Miriam Barbanti

Inhibition of intestinal bacterial translocation with rifaximin modulates lamina propria monocytic cells reactivity and protects against inflammation in a rodent model of colitis.

Background: A modification of the intestinal flora and an increased bacterial translocation is a common finding in patients with inflammatory bowel disease as well as in animal model of colitis. Rifaximin, a non-absorbable derivative of rifamycin, is an effective antibiotic that acts by inhibiting bacterial ribonucleic acid synthesis. Aims: In the present study, we investigated the effect of the administration of rifaximin (10, 30 and 50 mg/kg/day) or prednisolone (10 mg/kg/day) in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. Methods: Colitis was induced in mice by intrarectal administration of TNBS (1.5 mg/mouse in 50% ethanol) and disease severity assessed clinically and by histologic scoring of colon damage, determination of interleukin (IL)-2, IL-12, interferon (IFN)-γ and tumor necrosis factor (TNF)-α (protein and mRNA and myeloperoxidase (MPO) activity in the colon. Cytokines production by the lamina propria mononuclear cells (LPMC) and luminal bacteria were also measured. Results: Rifaximin administration (30 or 50 mg/kg/day) increased survival rates of colitic mice and reduced colitis severity as demonstrated by improvement of wasting syndrome, histologic scores, decrease in colon IL-2, IL-12, IFN-γ and TNF-α (protein and mRNA) levels, and diminished colon MPO activity. Rifaximin administration caused a significant reduction of colon bacterial translocation towards mesenteric lymph nodes. LPMC obtained from rifaximin-treated mice released significantly lower amount of IFN-γ in response to ex vivo stimulation with agonistic anti-CD3 and anti-CD28 antibodies. Rifaximin (50 mg/kg/day) significantly accelerates recovery in mice with established colitis. Conclusions: Luminal bacterial microflora plays a role in the pathogenesis of TNBS-induced colitis in mice. Rifaximin administration reduces the development of colitis and accelerates healing of established disease by preventing bacterial translocation.

Relative contribution of acetylated cyclo-oxygenase (COX)-2 and 5-lipooxygenase (LOX) in regulating gastric mucosal integrity and adaptation to aspirin.

In addition to inhibiting formation of prothrombotic eicosanoids, aspirin causes the acetylation of cyclooxygenase (COX)‐2. The acetylated COX‐2 remains active, and upon cell activation, initiates the generation of 15R‐HETE, a lipid substrate for 5‐lipoxygenase (LOX) leading to the formation of 15‐epi‐LXA4 (also termed “aspirin‐triggered lipoxin,” or ATL). Because ATL potently inhibits polymorphonuclear cell (PMN) function, we assessed the relative contribution of this lipid mediator in conjunction with another 5‐LOX product, the leukotriene (LT)B4, to the pathogenesis of acute damage and gastric adaptation to aspirin. Data presented herein indicate that acute injury and gastric adaptation to aspirin is associated with ATL generation. Administration of COX inhibitors (celecoxib, indomethacin, ketoprofen) to aspirin‐treated rats exacerbated acute injury and abolished adaptation to aspirin. Moreover, it inhibited ATL formation and caused a four‐ to fivefold increase in LTB4 synthesis. In contrast, licofelone, a COX/5‐LOX inhibitor, did not exacerbate acute gastric injury nor did it interfere with gastric adaptation to aspirin. Although licofelone blocked ATL and LTB4 formation in aspirin‐treated rats, it attenuated aspirin‐induced gastric PMN margination. These findings indicate that the balance between the production of LTB4 and ATL modulates PMN recruitment/function and gastric mucosal responses to aspirin.

Pregnane-X-receptor mediates the anti-inflammatory activities of rifaximin on detoxification pathways in intestinal epithelial cells.

The pregnane-X-receptor (PXR) is master gene overseeing detoxification of wide number of xenobiotics and is critical for maintenance of intestinal integrity. The intestinal expression of genes involved in cellular detoxification is down-regulated in patients with inflammatory bowel diseases (IBD). Rifaximin is a non-absorbable antibiotic endowed with a PXR agonistic activity. In the present study we have investigated whether rifaximin activates PXR in primary human colon epithelial cells and human colon biopsies and assessed whether this antibiotic antagonizes the effect of tumor necrosis factor (TNF)-α on expression of PXR and PXR-related genes. Present results demonstrate that primary colon epithelial cells express PXR and that their exposure to rifaximin induces the expression of genes involved in cellular detoxification. Exposure to TNFα reduces the expression of PXR mRNA as well as expression of its target genes. This inhibitory effect was prevented by that co-treatment with rifaximin. Knocking down the expression of PXR in colon epithelial cells by an anti-PXR siRNA, abrogated the counter-regulatory effects exerted by rifaximin on cell exposed to TNFα. Finally, ex vivo exposure of colon biopsies obtained from ulcerative colitis patients to rifaximin increased the expression of genes involved in xenobiotics metabolism. In aggregate, these data illustrate that rifaximin increases the expression of PXR and PXR-regulated genes involved in the metabolism and excretion of xenobiotics and antagonizes the effects of TNFα in intestinal epithelial cells and colon biopsies. These non-antibiotic effects of rifaximin could contribute to the maintenance of the intestinal barrier integrity against xenobiotics and products generated by luminal bacteria.

Inhibition of NF-κB by a PXR-dependent pathway mediates counter-regulatory activities of rifaximin on innate immunity in intestinal epithelial cells

A dysregulated interaction between intestinal epithelial cells (IEC) and components of innate immunity is a hallmark of inflammatory bowel diseases. Rifaximin is a poorly absorbed oral antimicrobial agent increasingly used in the treatment of inflammatory bowel diseases that has been demonstrated to act as a gut-specific ligand for the human nuclear receptor pregnane-X receptor (PXR). In the present study we investigated, whether activation of PXR in IEC by rifaximin, emanates counter-regulatory signals and modulates the expression of cytokines or chemokines mechanistically involved in dysregulated intestinal immune homeostasis documented in inflammatory bowel diseases. Our results demonstrate that primary IEC express PXR that regulate the pattern of cytokines and chemokines expressed. PXR silencing decreases TGF-β and IP-10 while increases the expression of TNF-α, IL-8, Rantes and increase the production of PGE2. This pattern is further exacerbated by treating anti-PXR siRNA cells with bacterial endotoxin (LPS). Exposure to rifaximin caused a robust attenuation of generation of inflammatory mediators caused by LPS and increased the generation of TGF-β. PXR silencing completely abrogated these anti-inflammatory effects of rifaximin. By Western blot analysis we found that rifaximin abrogates the binding of NF-κB caused by LPS. Finally, exposure of human colon biopsies from inflammatory bowel diseases patients to rifaximin reduced mRNA levels of IL-8, Rantes, MIP-3α and TNFα induced by LPS. Collectively, these data establish that rifaximin exerts counter-regulatory activities at the interface between enteric bacteria and intestinal epithelial cells. The ability of rifaximin to activate PXR contributes to the maintenance of the intestinal immune homeostasis.

Comparison of the Antithrombotic and Haemorrhagic Effects of Heparin and a New Low Molecular Weight Heparin in Rats

A new low molecular weight heparin (LMWH) and a conventional unfractionated heparin (H) were tested in rats for venous antithrombotic activity and bleeding tendency after intravenous administration. Both drugs showed antithrombotic activity, but LMWH at the low dosages tested (0.1 and 0.25 mg/kg) demonstrated significantly higher activity than H. In a rat bleeding time test (transection model) both heparins produced a prolonged bleeding time, but LMWH possessed significantly less potency than H at all the dosages tested. Ex vivo coagulation parameters (activated partial thromboplastin time and antiXa activity) were also evaluated: LMWH presented very low activity on the APTT test and a sustained antiXa activity, comparable to that of H.

Evidence for a hyperglycaemia-dependent decrease of antithrombin III-thrombin complex formation in humans.

SummaryIn the presence of increased levels of fibrinopeptide A, decreased antithrombin III biological activity, and thrombin-antithrombin III complex levels are seen in diabetic patients. Induced-hyperglycaemia in diabetic and normal subjects decreased antithrombin III activity and thrombin-antithrombin III levels, and increased fibrinopeptide A plasma levels, while antithrombin III concentration did not change; heparin was shown to reduced these phenomena. In diabetic patients, euglycaemia induced by insulin infusion restored antithrombin III activity, thrombin-antithrombin III complex and fibrinopeptide A concentrations; heparin administration had the same effects. These data stress the role of a hyperglycaemia-dependent decrease of antithrombin III activity in precipitating thrombin hyperactivity in diabetes mellitus.

Rifaximin: beyond the traditional antibiotic activity.

Rifaximin is a non-systemic oral antibiotic derived from rifampin and characterized by a broad spectrum of antibacterial activity against Gram-positive and -negative, aerobic and anaerobic bacteria. Rifaximin was first approved in Italy in 1987 and afterwards in many other worldwide countries for the treatment of several gastrointestinal diseases. This review updates the pharmacology and pharmacodynamics of rifaximin highlighting the different actions, beyond its antibacterial activity, such as alteration of virulence, prevention of gut mucosal adherence and bacterial translocation. Moreover, rifaximin exerts some anti-inflammatory effects with only a minimal effect on the overall composition of the gut microbiota. All these properties make rifaximin a good candidate to treat various gastrointestinal diseases.

Non-enzymatic glycation reduces heparin cofactor II anti-thrombin activity.

SummaryThe effects of non-enzymatic glycation on heparin cofactor II activity, at glucose concentrations which might be expected in physiological or diabetic conditions have been evaluated in this study. Radiolabelled glucose incorporation was associated with a loss of heparin cofactor anti-thrombin activity. The heparin cofactor heparin and dermatan sulfatedependent inhibition of thrombin was significantly reduced, showing a remarkable decrease of the maximum second order rate constant. This study shows that heparin cofactor can be glycated at glucose concentrations found in the blood, and that this phenomenon produces a loss of heparin cofactor-antithrombin activity. These data suggest, furthermore, a possible link between heparin cofactor glycation and the pathogenesis of thrombosis in diabetes mellitus.

Antithrombotic activity of Desmin 370. Comparison with a high molecular weight dermatan sulfate

Dermatans are endogenous glycosaminoglycans which catalyze thrombin-heparin cofactor II interaction (1) and may promote fibrinolysis by induction of PA release (2). In different animal models dermatans have been shown to prevent thrombus formation (3,4,5,6). Since they are less haemorrhagic than standard heparin (4,7), dermatans may represent a class of antithrombotic agents with reduced bleeding risk. Low molecular weight heparins have longer biological halflives than standard heparins (8). Recently it has been found that potentiation of thrombin-heparin cofactor II interaction by a low molecular weight dermatan sulfate lasted longer than that induced by a native dermatan sulfate, both in rats and in monkeys (9), suggesting that the two molecular weight forms have different pharmacokinetic profiles. The aim of the present study was to investigate whether the antithrombotic activity of Desmin 370, a new low molecular weight dermatan sulfate (IO, 11,12) and DS435, the native dermatan, was influenced by route of administration, In addition, the concentration-time curves of the two dermatans, given s.c., were evaluated ex vivo by a coagulation assay.

Antithrombotic and thrombolytic activity of sulodexide in rats

SummaryWe evaluated the ability of sulodexide, an extracted glycosaminoglycan, to prevent thrombus formation and to reduce a stabilized thrombus in a rat venous thrombosis model (vena cava ligature). Injection of sulodexide 10 min before induction of venous stasis, prevented thrombus formation in a dose-dependent manner (median effective dose 0.55 mg/kg). When given to rats with 6-h-old thrombi, sulodexide caused a marked reduction in thrombus size which reached 70% after 2 h with the highest dose tested (2 mg/kg). The effect of sulodexide on established thrombi appears to be due, at least in part, to a fibrinolysis-mediated mechanism, since it was significantly inhibited by ε-aminocaproic acid, a well-known antifibrinolytic drug. Treatment with sulodexide did not noticeably affect plasma levels of plasminogen activator and its specific inhibitor. We also showed that fluorescein-labelled sulodexide, when given to animals with 6-h-old thrombi, was present within the thrombi harvested 2 h later, but was then absent from blood. The fluorescence was mainly located in areas filled with amorphous material, that was identified as fibrin by staining with phosphotungstic acid-hematoxylin. No fluoresceinlabelled material could be detected in rats treated with fluorescein alone. These findings indicate that, besides preventing venous thrombus formation, sulodexide is able to promote thrombus dissolution by a mechanism that is partly related to local fibrinolysis stimulation.