Cystic fibrosis (CF) is a fatal genetic disorder characterized by chronic inflammation in the airways, involving an accumulation of neutrophils induced by increased production of interleukin (IL)-8 by pulmonary macrophages and airway epithelial cells. Increased airway inflammation leads to progressive lung damage and early death in patients with CF. There is a need for more specific therapies targeting sources of airway inflammation in CF. Vitamin D deficiency is frequent in the CF population, affecting up to 95% of patients. Serum vitamin D levels have been shown to positively correlate with lung function, and negatively correlate with inflammation and infection. Restoring vitamin D levels may be an effective anti-inflammatory therapy. There are numerous points in vitamin D metabolism or signalling that could possibly be impaired in CF. Anti-inflammatory function of vitamin D requires local conversion from the circulating metabolite 25-hydroxyvitamin D (25-OHD) to the active metabolite 1,25-dihydroxyvitamin D(1,25-diOHD), which then associates with the vitamin D receptor (VDR) to mediate effects on gene expression. We evaluated the potential of vitamin D metabolism and anti-inflammatory activity in monocyte-derived macrophages from both patients with CF and healthy controls, as well as in airway epithelial cell lines expressing either delf508/delf508 or wild-type CFTR. We observed that there was no difference between CF and non-CF MDM and epithelial cell lines in expression of genes relating to vitamin D metabolism or function. In response to 25-OHD3, MDM from CF patients and submerged airway epithelial cells with delf508/delf508 CFTR (CFBE41o-) dose-dependently down-regulated IL-8 induced by diffusible products of P. aeruginosa, demonstrating that CF-derived cells are both capable of synthesizing local 1,25-OHD3 and responding to it. In healthy controls, we observed two distinct populations of MDM: vitamin D "responders", that down-regulated IL-8 in response to both 25-OHD3 and 1,25-diOHD3, and vitamin D "non-responders" whose MDM did not down-regulate IL-8 in response to either metabolite. MDM from "responders" exhibited a greater magnitude in up-regulation of MAPK phosphatase 1 (MKP-1), the innate negative regulator of inflammation and vitamin D inducible gene, suggesting that the anti-inflammatory potency of vitamin D3 metabolites in macrophages appears to be individualized according to the magnitude of MKP-1 up-regulation. Furthermore, while submerged CFBE41o- down-regulated IL-8 in response to both 25-OHD3 and 1,25-diOHD3, CFBE41o- grown at the air-liquid interface (ALI) did not. However, in contrast to "responder" MDM, IL-8 down-regulation in submerged CFBE41o- was not associated with a concomitant up-regulation of MKP-1 mRNA, suggesting that the mechanism determining the response of epithelial cells to vitamin D may be different. Our results support the heterogeneity in response to vitamin D observed in the literature, highlighting the need to define populations who would be susceptible to vitamin D repletion anti-inflammatory therapy. Furthermore, the high-doses required for down-regulation of IL-8 challenge the current recommended levels of circulating vitamin D, which are based on optimal bone health. Overall, this thesis strengthens the need for a clinical trial testing high-dose vitamin D therapy in patients with CF and evaluating its effects on airway inflammation, lung function, and progression of lung disease. / La fibrose kystique (FK) est une maladie héréditaire potentiellement mortelle causée par une inflammation chronique des voies respiratoires. Elle est caractérisée par une accumulation de neutrophiles ayant été activés par une production accrue de l'interleukine (IL) -8 provenant des macrophages pulmonaires et des cellules épithéliales des voies respiratoires. Une augmentation de l'inflammation des voies respiratoires entraîne la détérioration progressive des poumons, et éventuellement le décès prématuré chez les patients atteints. Une déficience de vitamine D est fréquemment observée chez ceux atteints de FK, affectant jusqu'à 95% des patients. Des études démontrent que les taux sériques de vitamine D affectent positivement les fonctions pulmonaires, mais négativement l'inflammation et l'infection. Donc, rétablir les niveaux de vitamine D peut être une thérapie antiphlogistique efficace. La fonction anti-inflammatoire de la vitamine D nécessite une conversion locale du métabolite circulant 25-hydroxyvitamine D (25-OHD) au métabolite actif 1,25-dihydroxyvitamine D (1,25-diOHD). Cette dernière s'associe ensuite avec le récepteur de la vitamine D (VDR) afin de contrôler l'expression de plusieurs gènes. Nous avons étudié les effets de la vitamine D sur les réactions inflammatoires des macrophages dérivés de monocytes (MDM) de patients atteints de FK et de contrôles en santé, ainsi que dans des lignées cellulaires épithéliales des voies respiratoires exprimant soit la protéine du type CFTR delF508/delF508 ou du type naturel. Nous avons constaté qu'il n'y avait aucune différence dans l'expression des gènes liés au métabolisme et au fonctionnement de la vitamine D dans les MDM FK et non-FK, ainsi que les lignées de cellules épithéliales. En réponse à différentes doses de 25-OHD3, les MDM des patients FK et les cellules épithéliales des voies respiratoires immergées (CFBE41o-) ont eu un effet réducteur sur l'IL-8, produite par les produits diffusibles de Pseudomonas aeruginosa (P. aeruginosa). Cela a démontré que les cellules FK sont à la fois capables de synthétiser localement du 1,25-diOHD3 et d'y réagir. Chez les contrôles, nous avons observé deux populations distinctes de MDM : les vitamine D «répondeurs», qui régularise une baisse de l'IL-8 suite à un traitement de 25-OHD3 ou 1,25-diOHD3. Le deuxième groupe, les «non-répondeurs», ne voit aucun changement dans le niveau d'IL-8 suite au traitement d'aucun métabolite. Les MDM des «répondeurs» ont aussi démontré une hausse d'éxpression de l'ARNm de la phosphatase MAPK 1 (MKP-1), un régulateur négatif de l'inflammation induit par la vitamine D. Cela suggère que l'activité antiphlogistique des métabolites de la vitamine D dans les macrophages est définie en fonction de l'ampleur de la régulation de MKP-1. En outre, les cellules CFBE41o- immergées diminuent le niveau d'IL-8 en réponse au traitement de 25-OHD3 et 1,25-diOHD3, tandis que non chez les CFBE41o- cultivées à l'interface liquide/air (ALI). Cependant, à la différence des MDM "répondeur", la diminution d'IL-8 dans les CFBE41o- immergées n'a pas été associée à une hausse de l'ARNm MKP-1, ce qui laisse croire que le mécanisme de détermination de l'effet de la vitamine D sur les cellules épithéliales peut être différent. Nos résultats confirment l'hétérogénéité en réponse à la vitamine D observée dans la littérature, en soulignant la nécessité de définir les populations qui seraient sensibles à un traitement anti-inflammatoire excédent de vitamine D. Cependant, les dosages élevés nécessaires à la régulation de l'IL-8 vont à l'encontre des niveaux recommandés actuellement, basés sur une santé osseuse optimale. Dans l'ensemble, ces résultats soulignent la nécessité de tests cliniques de thérapie à dosage élevé de vitamine D chez les patients atteints de FK pour évaluer ses effets sur l'inflammation des voies respiratoires, la fonction pulmonaire, et la progression de la maladie pulmonaire.

Influence of KIR/HLA and FcγRIIIa genotypes on anti-HIV ADCC responses in HIV uninfected and infected slow progressor subjectsPilar ZanoniAbstractThirty-four million people are currently estimated to be infected with HIV. The need for a vaccine against HIV remains urgent. The RV144 vaccine trial reported partial protection against HIV infection. The RV144 vaccine regimen induced non-neutralizing antibodies (Abs) that engage cells of the innate immune system, such as natural killer (NK) cells, which may exert anti-viral activity by mediating antibody-dependent cell-mediated cytotoxicity (ADCC) against HIV-infected cells. The functional response of NK cells is determined by the integration of activating and inhibitory signals delivered to the NK cell through cell surface receptors, which include killer immunoglobulin-like receptors (KIR). Inhibitory KIRs (iKIR) bind human leukocyte antigen (HLA) molecules and are able to detect their downregulated expression on transformed or virally infected cells. Epidemiological evidence has linked certain KIR3DL1/HLA-B (3DL1/HLA-B) genotypes with protective outcomes in HIV-exposed seronegative (HESN) subjects and HIV infected slow progressors (SP). The mechanisms responsible for these protective outcomes remain unknown. Weak degranulation by 3DL1+ NK cells against autologous HIV-infected cells suggests that other activating signals may be required. Cross-linking NK cells with Ab-coated target cells via the NK cell surface Fc-receptor CD16a may provide these stimulatory signals. Using an assay that detects the delivery of Granzyme B (GzB) from NK effector cells to GzB substrate-coated target cells, I found that the source of NK effector cells influenced anti-HIV ADCC potency. In 47 uninfected subjects, NK cells from carriers of a protective 3DL1/Bw4 genotype known as *h/*y+B*57 had higher levels of ADCC activity than those from carriers of other 3DL1/Bw4 combinations or 3DL1/Bw6 homozygotes. This finding implicated NK cell education in ADCC potency. In contrast, in 47 HIV infected SP, NK cells from *h/*y+B*57 carriers had indistinguishable ADCC responses compared to NK cells from carriers of other 3DL1/Bw4 combinations or from 3DL1/Bw6 homozygotes suggesting that ADCC activity was sensitive to dysregulation by HIV infection. Since NK cells within a genotype group exhibited a range of ADCC activity, I examined the role of several factors other than 3DL1/HLA-B genotype that may influence ADCC potency. A polymorphism at amino acid 158 of CD16a results in greater affinity of the V than the F isoform for Ab. I assessed whether genetic variation in CD16a could contribute to differences in ADCC responses and report that percent ADCC target cell killing was independent of CD16a genotype. I also showed that self-iKIR to HLA-C contributed minimally to ADCC responses supporting our contention that interactions between KIR3DL1 and HLA-B*57 stand out from other educationally competent KIR/HLA combinations in educating NK cells for ADCC functional potential. My results suggest that HLA/KIR dependent NK cell education is a determinant of anti-HIV ADCC functional potential in uninfected individuals. Based on the absence of superior ADCC responses in SP carrying *h/*y+B*57 I propose that NK cell mediated ADCC activity is sensitive to and readily extinguished in the context of HIV infection. / Influence des génotypes KIR/HLA et FcγRIIIa sur les réponses ADCC anti-VIH chez des sujets non infectés par le VIH et les sujets infectés progresseurs lentsPilar ZanoniRésuméIl est estimé que 34 millions de personnes sont infectés par le VIH à ce jour. Il est donc urgent de développer un vaccin contre le VIH. Les essais du vaccin RV144 ont rapporté une protection partielle contre l'infection par le VIH. Ce vaccin induit des anticorps (Ac) non-neutralisants qui activent les cellules du système immunitaire inné, telles que les cellules natural killer (NK), qui pour leur part peuvent induire une activité anti-virale par la médiation de la cytotoxicité cellulaire anticorps-dépendante (ADCC) contre les cellules infectées par le VIH. La réponse fonctionnelle des cellules NK est déterminée par l'intégration de signaux activateurs ou inhibiteurs dans la cellule par le biais de récepteurs cellulaires de surface, incluant les récepteurs « killer immunoglobulin-like » (KIR). Les KIRs inhibiteurs (iKIR) se lient aux molécules de l'antigène leukocytaire humain (HLA) et sont capables de détecter leur expression diminuée sur les cellules transformées ou infectées par le virus. Des données épidémiologiques ont relié certains génotypes KIR3DL1/HLA-B (3DL1/HLA-B) à la protection chez des individus séronégatifs exposés au VIH (HESN) et chez des individus infectés au VIH à progression lente (SP). Les mécanismes menant à cette protection demeurent par contre inconnus. La faible dégranulation des cellules autologues infectées par le VIH par les cellules 3DL1 + NK suggère que d'autres signaux activateurs pourraient être requis. Le fait de lier des cellules NK avec des cellules-cibles recouvertes par des Ac via le récepteur de surface Fc-receptor CD16a pourrait induire ces signaux stimulateurs. En utilisant un test qui détecte le transfert du Granzyme B (GzB) des cellules effectrices NK aux cellules-cibles recouvertes du substrat GzB, j'ai découvert que la source de cellules effectrices NK influence le potentiel ADCC anti-VIH. Chez 47 sujets, les cellules NK provenant d'individus porteurs d'un génotype protecteur 3DL1/Bw4 connu (*h/*y+B*57) avaient des niveaux d'activité ADCC plus élevés que ceux observés chez les cellules provenant d'individus porteurs d'autres combinaisons 3DL1/Bw4 ou homozygotes pour 3DL1/Bw6. Ces résultats impliquent l'éducation des cellules NK dans le potentiel ADCC. Par contre, chez 47 individus SP infectés par le VIH, les cellules NK de porteurs de *h/*y+B*57 ont eu des réponses ADCC indétectables comparativement aux cellules NK de porteurs d'autres combinaisons 3DL1/Bw4 ou d'homozygotes pour 3DL1/Bw6, suggérant que l'activité ADCC est sensible à la dysrégulation par l'infection par le VIH. Comme les cellules NK incluses dans un groupe de génotype démontraient une activité ADCC variable, j'ai étudié le rôle de différents facteurs autres que le génotype 3DL1/HLA-B qui pourraient influencer le potentiel ADCC. Un polymorphisme à l'acide aminé 158 du CD16a résulte en une meilleure affinité de l'Ac avec l'isoforme V que l'isoforme F. J'ai donc déterminé si la variation du CD16a contribue aux différences de réponses ADCC et observé que le pourcentage de mort des cellules-cibles par ADCC ne dépendait pas du génotype CD16a. J'ai aussi démontré que les auto-iKIR de HLA-C contribuent un minimum aux réponses ADCC, supportant nos affirmations que les interactions entre KIR3DL1 et HLA-B*57 proviennent de combinaisons KIR/HLA pour l'éducation des cellules NK menant à un potentiel fonctionnel ADCC. Les résultats que j'ai obtenus suggèrent que l'éducation des cellules NK dépendante de HLA/KIR est déterminante pour le potentiel fonctionnel ADCC anti-VIH chez les individus non infectés. En me basant sur l'absence de réponses supérieure ADCC chez des individus SP porteurs de *h/*y+B*57, je suggère que l'activité ADCC médiée par les cellules NK est sensible à et facilement éliminée dans le contexte de l'infection par le VIH.

Hemojuvelin (HJV) is a membrane protein that controls the body iron metabolism. HJV mutations lead to juvenile hemochromatosis (JH), which arises from uncontrolled absorption of dietary iron from intestine and deposition of the metal into liver parenchymal cells. Hepatic iron overload progressively leads to inflammation, fibrosis or hepatocellular carcinoma. The molecular mechanisms responsible for iron induced liver injury are not fully characterized. Here, we investigated the potential contribution of hemojuvelin ablation to hepatic iron, lipid metabolism and its role in the pathogenesis of liver fibrosis. Wild-type (WT) and Hemojuvelin knockout mice (Hjv-/-) on C57BL/6J background were fed either a standard chow, a high fat, or a high-fat with 2% supplemented iron diet (HFD + Fe) for a time course experiment (0, 3, 6, 9, 12 weeks). Comparatively, Hjv-/- animals developed higher serum iron indices, liver iron deposition, and diminished hepcidin levels. Likewise, dramatic changes were observed between levels of iron uptake protein TfR1 and iron storage protein ferritin. Interestingly, HFD + Fe group showed significant reduction in body weight irrespective of genotype. Further, qPCR data revealed significant downregulation of Adiponectin receptor 2 (AdipoR2) in this group, in addition to upregulation of cholesterol biosynthesis with excess iron. Hjv-/- animals also develop higher serum titer for transaminases (ALT, AST), common markers of liver injury. However, animals failed to develop liver fibrosis. We speculate that this is related to the strain of the mice, which is genetically resistant to liver injury. / L'hémojuvéline (HJV) est une protéine membranaire qui contrôle le métabolisme systémique du fer. Les mutations de HJV conduisent à l'hémochromatose juvéline, qui découle de l'absorption incontrôlée du fer de l'alimentation et des dépots de ce métal dans les cellules parenchymateuses du foie. La surcharge hépatique en fer mène progressivement à l'inflammation, la fibrose et le carcinome hépatocellulaire. Les mécanismes moléculaires responsables des dommages au foie qui sont attribuables au fer ne sont pas entièrement caractérisés. Nous avons donc étudié la contribution potentielle de l'ablation de HJV sur le fer hépatique, le métabolisme des lipides et son rôle dans la pathogénèse de la fibrose hépatique. Des souris de type sauvage (WT) et déficiente pour HJV (Hjv-/-) dans la souche C57BL/6J ont été nourries avec un régime normal, riche en gras ou riche en gras et supplementé en fer (HFD + Fe) pour différentes périodes (0, 3, 6, 9, 12 semaines). Comparativement, les animaux Hjv-/- développent des indices de fer sérique plus élevés, des dépots de fer dans le foie, et une diminution de niveaux de hepcidine. Des changements dramatiques sont également observés entre les niveaux de la protéine de capture du fer, TfR1, et la protéine de stokage du fer, la ferritine. De manière intéressante, le groupe HFD+ Fe montre une réduction significative de la masse corporelle des animaux quel que soit leur génotype. De plus, les données de qPCR revèlent la régulation à la baisse significative du récepteur 2 de l'adiponectine (AdipoR2) dans ce groupe, en plus de la régulation à la hausse de la biosynthèse de cholestérol avec un excès de fer. Les animaux Hjv-/- montrent un titre sérique plus élevé de transaminases (ALT, AST), marqueurs communs de dommages du foie, bien que les animaux ne développent pas la fibrose hépatique. Nous spéculons que ceci est dû à la souche des souris qui est résistante aux dommages du foie.

The success of innate host defence to viral infection depends on the ability of innate immunecells to detect the invading pathogen culminating in the production of cytokines and chemokinesthat disrupt viral replication and shape the platform for innate and adaptive immune responses.Rapid induction of type I IFN is a central event in establishment of the antiviral response, whichis tightly regulated by extracellular and intracellular signals that activate transcription factors(NF-KB, AP-l and IRF). The synergistic action of these transactivators on the promoters of type IIFN genes triggers the immediate early IFN response. which is further amplified by JAK-ST ATsignaling proteins. In the IRF family, IRF-5 is the central mediator in modulating the productionof pro-inflammatory cytokines in TLR-dependent signalings, whereas IRF-3 and IRF-7 are themasters of type I IFN in both TLR-dependent and TLR-independent signalings. Upon activation,IRF-5 like IRF-3 undergoes phosphorylation, dimerization and translocation to the nucleus.However, the mechanism of IRF-5 cytoplasmic-to-nuclear translocation remains unknown. InTLR-independent signalings, RIG-I is critical for detecting intracellular RNA virus infection.RIG-I senses viral-derived RNA through its helicase domain and relays downstream signals viaits N-terminal caspase recruitment domain (CARD). The mitochondrial antiviral signaling(MAVS) adaptor links RIG-I to the downstream canonical IKKs and the IKK-related kinases,IKKE and TBKl, culminating in the activation ofNF-KB and IRF3, respectively. Previous studieshave demonstrated that cross-talk between the canonical IKKs and IKK-related kinasesinfluences NF-KB activation. TBKI was first described as a kinase interacting with the TRAFfamily member-associated NF-KB activator (TANK) adaptor protein that was able to activate NFKB.However, whether the canonical IKKs influence IRF-3 and IRF-7 activation represents acritical missing link in the understanding of TLR-independent / La réussite de la défense immunitaire face a l'infection virale dépend de la capacité des cellules immunitaires a détecter les pathogènes et a produire des cytokines et des chemokines qui interrompent la réplication virale et établissent la base des réponses immunitaires innée et acquise. La production d'interféron (IFN) de type I est un élément central dans la réponse antivirale et est régule étroitement par des signaux extracellulaires et intracellulaires qui activent les facteurs de transcription NF-KB, AP-l et IRF. L'action synergique de ces protéines sur les promoteurs des gènes IFN active la réponse rapide et immédiate des IFN, amplifiée par la signalisation JAKSTAT. Parmi les IRF, IRFS est le principal régulateurs de !'induction des cytokines pro inflammatoires par les TLRs alors qu'IRF3 et IRF7 sont les médiateurs de ractivation des IFN de manière dépendante et indépendante des TLRs. IRFS, comme IRF3, est active par phosphorylation, dimerisation et translocation dans le noyau OU il active la transcription de ses gènes cibles. Le mécanisme moléculaire de la translocation nucleaire de IRFS est inconnu. RIG-I joue un role crucial dans la détection de l' ARN viral indépendante des TLRs. RIG-I reconnait I' ARN d' origine virale par son domaine helicase et signale par son domaine CARD. L'adaptateur MAVS transmet l'activation de RIG-I aux kinases canoniques IKKs et aux kinases apparentées IKKE et TBKI qui activent NF-KB et IRF3, respectivement. Des études précédentes ont démontre que la communication croisée entre les kinases canoniques et apparentées module l'activation de NF-KB. TBKI a été décrit originellement comme une kinase qui interagit avec l'adaptateur TANK qui active NF-KB. Neanrnoins, le rôle des kinases canoniques dans l'activation d'IRF3 et IRF7 représente un lien crucial inexploré dans la compréhension de la signalisation dépendante des TLRs. Dans cette étude, nous avons caractérise un mutant d'IRFS

<p> The mammalian immune system has evolved a complex and diverse set of mechanisms to detect and respond to pathogens by recognizing conserved molecular structures and inducing protective immune responses. While many of these mechanisms are capable of sensing diverse molecular structures, a large fraction of pathogen sensors recognize nucleic acids. Pathogen-derived nucleic acids trigger nucleic acid sensors that typically induce anti-viral or anti-microbial immunity, however host-derived nucleic acids may also activate these sensors and lead to increased risk of inflammatory or autoimmune disease. Animal models and humans lacking key DNA nucleases, such as Trex1/Dnase3, accumulate intracellular DNA and develop progressive autoimmunity marked by increased Type-I Interferon (IFN) expression and inflammatory signatures. </p><p> Double-stranded DNA (dsDNA) is a potent inducer of the Type-I IFN response. Many of the sensors and signaling components that drive the IFN signature following simulation with transfected dsDNA (also called 'Interferon Stimulatory DNA' or 'ISD') remain unknown. We set out to identify novel components of the ISD pathway by developing a large-scale loss-of-function genetic perturbation screen of 1003 candidate genes. We interrogated multiple human and murine primary and immortalized cells, tested several Type-I IFN reporters, and considered multiple loss-of-function strategies before proceeding with an RNAi screen whereby mouse embryonic fibroblasts were stimulated with ISD and Type-IFN pathway activation was assessed by measuring Cxcl10 protein by ELISA. </p><p> Candidate genes for testing in the RNAi screen were curated from quantitative proteomic screens, IFN-beta and ISD stimulated mRNA expression profiles, and a selection of domain-based proteins including helicases, cytoplasmically located DNA-binding proteins and a set of potential negative regulators including phosphatases, deubiquitinases and known signaling proteins. </p><p> We identified a number of novel ISD pathway components including Abcf1, Ptpn1 and Hells. We validated hits through siRNA-resistant cDNA rescue, chemical inhibition or targeted knockout. Additionally, we evaluated protein-protein interactions of our strongest validated hits to develop a network model of the ISD pathway. In addition to the identification of novel ISD pathway components, our enriched screening data set may provide a useful resource of candidate genes involved in the response to cytosolic DNA.</p>

Abstract Not Available.

The contributions of CD18-integrins and selectins to neutrophil extravasation in an in vitro model blood vessel at physiological wall shear stresses were investigated in both human and canine systems. Our results demonstrate that extravasation can be conceptually divided into two consecutive steps: margination and migration. Monoclonal antibodies (MAb) against LECAM-1 significantly reduced margination, without effecting migration, in both systems. This percent inhibition was greater at the higher wall shear stress (WSS) of 1.85 dynes/cm$\sp2$ (dpcs, $\sim$70%) than at the lower WSS of 0.23 dpcs in the canine system and 0.36 dpcs in the human system ($\sim$30%). Chemotactic stimulation reduced adhesion to the same extent as the anti-LECAM-1 MAbs and showed no additive effect to these MAbs. In contrast, anti-CD18 MAbs reduced migration and total adhesion without effecting margination at these WSSs. The inhibitory effect of anti-CD18 MAbs on total adhesion was considerable at lower WSSs (25-40%) and minimal at WSS of 1.85 dpcs (10-20%). At lower WSSs, MAbs against CD18-integrins and LECAM-1 showed additive inhibition. Therefore these antigens are involved in independent adhesion mechanisms. Human neutrophils also marginated on human ELAM-1 transfected murine L-cells. This adhesion was almost completely blocked by anti-ELAM-1 MAb and significantly reduced by anti-LECAM-1 MAb or neutrophil activation. The adherence of activated neutrophils was further inhibited by anti-ELAM-1 MAb. In studies with human endothelial cells, the reduction in adhesion with anti-ELAM-1 MAb was significantly lower than with anti-LECAM-1 MAb. The combination of the two MAbs showed no additive effect, which was evidence for possible interactions between these two antigens. Finally neutrophil rolling was shown to be a LECAM-1/ELAM-1 mediated phenomenon, with CD18-integrins functioning in an accessory manner to slow the rolling velocity.

Endothelial cells line the inner walls of blood vessels and are constantly subjected to hemodynamic shear forces due to flowing blood. Both in vivo and in vitro studies have shown that local fluid shear stress may play an important role both in normal inflammatory responses as well as in the initial development and progression of atherosclerosis. This study describes their contribution in regulating the expression of adhesion molecules ICAM-1, VCAM-1 and E-selectin on endothelial cells, with the goal of understanding the link between local fluid mechanics, inflammatory response and atherogenesis. HUVEC monolayers exposed to different levels of controlled shear stress conditions were analyzed for the kinetics of expression of these adhesion molecules both in terms of gene transcription and surface protein expression. Results from the fluorescence analysis showed a transient increase in the surface expression of ICAM-1 upon exposure to shear stress (25 dynes/cm$\sp2$) for 12 hours that returned to basal levels within 24 hours. The messenger RNA (mRNA) levels for ICAM-1 also showed a transient increase after one to three hours of exposure to shear stress compared to matched control values. Subsequently, however, the mRNA levels decreased and within 6 hours dropped significantly below control values. VCAM-1 mRNA levels, however, decreased monotonically upon onset of flow at all values of shear stresses, and dropped well below basal levels within 6 hours. Neither molecule mRNA was present in detectable levels after 24 hours of flow. E-selectin mRNA levels appeared to be constant and comparable to control values for up to 6 hours after exposure to shear stress. These studies also demonstrate altered response of shear preconditioned endothelial cells to subsequent activation with inflammatory mediators such as IL-1$\beta$. Both ICAM-1 and VCAM-1 mRNA levels were comparable in the 4 and 6 hour IL-1$\beta$ treated cells under both flow and static conditions. However, ICAM-1 levels were higher after 24 hours of IL-1$\beta$ under flow compared to similarly activated static cells whereas VCAM-1 levels were lower. ICAM-1 surface expression was also significantly (3- to 4-fold, p $<$ 0.02) higher under these conditions, while VCAM-1 did not change as a result of flow preconditioning. The observed differences between static and pre-sheared cells discussed here suggests that in vitro studies involving vascular cells should mimic as closely as possible the representative fluid mechanical environment of the region they are modeling.

Gut associated lymphoid cells are modulated by gut hormones. We postulated that lymphokine-associated-killer (LAK) cell cytotoxicity of lymphocytes from the gut mucosa may be increased by substance P (SP). / Intestinal lamina propria mononuclear cells (LPMC) and colonic cancer cells were isolated from operative specimens by mechanical and enzymatic dissociation methods. Effector LAK cells were induced by culturing LPMC with 250 u/ml of recombinant interleukin-2 (IL-2). Substance P (10$ sp{-5}$M) was added to the culture. Targets consisted of fresh colon cancer cells, HT-29 and control cell lines. After 4 days of incubation, cytotoxicity was measured using a 4-hour $ sp{51}$Cr release assay. / LAK cells showed moderate cytotoxicity against HT-29 and none against fresh colon cancer cells. LAK cells + substance P showed moderate cytotoxicity against HT-29 and strong cytotoxicity against fresh cancer cells. Cytotoxicity $ pm$ SEM at various effector-to-target (E:T) ratios was: (* denotes p $<$ 0.05 compared with above).(UNFORMATTED TABLE OR EQUATION FOLLOWS) vbox{ halign{# hfil&& enspace# hfil cr &Effector&Target&N&5:1&10:1&25:1&50:1 cr cr&LAK&HT-29&7&2.8$ pm $0.5&11.6$ pm$1.0&12.3$ pm$2.3&17.8$ pm$4.2 cr&LAK+SP&HT-29&11&1.2$ pm $0.5& enspace 4.1$ pm $0.9& enspace 9.3$ pm$1.0&16.9$ pm$3.1 cr&LAK+SP&Fresh Ca&8&5.6$ pm$1.2&12.6$ pm$3.3&38.3$ pm$6.3*&59.4$ pm$9.5* cr&LAK&Fresh Ca&11&0.0$ pm$0.0*& enspace 0.1$ pm$0.1*& enspace 0.1$ pm$0.1*& enspace 0.5$ pm$0.5* cr}} TABLE/EQUATION ENDS)Substance P significantly increases LAK cell cytotoxicity against fresh colon cancer cells, but not against cultured cells.

The in situ microenvironmental organization of early precursor B cells in mouse bone marrow has been studied using three experimental models: (1) mutant mice with severe combined immunodeficiency (SCID), which develop pro-B cells but no pre-B and B cells; (2) SCID/myc transgenic mice having expanded pro-B cell populations, but no pre-B and B cells; (3) x-irradiated C3H/HeJ mice during early stages in the regeneration of pro-B cells in bone marrow seeded from a shielded marrow site. The in vivo localization of B220$ sp+$ cells was revealed by the binding of i.v. injected $ sp{125}$I-mAb 14.8 detected by light and electron microscope radioautography of femoral marrow sections. Many B220$ sp+$ pro-B cells were located in peripheral regions of SCID and SCID/myc bone marrow, often in clusters, associated with an electron dense extracellular matrix and with the processes of stromal reticular cells. Many B220$ sp+$ cells were associated with macrophages which contained numerous ingested bodies. Macrophage associations were more numerous in SCID/myc than in SCID mice, especially in the peripheral marrow regions. 3-5 day post-irradiation endocolonizing marrow contained increasing numbers of B220$ sp+$ cells in subosteal and peripheral regions, situated both within sinusoids and extravascularly, associated with stromal reticular cell processes and often close to nerve fibers. The results demonstrate that early B220$ sp+$ precursors begin to differentiate in peripheral marrow regions and develop intimate associations with reticular cells and macrophages. These findings suggest that through these associations, the bone marrow reticular cells promote the early development of the B cell lineage, while the bone marrow macrophages play a role in the elimination of aberrant precursor B cells.