Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 5 uppsatser.

The mammalian intestine contains a dense and diverse community of microorganisms. The resident microbiota makes contributions to host to promote proper immune system development and limit pathogen colonization. In this study, the effects of microbiota disruption with or without TLRs stimulation on intestinal permeability and immunity were examined in C57BL/6 mice receiving antibiotic treatment for 6 days and in antibiotics-treated mice received dead E. coli or S. aureus at day 4. The results showed that antibiotic treatment significantly decreased the total number of bacteria including specific aerobic group Enterobacteriaceae and Enterococcus, and specific anaerobic group Lactococcus/Bifidobacterium in intestinal mucosa and lumen. Although only a slight increase in the intestinal permeability and no change in caspase-3 activity of intestinal mucosa were observed after antibiotic treatment, the bacterial translocation (BT) to mesenteric lymph nodes (MLN) increased significantly. Subsequent experiments showed that antibiotic treatment decreased the mucosal killing activity and the expression of non-defensin family including RegIII£], RegIII£^, CRP-ductin and RELM£] but not the defensin family, and increased the translocation of pathogen K. pneumoniae significantly, suggesting that the increase of BT to MLN after antibiotic treatment is likely due to a reduction in gut immunity rather than an increase of intestinal permeability. Moreover, stimulation of TLR4 reversed the effect of antibiotic treatment, suggesting that the functioning of TLR4 in intestinal epithelium is required to prevent pathogenic invasion and maintain intestinal homeostasis.

Diss. (sammanfattning) Umeå : Univ., 2003. / Härtill 6 uppsatser.

nÃo hà / IntroduÃÃo: A esteatohepatite nÃo alcoÃlica (NASH) à um evento adverso do quimioterÃpico irinotecano extremamente relevante, pois associa-se a uma maior mortalidade apÃs ressecÃÃes hepÃticas. Atà o momento nÃo hà na literatura um modelo experimental bem estabelecido de NASH induzido por irinotecano. Objetivo: Estabelecer um modelo experimental de NASH induzida pelo irinotecano em camundongos que simule os aspectos histopatolÃgicos encontrados na clÃnica. Estudar os mecanismos e mediadores envolvidos na sua fisiopatologia incluindo citocinas inflamatÃrias, estresse oxidativo e participaÃÃo da translocaÃÃo bacteriana intestinal. MÃtodos: Camundongos machos do tipo Swiss, pesando entre 25 a 30g,foram divididos em grupos experimentais (n=8-10) e foram injetados com soluÃÃo salina (5 mL/kg, i.p.) ou Irinotecano nas doses 25, 50, 75 or 100 mg/kg, i.p, trÃs vezes por semana em dias alternados. Os animais foram entÃo pesados e sacrificados ao fim das semanas 1, 3, 5, 7 e 9. No sangue foram dosados os nÃveis de ALT e AST, leucograma, proteÃnas totais e realizada hemocultura da veia porta e do plexo ocular. Nas amostras do tecido hepÃtico, avaliaram-se os nÃveis de lipÃdeos totais, mieloperoxidase, malonaldeÃdo e glutationa (GSNP) seguida da anÃlise histopatolÃgica realizada apÃs preparaÃÃo da lÃmina pelos mÃtodos HE e Masson para avaliar e graduar os critÃrios histolÃgicos da NASH. As amostras de duodeno foram examinadas histologicamente (HE) para graduar a mucosite. ImunohistoquÃmica para IL-1, IL-18, NOSi, TNFα e TLR-4 foi realizada tanto nas amostras hepÃticas como duodenais. A anÃlise estatÃstica foi realizada empregando o teste t de Student para variÃveis nÃo pareadas ou o teste Mann-Whitney quando indicado. Resultados: A dose de 50mg/kg de irinotecano trÃs vezes por semana i.p. (IRI 50) se associou com perda de peso, leucopenia, hepatomegalia, diminuiÃÃo das proteÃnas plasmÃticas e aumento das enzimas hepÃticas (ALT e AST). IRI 50 alterou os parÃmetros de estresse oxidativo, elevando a expressÃo de malonaldeÃdo e diminuindo GSNP com inÃcio na primeira semana do experimento. IRI 50 tambÃm se associou a todas as alteraÃÃes histolÃgicas da NASH (esteatose, infiltraÃÃo neutrofÃlica e tumefaÃÃo celular e fibrose) analisadas na sÃtima semana quando comparado com controle salina. Nas amostras duodenais, observamos alteraÃÃes histolÃgicas de mucosite severa a partir da terceira semana. Houve tambÃm em IRI 50 aumento da imunoexpressÃo de IL-1, NOSi e TLR-4 tanto nas amostras do fÃgado como nas do duodeno na semana 7. Nas hemoculturas de sangue portal e orbitÃrio identificou-se o crescimento de enterobactÃrias gram-negativas desda a primeira semana do experimento. ConclusÃo: A administraÃÃo de irinotecano 50mg/kg i.p. trÃs vezes por semana por sete semanas consecutivas à um modelo experimental que reproduz todas as alteraÃÃes histopatolÃgicas da NASH induzida por irinotecano observadas na clÃnica. Existem evidÃncias da participaÃÃo concomitante das citocinas inflamatÃrias e do estresse oxidativo na patogÃnese da NASH em nosso modelo. A translocaÃÃo bacteriana intestinal com bacteremia portal por gram-negativos pode ser o insulto primeiro e o elo de conexÃo comum entre os diversos fatores fisiopatolÃgicos. / Introduction: The nonalcoholic steatohepatitis (NASH) is a relevant adverse effect of the chemotherapy with irinotecan, since it has been associated with increased surgical mortality after hepatic resections. So far there are no reports in the literature of a well established experimental irinotecan-induced NASH model. Aim: Develop an experimental model of irinotecan-induced NASH in mice that simulate the full histological alterations found in the clinical practice. Study the possible mechanisms and mediators involved into its pathophysiology including inflammatory cytokines, oxidative stress and the participation of intestinal bacterial translocation. Methods: Male swiss mice weighting between 35 and 30g were divided into experimental groups (n=8-10) and were injected with saline (5 mL/kg, i.p.) or irinotecan in the following dosages: 25, 50, 75 or 100 mg/kg, i.p, three times a week every other day. Mice were weighted and killed at the end of the weeks 1, 3, 5, 7 and 9. Measurements of ALT and AST, leukocytes, total proteins were performed in blood samples as well as cultures of portal vein and ocular plexus blood. In the hepatic tissue samples, total lipids, myeloperoxidase, malonaldehyde and nonproteic sulfidryl groups (NPSH) levels were analyzed, followed by histological evaluation, after slide preparation through HE and Masson methods. Duodenal samples were examined histologically for mucositis grading. Immunohistochemistry analysis for IL-1, IL-18, NOSi, TNFα e TLR-4 were performed in both hepatic and duodenal tissue samples. Statistical analysis was carried out through Studentâs t test or Mann-Whitney test, as indicated. Results: The dose of 50mg/kg of irinotecan three times a week i.p. (IRI 50) associated to weight loss, low white cell count, hepatomegaly, decreased total plasma proteins and elevated hepatic enzymes (AST and ALT). IRI 50 altered oxidative stress parameters, elevating elevating malonaldehyde and decreasing NSPG levels at hepatic tissue starting at week 1. IRI 50 also was associated to the full histological changes found in NASH (steatosis, neutrophilic infiltration and hepatocyte balloning) when analyzed at the seventh week and compared to saline group. The duodenal samples showed severe mucositis changes starting at week three. It was also demonstrated elevated immunostaining for IL-1, iNOS and TLR-4 in both liver and duodenal samples at week seven. The cultures were positive for gram negative intestinal bacteria in portal and orbital blood since the first experimental week. Conclusion: The administration of the irinotecan 50mg/kg i.p. three times a week for seven consecutive weeks is an experimental model that reproduces all the histological changes found in the irinotecan-associated NASH found in clinical practice. Concomitant participation of inflammatory cytokines and oxidative stress were found to play a role in NASH pathogenesis in this model. Intestinal gram-negative bacterial translocation leading to portal bacteremia may represent the very first hepatic insult and the connection between the pathogenic factors.

No

PGC-1 é uma família de coativadores de fatores de transcrição que controlam a expressão de diversos genes envolvidos na homeostase energética celular. As isoformas PGC-1 e estão presente em tecidos com alto metabolismo oxidativo e são capazes de aumentar biogênese mitocondrial, -oxidação de ácidos graxos e gliconeogênese em resposta à exposição ao frio, jejum e exercício. Inicialmente mostramos que macrófagos in vitro aumentaram a expressão de PGC-1 após 1h da exposição à zymosan. Com isso, hipotetizamos que PGC-1 poderia ter sua expressão aumentada em resposta a um insulto bacteriano. Para verificar nossa hipótese analisamos a expressão de PGC-1 em um modelo de pancreatite aguda (PA), caracterizada por uma forte resposta inflamatória estéril inicial, seguida, após poucos dias, por translocação bacteriana intestinal e infecção disseminada. PA foi induzida por infusão retrograda de taurocolato de sódio (2,5%). Também analisamos PGC-1 em um modelo de sepse por ligadura e perfuração cecal (CLP), cujo conteúdo intestinal é depositado no peritôneo, causando infecção grave local e disseminada. Animais tratados com Imipenem durante 48h após PA também foram analisados, bem como a interferência de PGC-1 ASO no processo de fagocitose. A expressão de PGC-1 e foi medida por PCR quantitativo. PA foi confirmada pelo aumento da amilase sérica e a inflamação sistêmica ratificada por leucocitose. PGC1 aumentou no baço e nos leucócitos circulantes 48h após PA e no lavado peritoneal 24h após PA e CLP. No entanto, PGC1 diminuiu no baço 24h após PA. Tratamento com Imipenem diminuiu PGC- 1. A diminuição de PGC-1 após transfecção com ASO levou à redução do processo de fagocitose. Assim, concluímos que ocorre aumento de PGC-1 na presença de bactérias e esse aumento está relacionado com fagocitose / PGC1 is a family of transcriptional coactivators that controls the expression of several genes involved in cell energy homeostasis. PGC1 isoforms and are present in tissues with high oxidative metabolism and are able to enhance mitochondrial biogenesis, -oxidation of fatty acids and gluconeogenesis in response to exposure to cold, fasting and exercise. Initial results showed macrophages in vitro present increased PGC-1 expression after 1h exposure to zymosan. Thus, we hypothesized that PGC-1 could be up-regulated in response to bacterial insult. We tested our hypothesis following PGC-1 expression in an acute pancreatitis (AP) model, characterized initially by a strong sterile inflammatory response, followed, few days later, by bacterial intestinal translocation and disseminated infection. AP was induced by retrograde infusion of sodium taurocholate (2.5%). We also analysed PGC-1 in a model of sepsis by cecal ligation and puncture (CLP), whose intestinal content is deposited in the peritoneum, causing a severe local and disseminated infection. Animals submitted to PA and treated with Imipenem for 48 hours were also analyzed, as well as the interference of PGC-1 ASO in phagocytosis process. PGC-1 and expression were measured by quantitative PCR. AP was confirmed by increased blood amylase and the systemic inflammation was noted by leukocytosis after 48h. PGC1 was increased in spleen and circulating leukocytes 48h after AP and in peritoneal lavage 24h after AP and CLP. On the other hand, PGC1 was decreased in spleen 24h after AP induction. Imipenem treatment decreased PGC-1. The decreased of PGC-1 after ASO transfection led to a reduction of phagocytosis process. Thus, we conclude there is a PGC-1 increase in bacterial presence and this increase is related to phagocytosis

PGC-1 é uma família de coativadores de fatores de transcrição que controlam a expressão de diversos genes envolvidos na homeostase energética celular. As isoformas PGC-1 e estão presente em tecidos com alto metabolismo oxidativo e são capazes de aumentar biogênese mitocondrial, -oxidação de ácidos graxos e gliconeogênese em resposta à exposição ao frio, jejum e exercício. Inicialmente mostramos que macrófagos in vitro aumentaram a expressão de PGC-1 após 1h da exposição à zymosan. Com isso, hipotetizamos que PGC-1 poderia ter sua expressão aumentada em resposta a um insulto bacteriano. Para verificar nossa hipótese analisamos a expressão de PGC-1 em um modelo de pancreatite aguda (PA), caracterizada por uma forte resposta inflamatória estéril inicial, seguida, após poucos dias, por translocação bacteriana intestinal e infecção disseminada. PA foi induzida por infusão retrograda de taurocolato de sódio (2,5%). Também analisamos PGC-1 em um modelo de sepse por ligadura e perfuração cecal (CLP), cujo conteúdo intestinal é depositado no peritôneo, causando infecção grave local e disseminada. Animais tratados com Imipenem durante 48h após PA também foram analisados, bem como a interferência de PGC-1 ASO no processo de fagocitose. A expressão de PGC-1 e foi medida por PCR quantitativo. PA foi confirmada pelo aumento da amilase sérica e a inflamação sistêmica ratificada por leucocitose. PGC1 aumentou no baço e nos leucócitos circulantes 48h após PA e no lavado peritoneal 24h após PA e CLP. No entanto, PGC1 diminuiu no baço 24h após PA. Tratamento com Imipenem diminuiu PGC- 1. A diminuição de PGC-1 após transfecção com ASO levou à redução do processo de fagocitose. Assim, concluímos que ocorre aumento de PGC-1 na presença de bactérias e esse aumento está relacionado com fagocitose / PGC1 is a family of transcriptional coactivators that controls the expression of several genes involved in cell energy homeostasis. PGC1 isoforms and are present in tissues with high oxidative metabolism and are able to enhance mitochondrial biogenesis, -oxidation of fatty acids and gluconeogenesis in response to exposure to cold, fasting and exercise. Initial results showed macrophages in vitro present increased PGC-1 expression after 1h exposure to zymosan. Thus, we hypothesized that PGC-1 could be up-regulated in response to bacterial insult. We tested our hypothesis following PGC-1 expression in an acute pancreatitis (AP) model, characterized initially by a strong sterile inflammatory response, followed, few days later, by bacterial intestinal translocation and disseminated infection. AP was induced by retrograde infusion of sodium taurocholate (2.5%). We also analysed PGC-1 in a model of sepsis by cecal ligation and puncture (CLP), whose intestinal content is deposited in the peritoneum, causing a severe local and disseminated infection. Animals submitted to PA and treated with Imipenem for 48 hours were also analyzed, as well as the interference of PGC-1 ASO in phagocytosis process. PGC-1 and expression were measured by quantitative PCR. AP was confirmed by increased blood amylase and the systemic inflammation was noted by leukocytosis after 48h. PGC1 was increased in spleen and circulating leukocytes 48h after AP and in peritoneal lavage 24h after AP and CLP. On the other hand, PGC1 was decreased in spleen 24h after AP induction. Imipenem treatment decreased PGC-1. The decreased of PGC-1 after ASO transfection led to a reduction of phagocytosis process. Thus, we conclude there is a PGC-1 increase in bacterial presence and this increase is related to phagocytosis

Early detection of infection, identification of microorganism, and correct choice of antibiotics are critical in the management of sepsis. Quantitative real-time polymerase chain reaction (RT-PCR) has the potential to improve the timeliness, sensitivity, and accuracy of detecting pathogens. In this study we utilize this method to detect the enteric bacterial counts in the blood from patients with systemic inflammatory response syndrome (SIRS) in the emergency department (ED). The universal primers utilized in RT-PCR are specific for 23S ribosomal DNA (rDNA) and wec F gene. The results show that in SIRS patients with positive culture results from specimen collected within 10 days after presenting to ED, and patients surviving for less than 28 days, the serum bacterial DNA load of enteric Gram negative bacilli is higher. In SIRS patients with shock, patients fulfilling both white blood cell counts and respiratory criteria of SIRS, and patients fulfilling both white blood cell counts and respiratory criteria of SIRS with Acute Physiology and Chronic Health Evaluation II score more than 20, the serum bacterial DNA load of enteric Gram negative bacilli and 28-day mortality are both higher. These results suggest that bacterial translocation may happen in patients with SIRS and may be related to higher mortality in patients with SIRS.

Σκοπός της μελέτης ήταν να εξετάσουμε, σε ένα πειραματικό μοντέλο πειραματικής ηπατεκτομής (70%) αφ’ ενός και παροδικής απόφραξης του ηπατοδωδεκαδακτυλικού συνδέσμου (χειρισμός Pringle) αφ’ ετέρου, το φαινόμενο της βακτηριακής μετακίνησης, της ενδοτοξιναιμίας, καθώς και τις μεταβολές οι οποίες συμβαίνουν στην εντερική χλωρίδα και τον εντερικό βλεννογόνο. Σε αυτά τα δύο μοντέλα προσπαθήσαμε κατ’ αρχήν να πιστοποιήσουμε την αυξημένη μετακίνηση των εντερικών βακτηρίων στους μεσεντέριους λεμφαδένες και την διαπίδυση ενδοτοξινών από τον αυλό του εντέρου στην πυλαία και την αορτή. Στα ίδια μοντέλα προσπαθήσαμε να αναστείλουμε τα ανωτέρω φαινόμενα με φαρμακολογικούς χειρισμούς, μελετώντας πρώτον τη χορήγηση λακτουλόζης από το στόμα, η οποία έχει την ικανότητα να δεσμεύει την ενδοτοξίνη και να σχηματίζει σύμπλεγμα που δεν απορροφάται από το έντερο, με σκοπό τη μείωση της ενδοαυλικής απορροφήσιμης ενδοτοξίνης και δεύτερον τη χορήγηση δυσαπορρόφητων αντιβιοτικών από το στόμα, με σκοπό την ποσοτική ελάττωση του πληθυσμού της μικροβιακής χλωρίδας στον αυλό του εντέρου. Τα αποτελέσματα κάθε φάσης της μελέτης της ηπατεκτομής (70%) και του χειρισμού Pringle είχαν ως εξής: Η ηπατεκτομή (70%), προάγει την εντερική βακτηριακή μετακίνηση στους μεσεντέριους λεμφαδένες και στο ήπαρ, αυξάνει την ενδοτοξιναιμία και επιφέρει ιστολογικές αλλαγές στο ύψος των λάχνων. Η χορήγηση λακτουλόζης δημιουργεί τις κατάλληλες συνθήκες που επιφέρουν την μείωση των θετικών καλλιεργειών ιδιαίτερα αυτών του ήπατος και ελαττώνει την ενδοτοξίνη στην πυλαία. Η αποστείρωση του εντέρου με τη χορήγηση δυσαπορρόφητων αντιβιοτικών, μειώνει την εντερική βακτηριακή μετακίνηση στους μεσεντέριους λεμφαδένες και την ενδοτοξιναιμία. Ο χειρισμός Pringle, προάγει την εντερική βακτηριακή μετακίνηση και την ενδοτοξιναιμία, ενώ η απόπτωση ελαττώνεται με την αποστείρωση του εντέρου και τη χορήγηση λακτουλόζης μόνο άμεσα μετά τον χειρισμό. Η εξασφάλιση της ανατομικής και λειτουργικής ακεραιότητας του εντερικού βλεννογόνου έχει ιδιαίτερη σημασία στη διατήρηση του βλεννογόνιου εντερικού φραγμού και αποτρέπει την ενδοτοξιναιμία και τη βακτηριακή μετακίνηση. Η αποκατάσταση της δομικής και λειτουργικής ακεραιότητας του εντερικού βλεννογόνου, στην εκτεταμένη ηπατεκτομή, με τη χορήγηση μη απορροφήσιμων αντιβιοτικών και λακτουλόζης, αναστέλλει σημαντικά τα ανωτέρω φαινόμενα. / The purpose of this study is to investigate on an experimental model of extended hepatectomy(70%) and temporary occlusion of the hepatoduodenal ligament (Pringle maneuver), the phenomenon of bacterial translocation, endotoxaemia as well as the changes that are taking place, both in the intestinal flora and intestinal mucosa. In both models we attempt to demonstrate the increase translocation of intestinal bacteria to mesenteric lymph nodes (MLNs) and increase of endotoxin in the systemic and portal circulation. Using the same models, we attempted to inhibit the above phenomena with pharmacological manipulation. We examined the decrease of the endotoxins translocation, by administering per.os lactulose, which has the property to bind the endotoxin formatting a nonabsorbable comlex in the gut lumen. We also investigated the effect of nonabsorbable antibiotics per. os on reducing the intestinal flora. Our results indicated that, extended hepatectomy (70%) increased translocation of intestinal bacteria to mesenteric lymph nodes and liver and increased the endotoxaemia inducing the decrease of intestinal villus height. The administering of lactulose created the proper conditions which decreased positive liver cultures and reduced the endotoxin concentrations in portal blood. Gut decontamination by administration of nonabsorbable antibiotics reduced the intestinal flora, the intestinal bacteria translocation to MLNs and endotoxaemia. Pringle maneuver promoted the intestinal bacterial translocation and endotoxaemia. Apoptosis decreases, by gut decontamination and administration of lactulose only immediately after Pringle maneuver. The anatomic and functional integrity reassurance, of intestinal mucosa, has strong significance for the preservation of intestinal mucosal barrier to prevent endotoxaemia and bacterial translocation. The structural and functional establishment of enteric mucosa integrity, after extended hepatectomy, by administration of nonabsorbable antibiotics and lactulose, inhibit considerably the above phenomena.

La consommation excessive d’alcool concerne environ 20% de la population adulte en France. Il est établi qu’une consommation excessive aigue d’alcool engendre une augmentation de la morbi-mortalité en cas d’infection ou de traumatisme. La consommation chronique d’alcool augmente le risque de cancers et d’infection.Toutes ces conséquences médicales sont intimement liées à une altération des défenses de l’hôte induite par l’alcool. L’ingestion d’alcool et ses métabolites engendre une modification de la flore digestive appelée dysbiose ainsi qu’une augmentation de la perméabilité digestive. De cet effet local découle une augmentation du passage d’endotoxines dans le système veineux portal et systémique. Ainsi l’ensemble des éléments impliqués dans la réponse immunologique sont impactés de façon locale (Foie) et systémique par cette endotoxinémie chronique. Deux axes de recherche ont été privilégiés dans cette thèse: La translocation bactérienne et les sous-populations monocytaires. L’originalité de ce travail est l’étude de la cinétique des modifications immunologiques, chez des sujets alcoolo-dépendants (AD) hospitalisés pour un sevrage en alcool durant 2 à 6 semaines.La translocation bactérienne (TB) a été étudiée par l’analyse itérative de marqueurs sériques témoins de celle-ci: l’ADN 16S ribosomial en PCR temps réel, les taux sériques de LBP (LPS-Binding-Protein) et de CD14 soluble par ELISA. Les prélèvements veineux ont été effectués à jeun chez des sujets AD à J0 de leur sevrage et après 4 puis 6 semaines de sevrage. Avant le sevrage (J0) les 3 marqueurs sont plus élevés que dans une population témoin (p<0.001). Après 6 semaines de sevrage, les taux de LBP (p=0.04) et sCD14 (p=0.001) diminuent de manière significative sans revenir aux taux de la population témoin. La consommation de cannabis dans le mois précédent le sevrage est associée à une plus grande diminution des marqueurs LBP et sCD14 au cours du sevrage en alcool.Notre étude confirme une majoration de la TB chez les sujets AD non sevrés et l’impact potentiel du cannabis sur celle-ci. Elle montre également que le délai de sevrage de 6 semaines ne permet pas un retour à la normale des marqueurs de la TB.La répartition, le phénotype et la fonctionnalité des sous-populations monocytaires sanguines ont été étudiés à J0 et après 14 jours de sevrage en alcool chez des sujets AD. Avant le sevrage (J0), la fréquence des monocytes classiques (CD14+CD16-) est diminuée alors que celle des non classiques (CD14dimCD16+) est augmentée chez les sujets AD comparativement aux sujets contrôles. La fréquence des monocytes exprimant les TLR-2 et -4 est réduite chez les sujets AD. La sécrétion basale des cytokines IL-1, IL-6 et TNF est comparable chez les sujets AD et contrôles. En revanche après stimulation in vitro des monocytes par les ligands des TLR-2 et 4, respectivement peptidoglycanes (PGN) et lipopolysaccharides (LPS), la sécrétion d’IL-6 et de TNF est augmentée chez les sujets AD. Le sevrage de 14 jours restaure partiellement la distribution des sous-populations monocytaires. Nos résultats indiquent que la consommation chronique d’alcool altère la distribution, le phénotype et la fonctionnalité des monocytes chez les sujets AD, ces altérations s’améliorent en 14 jours de sevrage mais ne reviennent pas à la normale.Mon travail de thèse confirme l’impact de la consommation chronique d’alcool sur la translocation bactérienne et sur la réponse immune chez l’homme. Il précise la nature et la cinétique de cet impact. Mes résultats suggèrent également que le délai de retour à la normale semble être supérieur à 6 semaines. De nouvelles études permettront de mettre en évidence une cinétique plus précise de ces améliorations biologiques. Les résultats permettent d’envisager des recommandations cliniques quant à une durée d’abstinence pré-opératoire en cas de chirurgie programmée par exemple. / Excessive alcohol consumption concerns about 20% of the French adult population. An acute excessive alcohol consumption named “binge drinking” causes an increase in mortality and morbidity in case of trauma or infection. Chronic alcohol consumption causes an increased risk and severity of infections and increased risk of cancer. All these medical consequences are linked to changes in host defense induced by alcohol consumption. Indeed, alcohol and its metabolites generate modifications of the gut microbiota and increased gut permeability. This local effect causes an increase in endotoxin translocation into portal and systemic blood. Thus, all elements involved in the immune response, and in particular the monocytes as cellular component acting as first line of defense of the immune system, are affected by this chronic endotoxemia. Two research axes were studied during my thesis: Microbial translocation and monocyte subsets. The originality of this work was to study the kinetic of immunological changes induced by alcohol withdrawal in alcohol-dependent (AD) subjects hospitalized for an alcohol withdrawal during 2-6 weeks.Microbial translocation (MT) was studied by iterative analysis of serum markers: Bacterial 16S rDNA levels were measured using qPCR, Lipopolysaccharides-Binding protein (LBP) and soluble CD14 were quantified using ELISA. Blood samples were collected in fasten AD subjects at D0 of alcohol withdrawal and after 4 and 6 weeks of alcohol withdrawal. At D0, the 3 markers of MT were higher in AD subjects than in healthy controls (P<0.001). After 6 weeks of alcohol withdrawal, the serum level of LBP (p=0.04) and sCD14 (p=0.001) were significantly decreased but did not reach rates of healthy controls (HC). Cannabis use during the last month before alcohol withdrawal was associated with a greater decrease in sCD14 and LBP upon alcohol withdrawal. My study confirms an increase in MT in AD subjects and the potential impact of cannabis on this phenomenon, and shows that a 6-weeks abstinence period is not sufficient to return to normal blood biological parameters.Whether and how blood monocyte subsets were impaired in AD patients were studied as well as their evolution after alcohol withdrawal. The CD14+CD16- subset was decreased whereas the CD14dimCD16+ subset was expanded (p<0,001) in AD compared to HC. The frequencies of TLR2- and TLR4-expressing monocytes were reduced in AD compared to HC. Although the basal production of IL-1, IL-6 and TNF by monocytes in AD was comparable to HC, the PGN- and LPS-mediated IL-6 and TNF production were increased in AD. Frequencies of IL-6-expressing monocytes were higher in AD than HC. Alcohol withdrawal partially restored the distribution of monocyte subsets and the frequency of IL-6-producing monocytes, and increased the frequency of TNF-producing cells in response to LPS and PGN stimulation to levels comparable to those in HC. Our findings indicate that chronic alcohol use alters the distribution as well as the phenotypic and functional characteristics of blood monocyte subsets, which are partially restored following 2 weeks of alcohol withdrawal. These studies confirm and specify the impact of chronic alcohol consumption on microbial translocation and on the immune response. Our results also suggest that a period superior to 6 weeks is necessary to reach normal biological parameters. News studies will be necessary to specify the exact kinetic of these biological improvements.These results allow to consider clinical recommendations for a period of abstinence before programmed surgery for example.