Probiotic Sonicates Selectively Induce Mucosal Immune Cells Apoptosis Through Ceramide Generation Via Neutral Sphingolyelinase

Background: Probiotics appear to be beneficial in inflammatory bowel disease, but their mechanism of action is incompletely understood. We investigated whether probiotic-derived sphingomyelinase mediates this beneficial effect. Methodology/Principal Findings: Neutral sphingomyelinase (NSMase) activity was measured in sonicates of the probiotic L.brevis (LB)and S. thermophilus (ST) and the non-probiotic E. coli EC) and E. faecalis (EF). Lamina propria mononuclear cells (LPMC) were obtained from patients with Crohn"s disease (CD) and Ulcerative Colitis (UC), and peripheral blood mononuclear cells (PBMC) from healthy volunteers, analysing LPMC and PBMC apoptosis susceptibility, reactive oxygen species (ROS) generation and JNK activation. In some experiments, sonicates were preincubated with GSH or GW4869, a specific NSMase inhibitor. NSMase activity of LB and ST was 10-fold that of EC and EF sonicates. LB and ST sonicates induced significantly more apoptosis of CD and UC than control LPMC, whereas EC and EF sonicates failed to induce apoptosis. Pre-stimulation with anti-CD3/CD28 induced a significant and time-dependent increase in LB-induced apoptosis of LPMC and PBMC. Exposure to LB sonicates resulted in JNK activation and ROS production by LPMC. NSMase activity of LB sonicates was completely abrogated by GW4869, causing a dose-dependent reduction of LB -induced poptosis. LB and ST selectively induced immune cell apoptosis, an effect dependent on the degree of cell activation and mediated by bacterial NSMase. Conclusions: These results suggest that induction of immune cell apoptosis is a mechanism of action of some probiotics and that NSMase-mediated ceramide generation contributes to the therapeutic effects of probiotics

Anti-inflammatory effects of pancreatitis associated protein in inflammatory bowel disease

Background and aims: Increased pancreatitis associated protein (PAP) mRNA has been reported in active inflammatory bowel disease (IBD). The aims of the current study were to characterise PAP production in IBD and the effects of PAP on inflammation. Patients and methods: Serum PAP levels were determined in healthy controls (n¿=¿29), inflammatory controls (n¿=¿14), and IBD patients (n¿=¿171). Ex vivo PAP secretion in intestinal tissue was measured in 56 IBD patients and 13 healthy controls. Cellular origin of PAP was determined by immunohistochemistry. The effects of exogenous PAP on nuclear factor ¿B (NF¿B) activation, proinflammatory cytokine production, and endothelial adhesion molecule expression were also analysed ex vivo. Results: Patients with active IBD had increased serum PAP levels compared with controls, and these levels correlated with clinical and endoscopic disease severity. Ex vivo intestinal PAP synthesis was increased in active IBD and correlated with endoscopic and histological severity of inflammatory lesions. PAP localised to colonic Paneth cells. Incubation of mucosa from active Crohn¿s disease with PAP dose dependently reduced proinflammatory cytokines secretion. PAP prevented TNF-¿ induced NF¿B activation in monocytic, epithelial, and endothelial cells and reduced proinflammatory cytokine mRNA levels and adhesion molecule expression. Conclusions: PAP is synthesised by Paneth cells and is overexpressed in colonic tissue of active IBD. PAP inhibits NF¿B activation and downregulates cytokine production and adhesion molecule expression in inflamed tissue. It may represent an anti-inflammatory mechanism and new therapeutic strategy in IBD

Heparin attenuates TNF-alpha induced inflammatory response through a CD11b dependent mechanism

Background In addition to its anticoagulant properties, heparin has anti-inflammatory effects, the molecular and mechanistic bases of which are incompletely defined. AIMS The current studies were designed to test the hypothesis that heparin abrogates the expression or function of leucocyte-endothelial adherence molecules which are fundamental to the acute inflammatory response. Methods The effects of heparin on tumour necrosis factor alpha (TNF-¿) induced leucocyte rolling, adhesion, and migration as well as vascular permeability were assessed in rat mesenteric venules using intravital microscopy. Expression of adhesion molecules was quantitated using a double radiolabelled monoclonal antibody (mAb) binding technique in vivo (P-selectin, intercellular cell adhesion molecule type 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1)) or flow cytometry (CD11a, CD11b, and L-selectin). Ex vivo binding of heparin to neutrophils was assessed by flow cytometry. RESULTS TNF-alpha induced a significant increase in leucocyte rolling, adhesion, and migration, and vascular permeability, coincident with a significant increase in expression of P-selectin, ICAM-1, and VCAM-1. Ex vivo assessment of blood neutrophils showed significant upregulation of CD11a and CD11b and significant downregulation of L-selectin within five hours of TNF-¿ administration. Heparin pretreatment significantly attenuated leucocyte rolling, adhesion, and migration but did not affect expression of cell adhesion molecules or vascular permeability elicited by TNF-¿ administration. Binding of heparin was significantly increased on blood neutrophils obtained five hours after TNF-¿ administration. Preincubation with an anti-CD11b mAb but not with an anti-CD11a or anti-L-selectin antibody significantly diminished heparin binding ex vivo

Hepatitis G virus infection in fulminant hepatic failure

Background¿RNA sequences of the recently identified hepatitis GB virus C (HGBV-C), also named hepatitis G virus (HGV), have been detected in patients with idiopathic fulminant hepatic failure (FHF) but the role of this agent in the disease remains controversial. Aims¿To investigate the presence and implications of HGV infection in a large series of Spanish patients with FHF. Patients¿Sixty eight patients with FHF, including 19 with idiopathic disease, were studied. In 28 cases, studies were performed before and after liver transplantation. For comparison 200 volunteer blood donors and 22 patients transplanted for chronic liver disease were also studied. Methods¿HGV RNA was measured in serum by reverse transcriptase polymerase chain reaction of the 5' non-coding region. Results¿Evidence of HGV infection was found in 3% (6/200) of blood donors and in 19% (13/68) of patients with FHF. HGV infection was more frequent in patients with hepatitis B (24%, 6/25) or hepatitis D (42%, 5/12), than in patients with idiopathic disease (11%, 2/19). Half of the patients with HGV infection used illicit intravenous drugs. Specific clinical features associated with HGV infection were not identified. A very high rate of infection with HGV was observed in patients who underwent liver transplantation, either for FHF (60%, 15/24) or chronic liver disease (45%, 9/20). Conclusions¿In our geographical area, HGV infection is relatively frequent in FHF, but it does not seem to play a major role in idiopathic cases

Hepatitis G virus infection in fulminant hepatic failure

Background¿RNA sequences of the recently identified hepatitis GB virus C (HGBV-C), also named hepatitis G virus (HGV), have been detected in patients with idiopathic fulminant hepatic failure (FHF) but the role of this agent in the disease remains controversial. Aims¿To investigate the presence and implications of HGV infection in a large series of Spanish patients with FHF. Patients¿Sixty eight patients with FHF, including 19 with idiopathic disease, were studied. In 28 cases, studies were performed before and after liver transplantation. For comparison 200 volunteer blood donors and 22 patients transplanted for chronic liver disease were also studied. Methods¿HGV RNA was measured in serum by reverse transcriptase polymerase chain reaction of the 5' non-coding region. Results¿Evidence of HGV infection was found in 3% (6/200) of blood donors and in 19% (13/68) of patients with FHF. HGV infection was more frequent in patients with hepatitis B (24%, 6/25) or hepatitis D (42%, 5/12), than in patients with idiopathic disease (11%, 2/19). Half of the patients with HGV infection used illicit intravenous drugs. Specific clinical features associated with HGV infection were not identified. A very high rate of infection with HGV was observed in patients who underwent liver transplantation, either for FHF (60%, 15/24) or chronic liver disease (45%, 9/20). Conclusions¿In our geographical area, HGV infection is relatively frequent in FHF, but it does not seem to play a major role in idiopathic cases

Hepatitis G virus infection in fulminant hepatic failure

Background¿RNA sequences of the recently identified hepatitis GB virus C (HGBV-C), also named hepatitis G virus (HGV), have been detected in patients with idiopathic fulminant hepatic failure (FHF) but the role of this agent in the disease remains controversial. Aims¿To investigate the presence and implications of HGV infection in a large series of Spanish patients with FHF. Patients¿Sixty eight patients with FHF, including 19 with idiopathic disease, were studied. In 28 cases, studies were performed before and after liver transplantation. For comparison 200 volunteer blood donors and 22 patients transplanted for chronic liver disease were also studied. Methods¿HGV RNA was measured in serum by reverse transcriptase polymerase chain reaction of the 5' non-coding region. Results¿Evidence of HGV infection was found in 3% (6/200) of blood donors and in 19% (13/68) of patients with FHF. HGV infection was more frequent in patients with hepatitis B (24%, 6/25) or hepatitis D (42%, 5/12), than in patients with idiopathic disease (11%, 2/19). Half of the patients with HGV infection used illicit intravenous drugs. Specific clinical features associated with HGV infection were not identified. A very high rate of infection with HGV was observed in patients who underwent liver transplantation, either for FHF (60%, 15/24) or chronic liver disease (45%, 9/20). Conclusions¿In our geographical area, HGV infection is relatively frequent in FHF, but it does not seem to play a major role in idiopathic cases