Lors de la conception de médicaments, le criblage in silico est de plus en plus utilisé et lesméthodes disponibles nécessitent d'être évaluées. L'évaluation de 8 méthodes a mis enévidence l'efficacité des méthodes de criblage in silico et des problèmes de construction de labanque d'évaluation de référence (DUD), la conformation choisie pour les sites de liaisonn'étant pas toujours adaptée à tous les actifs. La puissance informatique actuelle le permettant,plusieurs structures expérimentales ont été choisies pour tenter de mimer la flexibilité dessites de liaison. Un autre problème a été mis en évidence : les métriques d'évaluation desméthodes souffrent de biais. De nouvelles métriques ont donc été proposées, telles queBEDROC et RIE. Une autre alternative est proposée ici, mesurant la capacité prédictive d'uneméthode en actifs. Enfin, une petite molécule active sur le TNFα in vitro et in vivo sur souris aété identifiée par un protocole de criblage in silico. Ainsi, malgré le besoin d'amélioration desméthodes, le criblage in silico peut être d'un important soutien à l'identification de nouvellesmolécules a visée thérapeutique. / Since the introduction of virtual screening in the drug discovery process, the number ofvirtual screening methods has been increasing and available methods have to be evaluated.In this work, eight virtual screening methods were evaluated in the DUD database, showingadequate efficiency. This also revealed some shortcomings of the DUD database as thebinding site conformation used in the DUD was not relevant for all the actives.As computational power now permits to address this issue, classical docking runs have beenperformed on several X-ray structures, used to represent the binding site flexibility. This alsorevealed that evaluation metrics show some biases. New evaluation metrics have thus beenproposed, e.g. BEDROC and RIE. An alternative method was also proposed usingpredictiveness curves, based on compound activity probabilityFinally, a virtual screening procedure has been applied to TNFa. A small molecule inhibitor,showing in vitro and in vivo activity in mice, has been identified. This demonstrated the valueof virtual screening for the drug discovery process, although virtual screening methods needto be improved.

Introduction: The inflammation plays the essential role in the bone loss in juvenile idiopathic arthritis (JIA). Proinflammatory cytokines and also glucocorticoids (GCs) may activate bone resorption by osteoclasts. Simultaneously, bone formation can be attenuated, especially by inhibitors of proteins, which control the osteoblast differentiation. The aim was to verify the hypothesis that in patients with highly active JIA, reduction of bone formation via Wingless (Wnt) proteins inhibitors - Dickkopf 1 (Dkk-1) and sclerostin could be found. Except the densitometry measurements of bone and lean mass, we assessed markers of disease activity, bone metabolism and remodeling in young adult patients with JIA before and during 2 years of anti TNFα (tumour necrosis factor α) treatment, which decreases disease activity. Results: In patients with JIA before antiTNFα treatment, bone mineral density (BMD, g/cmš) was significantly reduced compared to controls. Values of BMD and body composition in JIA significantly depended on disease duration and GCs treatment. Serum concentration of sclerostin was significantly elevated in JIA compared to values in healthy controls. Values of the other monitored markers did not differ between JIA and controls. In patients with JIA, Dkk-1 correlated positively with C-reactive...

Le criblage virtuel est de plus en plus utilisé dans les programmes de recherche de nouveaux principes actifs. L’augmentation considérable du nombre de structures résolues a favorisé le recours aux méthodes basées sur la structure de la cible comme le docking. Néanmoins, le choix de la/des structure(s) à utiliser demeure une question d’actualité. Pour tenter d'apporter une réponse, les résultats des études de docking menées sur la banque d’évaluation de référence (DUD) ont été analysés en prenant en compte les propriétés des sites de liaisons des structures de référence. D’intéressants résultats ont été obtenus mettant en évidence l'influence du volume et de l’ouverture des sites actifs sur les performances des méthodes. Ces critères de sélection simples et peu coûteux peuvent servir pour l’optimisation de protocoles de docking.Alors qu’aucune petite molécule inhibitrice du TNFα n’est actuellement commercialisée, l’application d’un protocole hiérarchique de criblage virtuel/in vitro, a permis d’identifier des touches actives. L’une d’elle, de squelette benzènesulfonamide a fait l’objet de pharmacomodulation en vue d’obtenir des analogues optimisés. Vingt molécules inédites ont été synthétisées et testées in vitro et certaines ont montré une activité intéressante. L’ensemble des données obtenues apportent des éléments importants de relation structure-activité. Ces résultats peuvent être exploités pour la conception de molécules innovantes ciblant le TNFα ce qui serait une avancée prometteuse pour le traitement des pathologies liées à une surproduction de cette cytokine comme la polyarthrite rhumatoïde et la maladie de Crohn. / Virtual screening is widely used in drug discovery programs. The increasing number of resolved structures favored the use of Structure Based Virtual Ligand Screening methods like docking. Nevertheless, the choice of the structure(s) used as reference remains a topical issue when several are available. In this work, DUD database docking results were analyzed taking into account the properties of the query structure(s) binding sites. Interesting results were obtained highlighting the influence of active site volume and opening on methods performances. These simple and inexpensive “binding site properties-based” guidelines could be helpful to optimize future docking protocols.Despite important effort, no active small molecule targeting TNFα has been released so far. The use of a virtual/ in vitro hierarchical approach screening allowed identifying some active hits. Starting from one of them with a benzenesulfonamide structure, pharmacomodulation was achieved in order to obtain optimized analogs. Twenty new chemical derivatives with an original structure were synthesized and tested in vitro. Some of them exhibited an interesting activity. Moreover, data obtained provide important elements of structure-activity relationship. These results could constitute the basis for innovative small molecule TNFα-targeted therapeutics which would be a promising step for the treatment of diseases related to overproduction of this cytokine such as rheumatoid arthritis and Crohn's disease.

Introduction: The inflammation plays the essential role in the bone loss in juvenile idiopathic arthritis (JIA). Proinflammatory cytokines and also glucocorticoids (GCs) may activate bone resorption by osteoclasts. Simultaneously, bone formation can be attenuated, especially by inhibitors of proteins, which control the osteoblast differentiation. The aim was to verify the hypothesis that in patients with highly active JIA, reduction of bone formation via Wingless (Wnt) proteins inhibitors - Dickkopf 1 (Dkk-1) and sclerostin could be found. Except the densitometry measurements of bone and lean mass, we assessed markers of disease activity, bone metabolism and remodeling in young adult patients with JIA before and during 2 years of anti TNFα (tumour necrosis factor α) treatment, which decreases disease activity. Results: In patients with JIA before antiTNFα treatment, bone mineral density (BMD, g/cmš) was significantly reduced compared to controls. Values of BMD and body composition in JIA significantly depended on disease duration and GCs treatment. Serum concentration of sclerostin was significantly elevated in JIA compared to values in healthy controls. Values of the other monitored markers did not differ between JIA and controls. In patients with JIA, Dkk-1 correlated positively with C-reactive...

<p>Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification.</p><p>In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype.</p><p>In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21^WAF1/CIP1 (CDKN1A) and p27^KIP1 (CDKN1B).</p><p>TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.</p>

Transforming growth factor-β1 (TGFβ1) is a mediator of the fibrotic response through activation of quiescent cardiac fibroblasts to hypersynthetic myofibroblasts. Scleraxis (Scx) is a pro-fibrotic transcription factor that is induced by TGFβ1-3 and works synergistically with Smads to promote collagen expression. Ski is a negative regulator of TGFβ/Smad signaling through its interactions with Smad proteins at the promoter region of TGFβ regulated genes. To date, no studies have examined the direct DNA:protein transcriptional mechanisms that regulate Scx expression by TGFβ1-3 or Ski, nor the mechanisms that govern Ski expression by Scx. We hypothesize that Ski and Scx regulate one another, and form a negative feedback loop that represses gene expression and is a central regulator of the fibrotic response in cardiac myofibroblasts. Primary adult rat cardiac myofibroblasts were isolated via retrograde Langendorff perfusion. First passage (P1) cells were infected with adenovirus encoding HA-Ski, HA-Scx, or LacZ at the time of plating. Twenty-four hours later, cells were harvested for Western blot, quantitative real-time PCR (qPCR), and electrophoretic gel shift assays (EMSA). NIH-3T3 or Cos7 cells were transfected with equal quantities of plasmid DNA for 24 hours prior to harvesting for luciferase, qPCR, and EMSA analysis. Ski overexpression in P1 myofibroblasts resulted in a reduction in both Scx mRNA and protein levels. Overexpression of Scx had no effect on Ski expression. Luciferase reporter assays demonstrated that Scx was induced by TGFβ1 treatment in a concentration dependent manner. However, ectopic Smad2/3 expression was unable to transactivate the Scx promoter in a luciferase reporter assay. Inhibition of p44/42-MAPK signaling modestly counteracted the effect of TGFβ1 on Scx expression. Scx had no effect on Ski promoter expression, however, both tumor necrosis factor-α (TNFα) and p65 expression repressed the Ski promoter and correlated with reduced Ski mRNA levels. We conclude that Ski is a repressor of Scx and that Scx expression is partially mediated through a Smad-independent, p44/42-MAPK pathway in cardiac myofibroblasts. Furthermore, this study proposes a role for TNFα/p65 NF-κΒ signaling in the regulation of Ski gene expression in the cardiac myofibroblast. / October 2016

Les cellules épithéliales pulmonaires constituent la première ligne de défense face aux virus respiratoires via la sécrétion de mucus, de peptides, de cytokines et chimiokines qui déterminent l'élimination ou la progression de l’infection. Les principales cytokines antivirales produites par les cellules épithéliales alvéolaires (AEC) sont les interférons (IFN) type I (α/β) et III (λ). La liaison d’IFNβ à son récepteur induit une voie antivirale bien caractérisée qui aboutit à l’activation du complexe ISGF3 (STAT1, STAT2 et IRF9) qui permet la transcription de multiples gènes codant pour des protéines à activité antivirale et immunorégulatrice. Il a récemment été démontré que la costimulation des cellules épithéliales pulmonaires par l’IFNβ et le Tumor Necrosis Factor α (TNFα), également produit lors d’une infection, synergisent pour induire un état antiviral tardif distinct. D’autre part, il a été montré que la synergie entre le TNFα et l'IFNβ induit une voie de signalisation impliquant STAT2 et IRF9, mais indépendante de STAT1 permettant l’expression du gène DUOX2. Notre but est de déterminer l’importance de cette nouvelle voie de signalisation induite par la costimulation du TNFα+IFNβ, impliquant STAT2 et IRF9 indépendamment de STAT1 dans la régulation d’un programme transcriptionnel antiviral et immunorégulateur tardif. Notre premier objectif est de déterminer si des gènes antiviraux et immunorégulateurs qui sont induits par la costimulation par TNFα+IFNβ sont dépendants de la voie STAT2/IRF9, indépendamment de STAT1. En utilisant la technique de qRT-PCR, nous avons identifié 3 gènes immunorégulateurs, CXCL10, IDO et APOBEC3G, induits de manière synergique en réponse à TNFα+IFNβ dans les cellules A549, un modèle de cellules épithéliales pulmonaires. Afin de confirmer que ces gènes sont induits indépendamment de STAT1, nous avons validé leur expression dans la lignée cellulaire U3A déficiente en STAT1. Par l'utilisation d'ARN interférants (ARNi) dirigés contre STAT2 et IRF9, nous avons confirmé que l’induction de ces gènes est dépendante de STAT2 et IRF9. Finalement, l’analyse de l’activité du promoteur de CXCL10 en réponse à TNFα+IFNβ par des essais rapporteurs luciférases a permis de montrer que la régulation se fait au niveau transcriptionnel. Notre deuxième objectif, est de déterminer si STAT6 pourrait remplacer STAT1 dans la voie de signalisation induite par TNFα+IFNβ. En effet, STAT6 est un inducteur connu de l’expression de DUOX2 en réponse à IL4+IL13. Contrairement à notre hypothèse, l’inhibition de STAT6 par ARNi augmente l’expression de DUOX2 en réponse à TNFα+IFNβ suggérant que STAT6 est un régulateur négatif. Nos résultats ont permis de comprendre de manière plus détaillée les mécanismes mis en place dans le développement d’une réponse antivirale. D’autre part, l’étude de l’effet de l’IFNβ et du TNFα est également pertinente pour les maladies chroniques inflammatoires et autoimmunes. De plus, nos résultats illustrent un nouveau paradigme concernant les mécanismes de signalisation cellulaire impliqués dans la synergie entre deux cytokines qui pourrait être applicable à des combinaisons de cytokines autres que TNFα+IFNβ. / Lung epithelial cells are the first line of defense against respiratory viruses via mucus secretion, peptides, cytokines and chemokines that determine the progression of the infection. The main antiviral cytokines produced by alveolar epithelial cells (AEC) are the interferons (IFN) type I (α / β) and III (λ). IFNβ binding to its receptor induces an antiviral pathway that is well characterized and leads to activation of the ISGF3 complex (STAT1, STAT2 and IRF9) which allows the transcription of multiple genes encoding proteins with antiviral and immunoregulatory activity. It has recently been shown that the costimulation of lung epithelial cells by IFNβ and Tumor Necrosis Factor α (TNFα), also produced during infection, induces a separate and late antiviral state, through synergy. On the other hand, it has been shown that the synergy between IFNβ+TNFα induces a signaling pathway involving STAT2 and IRF9 independently of STAT1 permitting the expression of the DUOX2 gene. Our goal is to determine the importance of this new signaling pathway induced by costimulation of TNFα+IFNβ involving STAT2 and IRF9 regardless of STAT1 in regulating the antiviral immunoregulatory and late transcriptional program. Our first objective is to determine whether antiviral and immunomodulatory genes that are induced by costimulation TNFα+IFNβ are dependent on the STAT2/IRF9 way, indenpant of STAT1. Using the technique of qRT-PCR, we identified 3 immunoregulatory genes, CXCL10, IDO and APOBEC3G, synergistically induced in response to TNFα+IFNβ in A549 cells, a model of pulmonary epithelial cells. To confirm that these genes are induced independantly of STAT1, we validated their expression in the STAT1 deficient cell line, U3A. By the use of interfering RNA (siRNA) directed against STAT2 and IRF9, we confirmed that the induction of these genes is dependent STAT2 and IRF9. Finally, the analysis of the activity of CXCL10 promoter in response to TNFα+IFNβ by luciferase reporter assays has shown that the regulation is at the transcriptional level. Our second objective is to determine whether STAT6 could replace the STAT1 in the signaling pathway induced by TNFα+IFNβ. Indeed, STAT6 is a known inducer of the expression of DUOX2 in response to IL4+IL13. Contrarily to our hypothesis, inhibition of STAT6 by RNAi increases the expression of DUOX2 in response to TNFα+IFNβ suggesting that STAT6 is a negative regulator. Our results allow the understanding of the mechanisms in the development of an antiviral response in more detail. On the other hand, the study of the effect of IFNβ and TNFα is also relevant for chronic inflammatory and autoimmune diseases. In addition, our results illustrate a new paradigm for cell signaling mechanisms involved in the synergy between two cytokines that may be applicable to combinations of cytokines other than TNFα+IFNβ.

Epigenetic modifications were shown to play an essential role in tumorigenesis. Epigenetic mechanisms can alter transcription in several ways, through DNA methylation and/or through histone modification. DNA methylation at the TSS (transcriptional start site) has been implicated in tumor development and gene silencing. However, several examples of atypical methylation were shown. In Paper I we present the ICSBP/IRF8 gene that belongs to the IRF family and has characteristics of a tumor suppressor gene. The ICSBP/IRF8 is fully methylated in the promoter and TSS regions in U-937 and despite high expression of the gene. Presence of positive histone marks suggests that methylated DNA can be overridden by histone modification. In Paper II a panel of 13 MM (multiple myeloma) cell lines and 9 primary patient tumors were analysed for methylation status of the ICSBP/IRF8 gene. In most cell lines (8/13) the gene was partially or fully methylated and partial methylation was also observed in 1/9 primary tumors. In vitro methylation analysis and treatment with 5-aza-2’deoxycytidine (DAC) proved that the ICSBP/IRF8 gene is silenced by methylation and may be associated with the malignant phenotype. In Paper III and IV the NFκB signalling pathway was analysed and the role of ATRA and TNFα induction. In Paper III the data shows that activation of the NFκB pathway is essential in ATRA-induced terminal differentiation in the U-937 cell line and IκBα (S32A/S36A) inhibits ATRA-induced differentiation and G1 cell cycle arrest. This was accompanied by delayed down-regulation of several cyclins (A and E) and up-regulation of p21WAF1/CIP1 (CDKN1A) and p27KIP1 (CDKN1B). TNFα alone did not induce expression of RA-induced genes analysed in Paper IV. However, ATRA in combination with TNFα showed enhanced activation of RA-induced genes. TNFα triggers demethylation of H3K9me3/H3K9me2 and H3K4me3 at RAR/RXR target genes, which were not accompanied by changes in the level of H3K9-ac. This decrease in H3 methylation by TNFα may pave way for the later ATRA-induced gene transcription.

Σκοπός της παρούσας διατριβής ήταν η μελέτη της ασφάλειας και της αποτελεσματικότητας των αντι-TNFα βιολογικών παραγόντων, ινφλιξιμάβης και αδαλιμουμάβης, στους ασθενείς με ιδιοπαθή φλεγμονώδη νοσήματα του εντέρου (ΙΦΝΕ) στο Πανεπιστημιακό Γενικό Νοσοκομείο Πατρών. Προσπαθήσαμε να εντοπίσουμε τυχόν συσχετίσεις μεταξύ ομάδων ασθενών και εμφάνισης παρενεργειών ή κλινικής ανταπόκρισης. Μέθοδοι: Από τον Μάιο του 2013 ως τον Μάιο του 2014 καταγράφηκαν τα στοιχεία 63 ασθενών με μοναδικά κριτήρια εισαγωγής τη διάγνωσή τους με ΙΦΝΕ (νόσο του Crohn, ελκώδη κολίτιδα ή αδιευκρίνιστη κολίτιδα) και τη θεραπεία τους με ινφλιξιμάβη ή αδαλιμουμάβη. Κριτήριο αποτελεσματικότητας ήταν η κλινική ύφεση ή έξαρση της νόσου. Κριτήριο ασφαλείας ήταν η εμφάνιση ή όχι παρενεργειών. Αποτελέσματα: Το 65,1% των ασθενών (41/63) λάμβανε ινφλιξιμάβη. Μόλις το 20,6% των ασθενών (13/63) απαίτησε τροποποίηση του βιολογικού παράγοντα (αύξηση δόσης ή συχνότητας χορήγησης). Το 11,1% των ασθενών (7/63) διέκοψαν οριστικά τη θεραπεία εξαιτίας μη ανταπόκρισης ή απώλειας ανταπόκρισης και το 3,2% (2/63) εξαιτίας παρενεργειών. Το 7,9% των ασθενών (5/63) αναγκάστηκαν να διακόψουν παροδικά (λιγότερο από 1 έτος) τη θεραπεία λόγω μη ανταπόκρισης ή απώλειας ανταπόκρισης και το 9,5% (6/63) εξαιτίας παρενεργειών. Το 61,1% των ασθενών (33/54) που είχαν λάβει έστω και μια φορά ινφλιξιμάβη και το 38,1% των ασθενών (8/21) που είχαν λάβει έστω και μία φορά αδαλιμουμάβη εμφάνισε παρενέργειες. Οι πιο συχνές παρενέργειες ήταν οι λοιμώξεις (12/59, 20,3%) και οι αρθραλγίες (12/59, 20,3%) στους ασθενείς που λάμβαναν ινφλιξιμάβη, και οι λοιμώξεις (4/10, 40%) και η κεφαλαλγία (4/10, 40%) σε όσους λάμβαναν αδαλιμουμάβη. Το 69,8% των ασθενών (44/63) βρίσκονταν σε ύφεση. Συμπεράσματα: Η δράση των βιολογικών παραγόντων είναι στατιστικά σημαντικά καλύτερη στη νόσο του Crohn παρά στην ελκώδη κολίτιδα (81,4% vs 18,6%, αντίστοιχα βρίσκονταν σε κλινική ύφεση, p < 0,005). Για όσο περισσότερο χρόνο οι ασθενείς λαμβάνουν τους βιολογικούς παράγοντες και όσο νωρίτερα στην πορεία της νόσου τους ξεκινά η λήψη τους, τόσο καλύτερα ανταποκρίνονται. Κανένας θάνατος δεν παρουσιάστηκε. Οι παρενέργειες που αποτέλεσαν αιτία διακοπής (παροδικής ή οριστικής) εμφανίστηκαν μόνο σε ασθενείς που λάμβαναν ινφλιξιμάβη. Η αδαλιμουμάβη φαίνεται να έχει λιγότερες και λιγότερο σοβαρές παρενέργειες. Περαιτέρω μελέτες σε περισσότερους ασθενείς ή σε μεγαλύτερο βάθος χρόνου (περισσότερο από ένα έτος) είναι απαραίτητες για την εξαγωγή πιο ασφαλών συμπερασμάτων. / The aim of this work was to study the safety and efficacy of anti-TNFα biologic agents, infliximab and adalimumab, in patients suffering from inflammatory bowel disorders (IBD) at Patras University Hospital. We tried to associate clinical response or the occurrence of side-effects with several patient groups. Methods: From May 2013 to May 2014, 63 patients were recorded with the only inclusion criteria being their diagnosis with IBD (Crohn’s disease, ulcerative colitis or indeterminate colitis) and the treatment with infliximab or adalimumab. The efficacy was evaluated based on the presence of clinical remission or active disease. The safety was evaluated based on the presence or absence of side-effects. Results: 65.1% of all patients (41/63) received infliximab infusions. Only 20.6% of patients (13/63) required any adjustment to their biologic agent (dose increase or increased dose frequency). 11.1% of patients (7/63) permanently stopped treatment due to lack or loss of response and 3.2% (2/63) due to side-effects. 7.9% of patients (5/63) transiently stopped treatment (up to 1 year) due to lack or loss of response and 9.5% (6/63) due to side-effects. 61.1% of patients (33/54) who had been treated at least once with infliximab presented side-effects. 38.1% of patients (8/21) who had been treated at least once with adalimumab presented side-effects. The most common side-effects recorded in the group of patients that were treated with infliximab were infections (12/59, 20.3%) and arthralgias (12/59, 20.3%). The most common side-effects recorded in the group of patients that were treated with adalimumab were infections (4/10, 40%) and headache (4/10, 40%). 69.8 of patients (44/63) were in clinical remission. Conclusions: The efficacy of biologic agents is statistically significant better in Crohn’s disease than in ulcerative colitis (81.4% Vs 18.6% respectively were in clinical remission, p < 0.005). The longer the patients were being treated with the biologic agents and the sooner these agents were introduced in the course of the disease the better the clinical response was. No death was observed. The side-effects that led to treatment cessation (either permanent or transient) were only associated with the treatment with infliximab. The treatment with adalimumab seems to be associated with both fewer and less serious side-effects. Studies recording more patients and for a longer period of time (more than one year) need to be conducted in order to draw more clear conclusions.

These studies indicate that tumor necrosis factor-α (TNFα) alters albumin permeability and unlike bradykinin (BK) the increased albumin permeability lasts for the duration of the application. Neither agonist requires the presence of white blood cells or other blood-borne substances to produce this inflammatory response. These experiments were completed in the in situ, microcannulated, perfused venules of the mesentery in the anesthetized hamster. Albumin transport was measured using intravital fluorescence microscopy, TRITC-labeled albumin, and densitometric tracking. Further, by varying the intravascular pressure, the hydraulic (L(p)(1 - σ)) and diffusive permeability (P0) coefficients of these microvessels were determined. Both BK and TNFα produced an increase in albumin flux, which was dependent upon the dose and time domains. This response was present when the agonists were given by either intra- or extravascular presentation. Both hydraulic coupling and microvascular permeability were increased by BK and TNFα. TNFα increased albumin permeability rapidly and its effect lasted as long as TNFα was present, whereas the increased albumin transport by BK was biphasic. The results implicate a dynamic modification in the microvascular wall to these inflammatory agonists and the mechanism(s) for transduction in the endothelium are quite different.