. No statistical significance was seen in CV parameters. CC reduced ALT (P < 0.001) and increased PSA (P=0.023). CONCLUSIONS: CC was effective in increase hormonal response parametersand improved one sexual complaint (weaker erections). Despite body composition changes, CC did not improved metabolic profile and lowered LDL cholesterol. CC showed no adverse response in CV parameters. CC treatment for HG appears to be an effective alternative in young obese men wishing to preserve their fertility but long-term follow-up trials to better analyze the metabolic profile and CV outcomes are needed

Umbilical cord blood (UCB) is used as a source of haematopoietic stem cells (HSCs) for transplantation but shows defective homing to the bone marrow niche and delayed haematological reconstitution. Following transplantation, HSCs will home to the bone marrow in response to the CXCL12 chemokine, adhere to the bone marrow sinusoidal endothelial cells and then migrate into and lodge in bone marrow niches. In addition to CXCR4, a variety of molecules have been described as being important in these processes. In this laboratory, junctional adhesion molecule-A (JAM-A) was shown to be expressed on human UCB CD133⁺/CD34⁺ cells and regulated by hypoxia. In this thesis, further phenotypic studies show that this molecule is most highly expressed on human CD41a⁺ megakaryocytes and CD14⁺ monocytes/macrophages in UCB. JAM-A was also found to be expressed on all human UCB CD133⁺ cells, which have been shown by others to encompass the HSCs and early myeloid-lymphoid precursors and on the majority of CD34⁺ haematopoietic progenitor cells (HPCs). While it is also present on bone marrow sinusoidal endothelium (BMEC), JAM-A is not detected on cultured bone marrow mesenchymal stromal cells (MSCs). JAM-A blockade, silencing and overexpression experiments showed that JAM-A contributes to, but is not solely responsible for, the adhesion of CD34⁺ haematopoietic progenitor cells to IL-1β activated BMEC-60 cells and fibronectin. Lack of significance in cell migration suggested that JAM-A is more likely to act as an adhesion molecule or a regulator of adhesion rather than as a migratory molecule in such cells. Further functional studies using the proximity ligation assay highlight a potential association of JAM-A with CXCR4 and the adhesion molecules, tetraspanin CD82 and integrin β1. Mechanistic studies were commenced to establish if JAMA could modulate CXCR4 signalling following CXCL12 stimulation, but time constraints prevented these from being completed. These preliminary experiments which were carried out first in the Jurkat cell line lacking JAM-A or transduced to express JAM-A, however, suggest that JAM-A may modulate CXCL12-induced Rap1 phosphorylation and ERK1/2 phosphorylation. The former pathway is important for integrin function and the latter pathway is important in cell adhesion. The results described here, although requiring finalisation, support the hypothesis that JAM-A acts as an adhesion molecule and also may fine tune CXCR4 and integrin mediated functions on human CD34⁺ cells, thereby potentially regulating engraftment of these cells to the bone marrow niche.

The anatomy of the cranial peripheral nervous system has been studied for over a century, yet surprisingly little is known about how the nerves are guided to their targets. The study of the development of these nerves has important implications for our understanding of craniofacial anomalies and possible treatments for both injury and genetic disorders of nerve development such as Goldenhar-Gorlin syndrome. We have discovered that retinoic acid (RA) may play a role in the development of the trigeminal nerve. Inhibition of retinoic acid receptors (RAR) results in trigeminal nerves that become unbundled or defasciculated in the eye region. To further understand how RA is affecting trigeminal development we searched for genes downregulated in response to RAR inhibition by the inhibitor BMS-453 and have identified neurotrophin-3 (NT-3), activated leukocyte cell adhesion molecule (ALCAM) and Semaphorin 4B (Sema4B). We have analyzed the expression patterns of Sema4B and NT-3 by in situ hybridization and have found NT-3 expression in the eye and Sema4B in the embryonic target of the trigeminal nerve, lens of the eye and in the pharyngeal arches. ALCAM has been analyzed via qRT-PCR and its transcription is downregulated just prior to the observed defasciculation phenotype. The pattern of expression of these genes combined with known expression of NT-3 receptors allows us to suggest a model whereby RA signaling regulates Sema4B, ALCAM and NT-3, which support the survival, guidance and fasciculation of the trigeminal nerve. This work has the potential to better understanding of the complex nature of cranial nervous system development.

There is increasing evidence that chronic inflammation is tightly linked to diseases associated with endothelial dysfunction, including the induction of aberrant angiogenesis. While leukocytes have been described as mediators of inflammation-associated angiogenesis, the effects of direct chronic endothelial activation have not been addressed in this context. Using an uncleavable mutant of the transmembrane form of tumor necrosis factor-α (TNF-α), we have established models of stable TNF-α expression in endothelial cells in vitro and in transgenic mice in vivo. In the in vitro model, continuous endothelial activation leads to increased leukocyte cellular adhesion molecule expression and intracellular reactive oxygen species, hallmarks of a proinflammatory and dysfunctional endothelium. In addition, stable expression of TNF-α in endothelial cells increased angiogenic sprout formation in the presence but also in the absence of angiogenic growth factors. The partial neutralization of this effect by TNF-α antibodies and the inability of conditioned media from stable TNF-α-expressing endothelial cells to induce angiogenic activities in control endothelial cells suggest that this effect does not require expression of additional autocrine factors, but is an autonomous effect of the transmembrane TNF on the endothelial cells. Furthermore, using the Matrigel plug assay in vivo, increased angiogenesis was observed in endothelial TNF-α-expressing transgenic versus control mice. In conclusion, chronic inflammatory changes mediated by TNF-α can induce angiogenesis in vitro and in vivo, suggesting endothelial cell activation as a direct link between inflammation and angiogenesis. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Objective: We aimed to assess the prognostic value of circulating tumor cells (CTC) in patients with advanced gastric and gastroesophageal adenocarcinomas. Methods: The presence of CTC was evaluated in 62 patients with advanced gastric and gastroesophageal adenocarcinomas before systemic therapy and at follow-up through immunomagnetic enrichment for mucin 1- and epithelial cell adhesion molecule (EpCAM)-positive cells, followed by real-time RT-PCR of the tumor-associated genes KRT19 , MUC1 , EPCAM , CEACAM5 and BIRC5 . Results: The patients were stratified into groups according to CTC detection (CTC negative: with all marker genes negative; CTC positive: with at least 1 of the marker genes positive). Patients who were CTC positive at baseline had a significantly shorter median progression-free survival (PFS; 3.5 months, 95% CI: 2.9–4.2) and overall survival (OS; 5.8 months, 95% CI: 4.5–7.0) than patients lacking CTC (PFS 10.7 months, 95% CI: 6.9–14.4, p < 0.001; OS 13.3 months, 95% CI: 8.0–18.6, p = 0.003). Alterations in the marker profile during the course of chemotherapy were not predictive of clinical outcome or response to therapy. Yet, a favorable clinical response depended significantly on CTC negativity (p = 0.03). Conclusion: Our data suggest that the presence of CTC is a major predictor of outcome in patients with gastric and gastroesophageal malignancies.