Tsung-Chun Lee

Anti-apoptotic PI3K/Akt signaling by sodium/glucose transporter 1 reduces epithelial barrier damage and bacterial translocation in intestinal ischemia

Intestinal ischemia/reperfusion (I/R) causes mucosal barrier damage and bacterial translocation (BT), leading to septic complications. Previous in vitro studies showed that activation of sodium/glucose transporter 1 (SGLT1) prevented the epithelial apoptosis and permeability rise induced by microbial products. Our aim was to investigate whether luminal glucose uptake by SGLT1 protects against ischemia-induced epithelial cell death and barrier dysfunction, and to explore the glucose-mediated cellular survival pathways in vivo. Rat jejunum was luminally instilled with either vehicle, a pancaspase inhibitor ZVAD, or glucose prior to I/R challenge (occlusion of the superior mesenteric artery for 20 min and reperfusion for 60 min). Histopathology and apoptosis in the jejunum were examined by TUNEL staining and caspase-3 cleavage. Intestinal permeability was evaluated using in vivo assays measuring luminal-to-blood passage of fluorescein-dextran and portal drainage of enterally administered gadodiamide by magnetic resonance imaging. BT was determined by culturing liver and spleen homogenates. Immunofluorescent analysis and kinase assay were used to study PI3K/Akt signaling pathways. Intestinal I/R caused enterocyte apoptosis and villous destruction. Intestinal infusion with ZVAD decreased the I/R-triggered gut permeability rise and BT, suggesting that the barrier damage was partly dependent on cell apoptosis. Enteral instillation of glucose attenuated the epithelial apoptosis, barrier damage, and mucosal inflammation caused by I/R. Phloridzin (a SGLT1 inhibitor) reduced the protective effect of glucose in a dose-dependent manner. Enteral glucose increased the mucosal Akt kinase activity as evidenced by the augmented phosphorylation of exogenous GSK3. Enhanced membrane translocation and phosphorylation of Akt in epithelial cells were associated with elevated phosphorylation of mTOR, Bad, and FoxO1/3a following glucose uptake. Inhibition of PI3K/Akt signaling by LY294002 and wortmannin partially blocked the glucose-mediated rescue of cell apoptosis and barrier damage. In conclusion, SGLT1 glucose uptake alleviated I/R-induced barrier dysfunction and BT, partly by inhibiting epithelial apoptosis via activation of PI3K/Akt signaling.

Persistent gut barrier damage and commensal bacterial influx following eradication of Giardia infection in mice

BackgroundRecent studies of Giardia lamblia outbreaks have indicated that 40–80% of infected patients experience long-lasting functional gastrointestinal disorders after parasitic clearance. Our aim was to assess changes in the intestinal barrier and spatial distribution of commensal bacteria in the post-clearance phase of Giardia infection.MethodsMice were orogastrically inoculated with G. lamblia trophozoites (strain GS/M) or pair-fed with saline and were sacrificed on post-infective (PI) days 7 (colonization phase) and 35 (post-clearance phase). Gut epithelial barrier function was assessed by Western blotting for occludin cleavage and luminal-to-serosal macromolecular permeability. Gut-associated, superficial adherent, and mucosal endocytosed bacteria were measured by agar culturing and were examined by fluorescence in situ hybridization. Intracellular bacteria cultured from isolated mucosal cells were characterized by 16S rDNA sequencing. Neutrophil-specific esterase staining, a myeloperoxidase activity assay, and enzyme-linked immunosorbent assays for cytokine concentrations were used to verify intestinal tissue inflammation.ResultsTight junctional damage was detected in the intestinal mucosa of Giardia-infected mice on PI days 7 and 35. Although intestinal bacterial overgrowth was evident only during parasite colonization (PI day 7), enhanced mucosal adherence and endocytosis of bacteria were observed on PI days 7 and 35. Multiple bacterial strains, including Bacillus, Lactobacillus, Staphylococcus, and Phenylobacterium, penetrated the gut mucosa in the post-infective phase. The mucosal influx of bacteria coincided with increases in neutrophil infiltration and myeloperoxidase activity on PI days 7 and 35. Elevated intestinal IFNγ, TNFα, and IL-1β levels also were detected on PI day 35.ConclusionsGiardia-infected mice showed persistent tight junctional damage and bacterial penetration, accompanied by mucosal inflammation, after parasite clearance. These novel findings suggest that the host’s unresolved immune reactions toward its own microbiota, due to an impaired epithelial barrier, may partly contribute to the development of post-infective gut disorders.

Neutrophil priming by hypoxic preconditioning protects against epithelial barrier damage and enteric bacterial translocation in intestinal ischemia/reperfusion

Intestinal ischemia/reperfusion (I/R) induces mucosal barrier dysfunction and bacterial translocation (BT). Neutrophil-derived oxidative free radicals have been incriminated in the pathogenesis of ischemic injury in various organs, but their role in the bacteria-containing intestinal tract is debatable. Primed neutrophils are characterized by a faster and higher respiratory burst activity associated with more robust bactericidal effects on exposure to a second stimulus. Hypoxic preconditioning (HPC) attenuates ischemic injury in brain, heart, lung and kidney; no reports were found in the gut. Our aim is to investigate whether neutrophil priming by HPC protects against intestinal I/R-induced barrier damage and bacterial influx. Rats were raised in normoxia (NM) or kept in a hypobaric hypoxic chamber (380 Torr) 17 h/day for 3 weeks for HPC, followed by sham operation or intestinal I/R. Gut permeability was determined by using an ex vivo macromolecular flux assay and an in vivo magnetic resonance imaging-based method. Liver and spleen homogenates were plated for bacterial culturing. Rats raised in HPC showed diminished levels of BT, and partially improved mucosal histopathology and epithelial barrier function compared with the NM groups after intestinal I/R. Augmented cytokine-induced neutrophil chemoattractant (CINC)-1 and -3 levels and myeloperoxidase activity correlated with enhanced infiltration of neutrophils in intestines of HPC-I/R compared with NM-I/R rats. HPC alone caused blood neutrophil priming, as shown by elevated production of superoxide and hydrogen peroxide on stimulation, increased membrane translocation of cytosolic p47phox and p67phox, as well as augmented bacterial-killing and phagocytotic activities. Neutrophil depletion reversed the mucosal protection by HPC, and aggravated intestinal leakiness and BT following I/R. In conclusion, neutrophil priming by HPC protects against I/R-induced BT via direct antimicrobial activity by oxidative respiratory bursts and through promotion of epithelial barrier integrity for luminal confinement of enteric bacteria.

Male gender and renal dysfunction are predictors of adverse outcome in nonpostoperative ischemic colitis patients.

Objective Ischemic colitis (IC) spans a broad spectrum from self-limiting illness to intestinal gangrene and mortality. Prognostic factors specifically for nonpostoperative IC were not fully characterized. We aim to focus on nonpostoperative IC in patients with renal dysfunction and try to identify prognostic factors for adverse outcomes. Methods We conducted a retrospective analysis at a university-affiliated tertiary medical center in Taiwan. From January 2003 to August 2008, 25 men and 52 women (mean age: 66 y) had colonoscopic biopsy-proven IC without prior culprit surgery. We estimated glomerular filtration rate with simplified Modification of Diet in Renal Disease equation. Nine patients with glomerular filtration rate below 30 mL per minute per 1.73 m2 were classified as renal dysfunction group (including 7 dialysis patients). Adverse outcomes were defined as need for surgery and mortality. Predictors for adverse outcomes were captured by univariate and multivariate analysis. Research ethical committee approved the study protocol. Results Patients with renal dysfunction more often had: diabetes mellitus (56% vs. 16%, P=0.02), prolonged symptoms (6.8 d vs. 3.5 d, P=0.01), lower hemoglobin (11.1 g/dL vs. 13.4 g/dL, P=0.01), and more often right colonic involvement (56% vs. 19%, P=0.03). Renal dysfunction patients also had longer hospitalization days (median 15 d vs. 4 d, P=0.045). However, there was no statistical significance in the rate of either surgery or mortality between these 2 groups (P>0.05). Univariate analysis showed that renal dysfunction, sex, emergency department referral, presentation with abdominal pain were significant for adverse outcome (P<0.1). Multivariate analysis revealed that male sex conveyed 9.5-fold risk (P=0.01) and renal dysfunction conveyed 8.5-fold risk (P=0.03) for adverse outcomes. Conclusions Nonpostoperative IC patients with concurrent renal dysfunction had distinct clinical profiles. Multivariate analysis showed that male patients had 9.5-fold and renal dysfunction patients had 8.5-fold increased risk for adverse outcomes. Although IC is often self-limited, our data warrants special attention and aggressive therapy in treating these patients.

Differential diagnosis of Crohn's disease and intestinal tuberculous by enzyme-linked immunospot assay for interferon-gamma.

Differential Diagnosis of Crohns Disease and Intestinal Tuberculous by Enzyme-Linked Immunospot Assay for Interferon-γ

Inhibitory effect of acetylsalicylic acid on platelet function in patients with completed stroke or reversible ischemic neurologic deficit.

The purpose of our study was to investigate the effects of different doses of acetylsalicylic acid on platelet aggregation. Among inpatients of the National Taiwan University Hospital, 236 cases of completed stroke and seven cases of reversible ischemic neurologic deficit that were diagnosed by computed tomography of the brain and that had not ingested acetylsalicylic acid or acetylsalicylic acidlike drugs for greater than 2 weeks before admission were selected for this study. Thromboxane B2 and 6-keto-PGF1 alpha were measured by radioimmunoassay, threshold concentration of adenosine diphosphate was measured by Borns method, and circulating platelet aggregates were measured by the method of Wu and Hoak. Various single doses of acetylsalicylic acid (75, 300, or 600 mg) or 300 mg acetylsalicylic acid every 6 hours for four doses or one dose of 300 mg acetylsalicylic acid with 75 mg dipyridamole significantly suppressed the mean plasma thromboxane B2 concentrations and elevated the mean adenosine diphosphate threshold concentrations. Abnormal plasma thromboxane B2 concentrations, adenosine diphosphate threshold concentrations, or circulating platelet aggregate ratios were significantly normalized after administration of these regimens. The effects were not significantly different among treatment groups. Forty milligrams of acetylsalicylic acid seemed to have less platelet-inhibitory effect. A single dose of 75 mg acetylsalicylic acid significantly inhibited platelet hyperfunction and effectively corrected the abnormal plasma thromboxane B2 concentrations, adenosine diphosphate threshold concentrations, and circulating platelet aggregate ratios. Higher doses did not enhance the inhibitory effect. In addition, this single dose of acetylsalicylic acid did not significantly suppress plasma 6-keto-PGF1 alpha. We conclude that 75 mg acetylsalicylic acid per day is adequate to inhibit platelet hyperfunction.

Combined use of aspirin and heparin inhibits in vivo acute carotid thrombosis.

Background and Purpose Carotid atherosclerotic thrombosis is an important cause of ischemic stroke in Western countries. The therapeutic efficacy of either aspirin or heparin alone in this setting is still controversial. Recently we developed a simple model, the “clamp” method, to induce acute carotid mural thrombosis in vivo in guinea pigs. In this study, we used this model to evaluate the antithrombotic effects of aspirin, heparin, and their combination. Methods Sixty-four male guinea pigs were divided equally into control, aspirin, heparin, and combined groups. Physiological saline, aspirin (5 mg/kg body wt), heparin (200 units/kg body wt), or a combination of aspirin and heparin, respectively, was injected via the jugular vein before the use of the clamp method. Thirty minutes after the injection of saline or drug(s), Péans forceps was used to clamp the carotid artery at a tangent angle for 3 minutes. One hour later, the carotid artery was resected and prepared for observation under a scanning electron microscope or light microscope to evaluate the degree of mural thrombosis. Results The results showed that the combination of aspirin and heparin had an excellent effect in inhibiting in vivo acute carotid thrombosis (p<0.001) and was significantly better than the effect of aspirin alone (p<0.01) or heparin alone (p<0.01). Conclusions Our study clearly demonstrated that the combined use of aspirin and heparin produced a much better antithrombotic effect than either agent alone at sites of carotid endothelial injury when given before the injury. This combined regimen may be useful clinically in acute carotid thrombosis secondary to carotid diseases or carotid endarterectomy.

Eritoran suppresses colon cancer by altering a functional balance in Toll-like receptors that bind lipopolysaccharide

Colorectal carcinogenesis is affected by overexpression of the lipopolysaccharide (LPS) receptors CD14 and TLR4, which antagonize each other by affecting epithelial cell proliferation and apoptosis. Eritoran is an investigational drug for sepsis treatment that resembles the lipid A moiety of LPS and therefore acts as a TLR4 inhibitor. In the present study, we explored the potential therapeutic uses and mechanisms of action of eritoran in reducing colon cancer progression. Eritoran administration via intracolonic, intragastric, or intravenous routes significantly reduced tumor burden in a chemically induced mouse model of colorectal carcinoma. Decreased proliferation and increased apoptosis were observed in mouse tumor cells after eritoran treatment. In vitro cultures of mouse primary tumor spheroids and human cancer cell lines displayed increased cell proliferation and cell-cycle progression following LPS challenge. This effect was inhibited by eritoran and by silencing CD14 or TLR4. In contrast, apoptosis induced by eritoran was eliminated by silencing CD14 or protein kinase Cζ (PKCζ) but not TLR4. Lastly, LPS and eritoran caused hyperphosphorylation of PKCζ in a CD14-dependent and TLR4-independent manner. Blocking PKCζ activation by a Src kinase inhibitor and a PKCζ-pseudosubstrate prevented eritoran-induced apoptosis. In summary, our work offers a preclinical proof of concept for the exploration of eritoran as a clinical treatment, with a mechanistic rationale to reposition this drug to improve the management of colorectal cancer. Cancer Res; 76(16); 4684-95. ©2016 AACR.

Unusual presentation of mesenteric vasculitis as isolated dissection of the superior mesenteric artery

To the editor, Isolated dissection of the main trunk of the superior mesenteric artery (SMA) without aortic dissection is rare. Only 56 cases have been reported [1]. We herein report an unusual case of mesenteric vasculitis presenting as isolated dissection of the SMA, in which the initial presentation was masked by acute calculous cholecystitis. A 57-year-old man visited our emergency department for severe right upper quadrant abdominal pain. Physical examination revealed a palpable and tender gallbladder. Laboratory data showed leukocytosis and an elevated C-reactive protein. Abdominal ultrasonography and computed tomography (CT) scan demonstrated a gallstone impacted at the neck of the distended gallbladder. Emergency laparoscopic cholecystectomy was performed under the impression of acute calculous cholecystitis, which was confirmed by pathological examinations of the resected gallbladder. On the second postoperative day, the patient complained of diffuse abdominal pain and passage of bloody stool. Repeated abdominal CT scans disclosed thickened walls of the small intestines and partial thrombosis of the SMA. Review of preoperative CT scans showed an isolated dissection of the SMA with an intimal flap in the main trunk of the SMA (Fig. 1a, arrow). Angiography of the SMA demonstrated strictures and dilatations of the SMA in segmental and focal distributions (Fig. 1b). Mesenteric vasculitis complicated by isolated dissection and partial thrombosis of the SMA, leading to ischemic bowel disease, was diagnosed. Detailed inquiry of his past medical history revealed several episodes of abdominal pain during preceding 2 years and a history of hypertension with regular medical treatment. Meanwhile, laboratory tests were negative for anti-nuclear antibody, rheumatoid factor, anti-neutrophil cytoplasmic antibody, cryoglobulin and serological markers of viral hepatitis. Workups for involvements of vasculitis in other organ systems were negative. Because there was no sign of intestinal infarction, he was managed nonoperatively with corticosteroid, cyclophosphamide and antiplatelets for the SMA vasculitis associated with thrombosis. He made an uneventful recovery and had been free of symptoms during 2 years’ follow-up. Isolated dissection of the SMA is rare. The pathogenesis of isolated dissection of the SMA remains obscured. No definite correlation with cystic media necrosis, fibromuscular dysplasia, or atherosclerosis is found [2]. In diagnosing isolated dissection of the SMA, contrast enhanced CT scan is the method of choice [3]. It helps identifying the occlusive vascular lesions, which in delay may result in intestinal infarction and mortality. Meticulous observations on the morphology and enhancement patterns of the mesenteric vessels are keys to accurate diagnosis. In early stage of dissection of the SMA, ischemic changes of the intestine may be indistinct. Suzuki et al. [1] suggested blurred perivascular fat planes might be a diagnostic hint. The treatment for isolated dissection of the SMA includes anticoagulation therapy in cases without intestinal infarction. Successful cases of endovascular stenting and of vascular bypass surgery were reported as well [4, 5]. Corticosteroid reT.-C. Lee Department of Internal Medicine, National Taiwan University Hospital Yun-Lin Branch, Yun-Lin, Taiwan

UNUSUAL CAUSE OF EMESIS IN AN OCTOGENARIAN: ORGANOAXIAL GASTRIC VOLVULUS ASSOCIATED WITH PARAESOPHAGEAL DIAPHRAGMATIC HERNIA

a restoration of b cell response to GIP in elderly patients with diabetes mellitus. These studies were conducted in five elderly patients with diabetes mellitus (mean age standard error 72 2; BMI 29 1 kg/m, hemoglobin A1c 6.4 0.1%). Patients were treated with metformin, which was withdrawn 3 days before the first study. At baseline, subjects underwent a 3-hour hyperglycemic glucose clamp (glucose 5.4 mM above basal). For 120 to 180 minutes, GIP was infused in a primed continuous manner at a rate of 2.0 pM/kg per minute. Subsequently, patients were given GLP-1 (433 49 ng/kg per hour) for 6 weeks by continuous subcutaneous infusion into the periumbilical region using a MiniMed infusion pump (Sylmar, CA). After 6 weeks, the hyperglycemic glucose clamp study was repeated. Insulin and glucose values were measured as previously described. During a hyperglycemic clamp, insulin values rise continuously. A formula that allows prediction of insulin values during the clamp was developed. Mean incremental insulin values from 150 to 180 minutes were calculated from the difference of the actual 150to 180minute levels minus the anticipated 150to 180-minute insulin levels, using the formula