Enterocyte Injury Research Articles

Association between endotoxemia and enterocyte injury and clinical course in patients with gram-positive septic shock: A posthoc analysis of a prospective observational study.

Endotoxemia often occurs in patients with gram-positive infections. The possible mechanism is thought to be bacterial translocation after enterocyte hypoperfusion injury. However, the association between endotoxemia and enterocyte injury among patients with gram-positive septic shock has never been assessed. The aim of this study was to evaluate the association between endotoxemia and enterocyte injury in gram-positive septic shock patients and to evaluate the association among endotoxemia, subsequent clinical course, and other related factors.This was a posthoc analysis of a prospective observational study that evaluated the capability of intestinal fatty acid-binding protein (I-FABP), an indicator of enterocyte injury, to predict mortality. Among 57 patients in septic shock, those whose causative microorganisms were gram positive were included. The correlation between endotoxin activity (EA), which indicates endotoxemia, and I-FABP levels upon admission to the intensive care unit (ICU), the clinical course, and other related factors were evaluated.A total of 21 patients were examined. One-third of the patients presented with high EA levels at the time of ICU admission. However, there was no significant correlation between EA and I-FABP levels (Spearman ρ = 0.002, P = .993). Additionally, high EA levels were not associated with abdominal complications after ICU admission or mortality. Similarly, high EA levels were not associated with severity scores, inotropic scores, or lactate levels upon ICU admission, which were previously reported to be factors related to high EA levels.In this posthoc analysis, no correlation was observed between endotoxemia and enterocyte injury among patients in gram-positive septic shock. Additionally, high EA levels were not associated with the clinical course and reported factors related to endotoxemia. Although our results need to be validated in a large prospective cohort study, hypoperfusion enterocyte injury might not be a cause of endotoxemia in these patients. Thus, if there is no correlation between EA and I-FABP levels, other mechanisms that induce high EA levels among patients with gram-positive septic shock should be elucidated.

Association Between Macroscopic Tongue Ischemia and Enterocyte Injury and Poor Outcome in Patients With Septic Shock: A Preliminary Observational Study.

A correlation between sublingual and intestinal mucosa microcirculation, and ischemic necrosis of the tongue as a sign of poor prognosis has been reported. However, an association between tongue ischemia and intestinal health and subsequent outcome has never been studied. This preliminary prospective observational study evaluated the association between macroscopic tongue ischemia and enterocyte injury and poor outcome in patients with septic shock. In this study, 57 adults with septic shock on mechanical ventilators were enrolled. Macroscopic tongue ischemia upon intensive care unit (ICU) admission was assessed by two independent intensivists. We used intestinal fatty-acid binding protein (I-FABP) as a biomarker of enterocyte injury and evaluated the association with tongue ischemia. Demographic variables, risk factor data, and 28-day mortality information were also collected. Compared with patients with normal tongues (n = 45), those with ischemic tongues (n = 12) had a significantly higher Acute Physiology and Chronic Health Evaluation II score (29.0 [25.0-34.0] vs. 36.5 [30.5-44.5], P = 0.017), lactate level (2.8 [2.0-5.0] vs. 9.3 [4.5-10.6], P = 0.002), and I-FABP level (1.9 [0.8-4.0] vs. 54.4 [19.5-159.3], P < 0.001) and the all-cause 28-day mortality was significantly higher (7% vs. 83%, P < 0.001). In conclusion, macroscopic tongue ischemia at ICU admission was associated with enterocyte injury and poor outcome in patients with septic shock. Although there is a disadvantage in that assessment of the tongue was subjective, tongue ischemia could be used to gauge the severity of intestinal injury and to estimate poor outcome in the clinical setting.

Intestinal fatty acid-binding protein as a predictor of prognosis in postoperative cardiac surgery patients.

During the perioperative period of cardiac disease, as many risk factors exist, such as primary cardiac diseases, the use of vasopressors, ischemia-reperfusion injury during cardiopulmonary bypass (CPB), and surgical stress, the gut suffered from ischemia, anoxia and oxidative stress, which would lead to the enterocyte injury. The aim of this study was to explore whether serum intestinal fatty acid-binding protein (IFABP), which is excreted specifically from damaged intestinal enterocytes, as a predictor of prognosis in postoperative cardiac surgery patients.From January 2017 to December 2017, 40 postoperative cardiac surgery patients were enrolled in this observational study. Serum IFABP levels and prognostic biomarkers were recorded at intensive care unit (ICU) admission.The serum IFABP levels were significantly higher in postoperative cardiac surgery patients who complicated with multiple organ dysfunction syndrome (MODS) (median, 883.20 pg/mL vs 426.10 pg/mL; P < .001), infective complications (median, 917.70 pg/mL vs 409.40 pg/mL; P < .001), or who stayed in ICU beyond 4 days (median, 807.65 pg/mL vs 426.10 pg/mL; P < .001). Moreover, in patients who suffered from right ventricular dysfunction, the serum IFABP levels were significantly higher (median, 737.85 pg/mL vs 445.55 pg/mL; P = .016). The serum IFABP levels also showed great precision for the prediction of MODS (the area under curve, AUC 0.923), infective complications (AUC 0.961) and ICU stay beyond 4 days (AUC 0.853). And it correlated significantly with the acute physiology and chronic health evaluation (APACHE) II score (P < .05), sequential organ failure assessment (SOFA) score (P < .05), and acute gastrointestinal injury (AGI) grade (P < .001).The serum IFABP level at ICU admission is a valuable, convenient, and objective early predictor of prognosis in postoperative cardiac surgery patients.

Intestinal fatty acid–binding protein levels in patients with chronic renal failure

BackgroundIntestinal fatty acid–binding protein (I-FABP), a biomarker of enterocyte injury, has been reported to be a diagnostic marker of intestinal ischemia and a prognostic marker in critically ill patients. However, the kinetics of I-FABP in renal failure patients is unknown. We sought to identify I-FABP levels in patients with chronic kidney disease (CKD) and end-stage kidney disease (ESKD) on hemodialysis (HD) and to identify the manner in which the I-FABP levels change. Materials and methodsAdult patients who were admitted for elective cardiac surgery with either normal renal function (NRF), CKD, or ESKD on HD were enrolled. Serum I-FABP levels in NRF and CKD patients and in ESKD patients before and after HD were determined. ResultsA total of 124 patients were evaluated: 47 NRF, 53 CKD, and 24 ESKD. The I-FABP levels of the CKD patients and pre-HD ESKD patients were significantly higher than those of the NRF patients (P = 0.018 and P <0.001, respectively). I-FABP levels were significantly negatively correlated with the estimated glomerular filtration rate in NRF and CKD patients (Spearman's ρ = −0.313, P = 0.002). In addition, I-FABP levels in ESKD patients were significantly lower after HD than those before HD (P <0.001). ConclusionsI-FABP levels in CKD and pre-HD ESKD patients were significantly higher than those in NRF patients. In addition, I-FABP was significantly eliminated by HD in patients with ESKD. Clinicians and researchers should consider this aspect of I-FABP when using it as a diagnostic and prognostic marker in patients with renal insufficiency.

Intestinal fatty acid-binding protein level as a predictor of 28-day mortality and bowel ischemia in patients with septic shock: A preliminary study

PurposeWe sought to evaluate the levels of intestinal fatty acid-binding protein (I-FABP), a biomarker of enterocyte injury, as a predictor of 28-day mortality and bowel ischemia in septic shock patients. Material and methodsIn this preliminary prospective observational study, 57 adult septic shock patients under mechanical ventilation were enrolled. Serum I-FABP levels and prognostic biomarkers were recorded upon intensive care unit (ICU) admission. ResultsThe overall 28-day mortality rate of participants was 23% (13/57). Non-survivors displayed significantly higher lactate (p=0.009), I-FABP (p=0.012), and N-terminal pro-B-type natriuretic peptide (p=0.039) levels compared to survivors. Only I-FABP was associated with 28-day mortality (odds ratio, 1.036; 95% confidence interval, 1.003–1.069; p=0.031) in a multiple logistic regression analysis adjusted for the Acute Physiology and Chronic Health Evaluation II score. When divided into low and high I-FABP groups based on the optimum cut-off value of 19.0ng/mL for predicting 28-day mortality, high-I-FABP patients had a significantly higher incidence of non-occlusive mesenteric ischemia (NOMI) (2% [1/43] vs 29% [4/14]; p=0.011). ConclusionsI-FABP level at ICU admission can serve as a predictor of 28-day mortality in septic shock patients and is associated with the incidence of NOMI.

Urinary Intestine Fatty Acid Binding Protein is Associated with Poor Outcome of Pneumonia Patients in Intensive Care Unit.

Urinary intestine fatty acid binding protein (U-IFABP) is a biomarker for gut injury. Previous studies showed that enterocyte damage in critically ill patients was common and appeared to be associated with poor prognosis. However, the impact of enterocyte damage on the outcome of critically ill patients with pneumonia has not yet been well investigated. The aim of the study is to evaluate the prognostic value of U-IFABP in critically ill patients with pneumonia. A prospective observational study was performed in the intensive care unit (ICU) from September 1, 2013 to April 30, 2014. Pneumonia patients were divided into survival and non-survival groups. U-IFABP was measured using enzyme linked immunosorbent assay for 7 consecutive days after admission to ICU and expressed as U-IFABP/urine creatinine ratio. The prognostic value was tested by Receiver Operator Characteristic (ROC) curves and Kaplan-Meier curves. A total of 32 pneumonia patients with endotracheal intubation were enrolled. U-IFABP/Cr levels were significantly higher in non-survivors than in survivors at day 1 (p = 0.033), day 4 (p = 0.018), day 5 (p = 0.008), day 6 (p = 0.006) and day 7 (p = 0.008) after ICU admission. The areas under ROC curve in predicting mortality were 0.755 (D1), 0.781 (D4), 0.812 (D5), 0.823 (D6), and 0.812 (D7). Moreover, pneumonia patients with day 7 U-IFABP/Cr above the cutoff of 28.9 pg/100 µL had a significantly lower survival rate (p = 0.043). Enterocyte injury was common in critically ill patients with pneumonia. The severity of enterocyte injury, as evidenced by the U-IFABP/Cr, was associated with the patient's mortality. U-IFABP/Cr may serve as a significant prognostic factor for patients with pneumonia admitted to ICU. Further studies with larger populations are needed to verify these issues.

Soluble Dextrin Fibers Alter the Intestinal Microbiota and Reduce Proinflammatory Cytokine Secretion in Male IL-10–Deficient Mice1–3

Background: Prebiotic fibers stimulate the growth and activity of the gut microbiota. Interleukin 10–deficient (IL-10−/−) mice develop a colitis that is influenced by the gut microbial composition.Objective: The purpose of this study was to determine the effect of prebiotic fibers on the intestinal microbiota and immune function in IL-10−/− mice.Methods: At 4 wk of age, male IL-10−/− mice (n = 8/group) were randomly assigned to 5 diets: unpurified diet with cellulose (4%; control), corn-derived hydroxypropylated new resistant starch (NRS) (2% NRS + 2% cellulose), soluble fiber dextrin from tapioca (SFD-t) (4%), soluble fiber dextrin from corn (SFD-c) (4%), or soluble corn fiber (4%) for 12 wk. Growth, small intestinal permeability, histologic injury, intestinal cytokine secretion, and microbiota composition by 16S ribosomal RNA pyrosequencing of stool were measured. ANOVA and principal component analysis were applied to assess the fibers' effects.Results: There were no significant differences in mouse growth, intestinal weight, length, or gut permeability over the 12 wk feeding period. Mice fed dextrin-based diets secreted 47–88% less colonic IL-1β, tumor necrosis factor α, and IL-23 (SFD-t diet) and IL-12 heterodimer p70, IL-6, and chemokine ligand 1 (CXCL1) (SFD-c diet) (P < 0.05) than did the control group, whereas NRS-fed mice secreted 55–77% less IL-6 and CXCL1 (P < 0.05). Both SFD-t– and SFD-c–fed mice had a 70–75% lower abundance of Lactobacillaceae than control mice. The SFD-t diet group had a lower enterocyte injury score (P < 0.04) than did control mice, and this was associated with increased abundance of butyrate producers, including Incertae sedis XIV, Lachnospiraceae, and Ruminococcaceae (P < 0.001).Conclusions: These results demonstrate that soluble prebiotic fibers selectively stimulate the growth of a distinctive gut microbiota in IL-10−/− mice. SFD-t induced the growth of butyrate-producing microbes and was effective in reducing proinflammatory cytokine secretion and enterocyte injury in this mouse model of colitis.

Prognostic Value of Enterocyte Injury in Critically Ill Patients

Prognostic Value of Enterocyte Injury in Critically Ill Patients

Candida Species Differ in Their Interactions With Immature Human Gastrointestinal Epithelial Cells

Life-threatening gastrointestinal (GI) diseases of prematurity are highly associated with systemic candidiasis. This implicates the premature GI tract as an important site for invasion by Candida. Invasive interactions of Candida spp. with immature enterocytes have heretofore not been analyzed. Using a primary immature human enterocyte line, we compared the ability of multiple isolates of different Candida spp. to penetrate, injure, and induce a cytokine response from host cells. Of all the Candida spp. analyzed, C. albicans had the greatest ability to penetrate and injure immature enterocytes and to elicit IL-8 release (p < 0.01). In addition, C. albicans was the only Candida spp. to form filamentous hyphae when in contact with immature enterocytes. Similarly, a C. albicans mutant with defective hyphal morphogenesis and invasiveness had attenuated cytotoxicity for immature enterocytes (p < 0.003). Thus, hyphal morphogenesis correlates with immature enterocyte penetration, injury, and inflammatory responses. Furthermore, variability in enterocyte injury was observed among hyphal-producing C. albicans strains, suggesting that individual organism genotypes also influence host-pathogen interactions. Overall, the finding that Candida spp. differed in their interactions with immature enterocytes implicates that individual spp. may use different pathogenesis mechanisms.

Pharmacologic Targeting or Genetic Deletion of Mitochondrial Cyclophilin D Protects from NSAID-Induced Small Intestinal Ulceration in Mice

Small intestinal ulceration is a frequent and potentially serious condition associated with nonselective cyclooxygenase 1/2 inhibitors (nonsteroidal anti-inflammatory drugs, NSAIDs) including diclofenac (DCF). An initial topical effect involving mitochondria has been implicated in the pathogenesis, but the exact mechanisms of NSAID-induced enteropathy are unknown. We aimed at investigating whether DCF caused enterocyte demise via the mitochondrial permeability transition (mPT) and whether inhibition of critical mPT regulators might protect the mucosa from DCF injury. Cultured enterocytes (IEC-6) exposed to DCF readily underwent mPT-mediated cell death. We then targeted mitochondrial cyclophilin D (CypD), a key regulator of the mPT, in a mouse model of NSAID enteropathy. C57BL/6J mice were treated with an ulcerogenic dose of DCF (60 mg/kg, ip), followed (+ 1 h) by a non-cholestatic dose (10 mg/kg, ip) of the CypD inhibitor, cyclosporin A (CsA). CsA greatly reduced the extent of small intestinal ulceration. To avoid potential calcineurin-mediated effects, we used the non-immunosuppressive cyclosporin analog, D-MeAla(3)-EtVal(4)-cyclosporin (Debio 025). Debio 025 similarly protected the mucosa from DCF injury. To exclude drug-drug interactions, we exposed mice genetically deficient in mitochondrial CypD (peptidyl-prolyl cis-trans isomerase F [Ppif(-/-)]) to DCF. Ppif-null mice were largely protected from the ulcerogenic effects of DCF, whereas their wild-type littermates developed typical enteropathy. Enterocyte injury was preceded by upregulation of the proapoptotic transcription factor C/EBP homologous protein (Chop). Chop-null mice were refractory to DCF enteropathy, suggesting a critical role of endoplasmic reticulum stress induced by DCF. In conclusion, mitochondrial CypD plays a key role in NSAID-induced enteropathy, lending itself as a potentially new therapeutic target for cytoprotective intervention.

Protection from diclofenac-induced small intestinal injury by the JNK inhibitor SP600125 in a mouse model of NSAID-associated enteropathy

Small intestinal ulceration, bleeding, and inflammation are major adverse effects associated with the use of diclofenac (DCF) or other nonsteroidal anti-inflammatory drugs (NSAIDs). The underlying mechanisms of DCF enteropathy are poorly understood, but there is increasing evidence that topical effects are involved. The aim of this study was to explore the role of c-Jun-N-terminal kinase (JNK) in DCF-induced enterocyte death because JNK not only regulates mitochondria-mediated apoptosis but also is a key node where many of the proximal stress signals converge. Male C57BL/6J mice were injected intraperitoneally with DCF or vehicle (Solutol HS-15), and the extent of small intestinal ulceration was determined. A single dose of DCF (60 mg/kg) produced numerous ulcers in the third and fourth quartiles of the jejunum and ileum, with maximal effects after 18 h and extensive recovery after 48 h. To study the molecular pathways leading to enterocyte injury, we isolated villi-enriched mucosal fractions from DCF-treated mice. Immunoblot studies with a phosphospecific JNK antibody revealed that JNK1/2 (p46) was activated at 6 h, leading to phosphorylation of the downstream target c-Jun. The levels of the JNK-regulated proapoptotic transcription factor C/EBP homologous protein (CHOP) were also increased after DCF. The selective JNK inhibitor SP600125 (30 mg/kg ip), given both 1 h before and 1 h after DCF, blocked JNK kinase activity and afforded significant protection against DCF enteropathy. In conclusion, these data demonstrate that the JNK pathway is critically involved in the pathogenesis of DCF-induced enteropathy and suggest a potential application of JNK inhibitors in the prevention of NSAID-induced enteropathy.

Effects and mechanism of aspirin on enterocyte injury

Effects and mechanism of aspirin on enterocyte injury

A Critical Role for TLR4 in the Pathogenesis of Necrotizing Enterocolitis by Modulating Intestinal Injury and Repair

Necrotizing enterocolitis (NEC) is the leading cause of death from gastrointestinal disease in preterm infants and is characterized by translocation of LPS across the inflamed intestine. We hypothesized that the LPS receptor (TLR4) plays a critical role in NEC development, and we sought to determine the mechanisms involved. We now demonstrate that NEC in mice and humans is associated with increased expression of TLR4 in the intestinal mucosa and that physiological stressors associated with NEC development, namely, exposure to LPS and hypoxia, sensitize the murine intestinal epithelium to LPS through up-regulation of TLR4. In support of a critical role for TLR4 in NEC development, TLR4-mutant C3H/HeJ mice were protected from the development of NEC compared with wild-type C3H/HeOUJ littermates. TLR4 activation in vitro led to increased enterocyte apoptosis and reduced enterocyte migration and proliferation, suggesting a role for TLR4 in intestinal repair. In support of this possibility, increased NEC severity in C3H/HeOUJ mice resulted from increased enterocyte apoptosis and reduced enterocyte restitution and proliferation after mucosal injury compared with mutant mice. TLR4 signaling also led to increased serine phosphorylation of intestinal focal adhesion kinase (FAK). Remarkably, TLR4 coimmunoprecipitated with FAK, and small interfering RNA-mediated FAK inhibition restored enterocyte migration after TLR4 activation, demonstrating that the FAK-TLR4 association regulates intestinal healing. These findings demonstrate a critical role for TLR4 in the development of NEC through effects on enterocyte injury and repair, identify a novel TLR4-FAK association in regulating enterocyte migration, and suggest TLR4/FAK as a therapeutic target in this disease.

Na+,K+-ATPase activity is inhibited in cultured intestinal epithelial cells by endotoxin or nitric oxide

Na+K+-ATPase is an important enzyme serving vital functions in various mammalian tissues, including the intestine. We have previously documented that endotoxin (LPS) and nitric oxide (NO) can induce enterocyte injury in vitro. To examine whether alterations Na+,K+-ATPase activity might be involved in LPS- or NO-induced enterocyte dysfunction, we carried out four series of experiments. The first set of experiments documented that LPS decreases IEC-6 Na+,K+-ATPase activity at concentrations as low as 0.10 microg/ml. The second set of experiments tested whether exposure of IEC-6 cells to the exogenous NO donor, S-Nitroso-N-acetylpenicillamine (SNAP), would decrease IEC-6 Na+,K+-ATPase activity. The results of these experiments documented that SNAP significantly decreased IEC-6 Na+,K+-ATPase activity in a dose-dependent fashion at a threshold inhibitory concentration of 0.1 mM, and there was an inverse correlation between Na+,K+-ATPase activity and NO concentrations in the medium. Since enterocytes contain iNOS, and LPS can increase iNOS activity, the third set of experiments examined the relationship between LPS-induced inhibition of Na+),K+-ATPase activity and NO production by the IEC-6 cells. These results showed that LPS increased IEC-6 NO production in both a dose- and time-dependent fashion and an inverse correlation existed between LPS-induced NO production and decreased Na+,K+-ATPase activity. Addition of the NOS inhibitor, L-NNA, prevented the LPS-induced decrease in Na+,K+ATPase activity, suggesting that NO is involved in the decrease of Na+,K+-ATPase activity observed in the IEC-6 cells incubated with LPS. One mechanism by which the increased NO concentrations could have contributed to the decrease in Na+,K+ATPase activity, after the addition of LPS or SNAP, is via the production of peroxynitrite during the reaction of NO with superoxide. This notion was supported by studies showing that SNAP- and LPS-induced decreases in IEC-6 Na+,K+-ATPase activity could be blocked by adding superoxide dismutase to the medium. The last set of experiments tested whether the inhibition of Na+,K+-ATPase activity with the specific Na+,K+-ATPase inhibitor ouabain would increase the permeability of an IEC-6 monolayer. IEC-6 monolayer permeability was increased by ouabain, but only at a high concentration. In conclusion, these studies indicate that LPS or the NO donor, SNAP, inhibit Na+,K+-ATPase activity and this inhibition is at least partly related to peroxynitrite production. These studies also suggest that LPS-induced NO production by the IEC-6 cells decreases IEC-6 Na+,K+-ATPase activity in an autocrine fashion.

Evaluation of diarrhea in the HIV-infected patient

The evaluation of HIV-infected patients with on clinical-pathological correlations. Distinguishing features of diarrhea in HIV infection are the frequent coexistence of multiple pathogens, heterogeneity in clinical syndromes and organisms, disease chronicity, and frequent need for mucosal biopsy for diagnosis. Pathogenic mechanisms for diarrhea include primary enterocyte injury, altered secretion of inflammatory mediators or secretory enterotoxins, ileal dysfunction, exudative enteropathy, and altered motility. Diarrhea also may result from drug toxicity. Clinical evaluation is mandated for patients whose symptoms are persitent, increasing in severity or associated with fever and/or weight loss, and the yield varies, based on CD4+ lymphocyte counts. The 3 steps in the evaluating diarrhea in HIV infection are: 1) to distinguish between acute and chronic diarrhea, 2) to localize the site of disease, and 3) to make an etiologic diagnosis. Techniques for successful endoscopic evaluation include the use of pediatric colonoscope to perform upper gastrointestinal endoscopy, examining the topography of the small intestinal mucosa, obtaining multiple biopsies, and saving tissue for electron microscopy.

Rotavirus Infection Induces Cytoskeleton Disorganization in Human Intestinal Epithelial Cells: Implication of an Increase in Intracellular Calcium Concentration

Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca(2+)-dependent microtubule disassembly and Ca(2+)-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.

The effect of hypoxia/reoxygenation on the cellular function of intestinal epithelial cells.

Previously, using in vivo models, we have demonstrated that ischemia/reperfusion can increase intestinal mucosal permeability, promote bacterial translocation, and induce gut cytokine production. Because of the cellular heterogeneity of the gut, however, studies investigating the direct effects of hypoxia/reoxygenation on intestinal epithelial cells are confounded in in vivo model systems. Consequently, this study examines oxidant-mediated enterocyte injury using an in vitro intestinal enterocyte hypoxia/reoxygenation model system. Two intestinal epithelial cell lines, IEC-6 and Caco-2, were seeded onto 3-microm filters in a Transwell bicameral system and grown until tight junction integrity was established. Cells were subjected to hypoxia in a sealed chamber with 95% nitrogen and 5% carbon dioxide and incubated at 37 degrees C for 60 or 90 minutes. Reoxygenation was initiated by replacing the media and putting the cells in an environment of room air plus 5% carbon dioxide. Permeability and bacterial translocation were assayed by measuring the phenol red concentration and culturing the bacteria that crossed the cell monolayer and reached the basal chamber of the bicameral system. Monolayer tight junction integrity was monitored by serial measurements of transepithelial electrical resistance (TEER), and cell viability was assessed by trypan blue dye. IEC-6 cell monolayers subjected to 60 or 90 minutes of hypoxia showed significantly higher permeability to phenol red, with 54+/-5% and 57+/-5% of the dye crossing the monolayers, respectively, compared with normoxic control (38+/-2%; p < 0.01). Caco-2 cell monolayers also had increased permeability to phenol red, with 24+/-6% and 20+/-4% of the phenol red crossing the monolayer after 60 or 90 minutes of hypoxia, respectively, compared with 8+/-3% in the normoxic controls (p < 0.01). At 3 hours after challenge with Escherichia coli, the monolayers subjected to 60 or 90 minutes of hypoxia had significantly increased bacterial translocation (IEC-6 cells, p < 0.05; Caco-2 cells, p < 0.01) compared with controls. The increased permeability of the hypoxic Caco-2 and IEC-6 monolayers was associated with a decrease in TEER beginning as early as 1 hour after reoxygenation (p < 0.01). Cell viability, however, was not decreased. These results indicate that hypoxia/reoxygenation can directly impair cellular function as manifested by increased monolayer permeability to phenol red, increased E. coli bacterial translocation, and a decrease in TEER values.

Polarized Caco-2 cells

Reactive oxygen metabolites are implicated in gastrointestinal disease and enterocyte injury associated with ischemia-reperfusion, bacterial translocation, inflammatory bowel disease, and necrotizing enterocolitis. The ileal-like, human colon carcinoma cell line, Caco-2, was used to investigate oxidative damage. After challenging Caco-2 cells with reactive oxygen metabolites, the permeability, viability, and energy charge of Caco-2 cells were assessed. Permeability was determined by transepithelial electrical potential and flux of small molecules. Viability was determined by release of 51Cr. Cell energy was evaluated by determining adenylate energy charge. The source of reactive oxygen metabolites, with the exception of menadione, did not affect viability of Caco-2 cells; cell permeability was increased. The increased varied with the source and location of the reactive oxygen metabolite. There was no change in energy charge. This study suggests that reactive oxygen metabolites could cause enterocyte damage and that the source of the reactive oxygen metabolite is an important variable in determining the extent of damage. Antioxidants might prevent injury.

Effect of the cyanobacterial (blue-green algal) toxins from Microcystis aeruginosa on isolated enterocytes from the chicken small intestine

Livestock deaths, and clinical reports of human injury, follow the consumption of toxic blue-green algae. The experiments described show that isolated intestinal enterocytes from chicks are both deformed and killed by exposure to Microcystis toxins, in a dose-dependent and time-dependent manner. The enterocytes were protected from toxicity by deoxycholate, bromosulphothalein and rifampicin. It was concluded that the gastroenteritis clinically associated with accidental Microcystis ingestion is likely to reflect enterocyte injury by Microcystis toxins, and that the therapeutic use of bile acids or transport inhibitors may be of value in treatment.

Rat enterocyte injury by oxygen-dependent processes

Neutrophils migrate to areas of inflammation and, when stimulated, produce O2−, H2O2, and other reactive O2 metabolites. To assess the effects of stimulated neutrophils on enterocytes, rat enterocytes were incubated with peripheral neutrophils. To assess cell viability, trypan blue exclusion and lactate dehydrogenase and protein release were measured. When 106 enterocytes/mL were incubated with 2.5 × 105 neutrophils/mL stimulated with phorbol myristate acetate, trypan blue exclusion decreased and lactate dehydrogenase and protein release increased. With the addition of 0.10 mg/mL of Superoxide dismutase, trypan blue exclusion further decreased and lactate dehydrogenase and protein release increased. This suggests that H2O2- or H2O2/ O2 -derived metabolites are more damaging to isolated enterocytes than O2−1. To test this hypothesis, enterocytes were incubated with xanthine and increasing concentrations of xanthine oxidase in the presence and absence of Superoxide dismutase. With increasing concentrations of xanthine oxidase, the cell number decreased and protein release increased. With the addition of Superoxide dismutase, fewer cells were present, suggesting that cell lysis occurred. Protein release was further increased by the addition of Superoxide dismutase. Enterocytes were then incubated with leucine and increasing concentrations of amino acid oxidase. With increasing concentrations of amino acid oxidase, trypan blue exclusion decreased and protein and lactate dehydrogenase release increased. These effects were ameliorated by the addition of 500 IU catalase/mL. These data suggest that O2− and H2O2, whether created by stimulated neutrophils or an enzymegenerating system, are damaging to isolated enterocytes. Superoxide dismutase did not offer enterocytes protection.