Chorioamnionitis-induced fetal gut injury is mediated by direct gut exposure of inflammatory mediators or by lung inflammation.

Intra-amniotic exposure to proinflammatory agonists causes chorioamnionitis and fetal gut inflammation. Fetal gut inflammation is associated with mucosal injury and impaired gut development. We tested whether this detrimental inflammatory response of the fetal gut results from a direct local (gut derived) or an indirect inflammatory response mediated by the chorioamnion/skin or lung, since these organs are also in direct contact with the amniotic fluid. The gastrointestinal tract was isolated from the respiratory tract and the amnion/skin epithelia by fetal surgery in time-mated ewes. Lipopolysaccharide (LPS) or saline (controls) was selectively infused in the gastrointestinal tract, trachea, or amniotic compartment at 2 or 6 days before preterm delivery at 124 days gestation (term 150 days). Gastrointestinal and intratracheal LPS exposure caused distinct inflammatory responses in the fetal gut. Inflammatory responses could be distinguished by the influx of leukocytes (MPO(+), CD3(+), and FoxP3(+) cells), tumor necrosis factor-α, and interferon-γ expression and differential upregulation of mRNA levels for Toll-like receptor 1, 2, 4, and 6. Fetal gut inflammation after direct intestinal LPS exposure resulted in severe loss of the tight junctional protein zonula occludens protein 1 (ZO-1) and increased mitosis of intestinal epithelial cells. Inflammation of the fetal gut after selective LPS instillation in the lungs caused only mild disruption of ZO-1, loss in epithelial cell integrity, and impaired epithelial differentiation. LPS exposure of the amnion/skin epithelia did not result in gut inflammation or morphological, structural, and functional changes. Our results indicate that the detrimental consequences of chorioamnionitis on fetal gut development are the combined result of local gut and lung-mediated inflammatory responses.

Noninvasive measurement of intestinal epithelial damage at time of refeeding can predict clinical outcome after necrotizing enterocolitis.

BACKGROUND: Reintroduction of enteral nutrition in neonates with necrotizing enterocolitis (NEC) should take place when the gut is ready for its normal function. Too early a start of oral feeding might lead to disease relapse, whereas prolonged discontinuation of enteral nutrition is associated with impaired gut function and parenteral nutrition-related complications. This study evaluated whether noninvasive urinary measurement of intestinal fatty acid binding protein (I-FABP) at the time of refeeding can predict clinical outcome in neonates with NEC. METHODS: Urinary I-FABP concentrations were measured in 21 infants with NEC just before reintroducing enteral nutrition. Poor outcome was defined as unsuccessful reintroduction of enteral feeding (EF), (re)operation for NEC, or death related to NEC after reintroduction of EF. RESULTS: Median urinary I-FABP levels in neonates with poor outcome (n = 5) were significantly higher as compared with I-FABP levels in neonates with good outcome (n = 16) (P < 0.01). A clinically significant cutoff value of 963 pg/ml was found to discriminate between infants with poor outcome and those with good outcome (sensitivity 80%, specificity 94%). CONCLUSION: Noninvasive urinary I-FABP measurement at time of refeeding differentiates neonates with poor outcome from neonates with good outcome in NEC. Urinary I-FABP measurement may therefore be helpful in the timing of EF in neonates with NEC.

Preventing enterocyte damage by maintenance of mean arterial pressure during major nonabdominal surgery in children.

Loss of the gut barrier, which is related to hypotension and gastrointestinal hypoperfusion during surgery, has been implicated as a critical event in postoperative complications development. This study aims at preventing gut barrier loss by maintenance of mean arterial pressure (MAP) in patients undergoing major nonabdominal surgery. In 20 previously included children undergoing spinal fusion surgery, the critical MAP value, which should be maintained to prevent enterocyte damage, was determined. In the following 12 children, MAP was kept above the critical value during surgery. Gut mucosal barrier loss was assessed by plasma intestinal fatty acid-binding proteins levels, a marker for enterocyte damage. Gastrointestinal perfusion was measured by gastric tonometry. First, we determined that the MAP should be maintained greater than 60 mmHg to prevent enterocyte damage. Next, maintenance of the MAP above this critical value during surgery resulted in adequate intestinal perfusion and preservation of enterocyte integrity, represented by intestinal fatty acid-binding protein levels within the reference range. This study shows that maintenance of the MAP at greater than 60 mmHg is associated with adequate intestinal perfusion and reduced enterocyte loss in children undergoing major nonabdominal surgery. These data stress the importance and benefits of good circulatory management during major surgery.

Rapid lamina propria retraction and zipper-like constriction of the epithelium preserves the epithelial lining in human small intestine exposed to ischaemia-reperfusion.

To ensure a sufficient barrier between a host and noxious luminal content, the intestinal epithelium must be equipped with efficient mechanisms to limit damage to the epithelial lining. Using a human model, we were able to investigate these mechanisms in the human gut exposed to ischaemia-reperfusion (IR) over the time course of 150 min. In 10 patients a part of jejunum, to be removed for surgical reasons, was selectively exposed to IR. Control tissue was collected, as well as tissue exposed to 30 min of ischaemia with 0, 30 or 120 min of reperfusion. Haematoxylin/eosin staining demonstrated the appearance of subepithelial spaces following 30 min of ischaemia, while the epithelial lining remained intact at this stage. Western blot for myosin light chain kinase (MLCK) revealed a significant increase in protein levels after ischaemia (p < 0.01), and selective staining of MLCK and phosphorylated MLC (pMLC) in lamina propria muscle fibres indicated that appearance of subepithelial spaces was a consequence of active villus contraction. Early during reperfusion, accumulation of pMLC was observed exclusively at the basal side of enterocytes that had lost contact with the collagen-IV-positive basement membrane. These epithelial sheets were pulled together like a zipper, even before these cells were shed. This constriction, verified by increased F-actin and pMLC double staining, accounted for a 45% reduction in virtual wound surface (p < 0.001) at 30 min of reperfusion. In addition, these mechanisms were involved in resealing remaining small epithelial defects, resulting in a fully restored epithelial lining within 120 min of reperfusion. In conclusion, we show in a human in vivo model that the human jejunum has the ability to preserve the epithelial lining during intestinal IR by rapid lamina propria contraction and zipper-like constriction of epithelial cells that are to be shed into the lumen. These newly described phenomena limit exposure to noxious luminal content.

Early diagnosis of intestinal ischemia using urinary and plasma fatty acid binding proteins.

OBJECTIVE: This study aims at improving diagnosis of intestinal ischemia, by measuring plasma and urinary fatty acid binding protein (FABP) levels. METHODS: Fifty consecutive patients suspected of intestinal ischemia were included and blood and urine were sampled at time of suspicion. Plasma and urinary concentrations of intestinal FABP (I-FABP), liver FABP (L-FABP) and ileal bile acid binding protein (I-BABP) were measured using enzyme-linked immunosorbent assays. RESULTS: Twenty-two patients suspected of intestinal ischemia were diagnosed with intestinal ischemia, 24 patients were diagnosed with other diseases, and 4 patients were excluded from further analysis fulfilling exclusion criteria. Median plasma concentrations of I-FABP and L-FABP and urinary concentrations of all 3 markers were significantly higher in patients with proven intestinal ischemia than in patients suspected of intestinal ischemia with other final diagnoses (plasma I-FABP; 653 pg/mL vs. 109 pg/mL, P = 0.02, plasma L-FABP; 117 ng/mL vs. 25 ng/mL, P = 0.006, urine I-FABP; 3377 pg/mL vs. 115 pg/mL, P = 0.001, urine L-FABP; 1,199 ng/mL vs. 37 ng/mL, P =0.004, urine I-BABP; 48.6 ng/mL vs. 0.6 ng/mL, P = 0.002). Positive and negative likelihood ratios significantly increased positive posttest probability and decreased negative posttest probability on intestinal ischemia. In patients with intestinal ischemia a trend to higher plasma I-BABP levels was observed when the ileum was involved (18.4 ng/mL vs. 2.9 ng/mL, P = 0.05). CONCLUSION: Plasma and especially urinary I-FABP and L-FABP levels and urinary I-BABP levels can improve early diagnosis of intestinal ischemia. Furthermore, plasma I-BABP levels can help in localizing ileal ischemia.

A pilot study on potential new plasma markers for diagnosis of acute appendicitis.

BACKGROUND: Diagnosis of acute appendicitis (AA) remains a surgical dilemma, with negative appendectomy rates of 5% to 40% and perforation suggestive for late operative intervention in 5% to 30%. The aim of this study is to evaluate new plasma markers, representing early neutrophil activation, to improve diagnostic accuracy in patients suspected for AA. MATERIALS AND METHODS: Fifty-one patients who underwent surgery for AA were included (male-female = 28:23), and blood was sampled. Plasma concentrations of 2 neutrophil proteins were measured: lactoferrin (LF) and calprotectin (CP). Controls consisted of 27 healthy volunteers. C-reactive protein (CRP) and white blood cell count (WBC) concentrations were measured for routine patient care. RESULTS: Median plasma concentrations for LF and CP were significantly higher in 51 patients with proven AA (665 and 766 ng/mL, respectively) than in 27 healthy volunteers (198 and 239 ng/mL, respectively, P < .001). No clinically relevant correlation exists between the plasma levels of LF and CP and the conventional laboratory tests for CRP and WBC. CONCLUSIONS: Circulating LF and CP levels are significantly elevated in patients with appendicitis and are detectable in plasma using relatively simple and low-cost enzyme-linked immunosorbent assays. Furthermore, plasma levels of LF and CP give additional information to conventional markers WBC and CRP, making them potential new markers for AA diagnosis.

Non-invasive assessment of barrier integrity and function of the human gut.

Over the past decades evidence has been accumulating that intestinal barrier integrity loss plays a key role in the development and perpetuation of a variety of disease states including inflammatory bowel disease and celiac disease, and is a key player in the onset of sepsis and multiple organ failure in situations of intestinal hypoperfusion, including trauma and major surgery. Insight into gut barrier integrity and function loss is important to improve our knowledge on disease etiology and pathophysiology and contributes to early detection and/or secondary prevention of disease. A variety of tests have been developed to assess intestinal epithelial cell damage, intestinal tight junction status and consequences of intestinal barrier integrity loss, i.e. increased intestinal permeability. This review discusses currently available methods for evaluating loss of human intestinal barrier integrity and function.

Non-invasive markers for early diagnosis and determination of the severity of necrotizing enterocolitis.

OBJECTIVES: To improve diagnosis of necrotizing enterocolitis (NEC) by noninvasive markers representing gut wall integrity loss (I-FABP and claudin-3) and gut wall inflammation (calprotectin). Furthermore, the usefulness of I-FABP to predict NEC severity and to screen for NEC was evaluated. METHODS: Urinary I-FABP and claudin-3 concentrations and fecal calprotectin concentrations were measured in 35 consecutive neonates suspected of NEC at the moment of NEC suspicion. To investigate I-FABP as screening tool for NEC, daily urinary levels were determined in 6 neonates who developed NEC out of 226 neonates included before clinical suspicion of NEC. RESULTS: Of 35 neonates suspected of NEC, 14 developed NEC. Median I-FABP, claudin-3, and calprotectin levels were significantly higher in neonates with NEC than in neonates with other diagnoses. Cutoff values for I-FABP (2.20 pg/nmol creatinine), claudin-3 (800.8 INT), and calprotectin (286.2 microg/g feces) showed clinically relevant positive likelihood ratios (LRs) of 9.30, 3.74, 12.29, and negative LRs of 0.08, 0.36, 0.15, respectively. At suspicion of NEC, median urinary I-FABP levels of neonates with intestinal necrosis necessitating surgery or causing death were significantly higher than urinary I-FABP levels in conservatively treated neonates. Of the 226 neonates included before clinical suspicion of NEC, 6 developed NEC. In 4 of these 6 neonates I-FABP levels were not above the cutoff level to diagnose NEC before clinical suspicion. CONCLUSIONS: Urinary I-FABP levels are not suitable as screening tool for NEC before clinical suspicion. However, urinary I-FABP and claudin-3 and fecal calprotectin are promising diagnostic markers for NEC. Furthermore, urinary I-FABP might also be used to predict disease severity.

Protection against early intestinal compromise by lipid-rich enteral nutrition through cholecystokinin receptors.

OBJECTIVE: Early gut wall integrity loss and local intestinal inflammation are associated with the development of inflammatory complications in surgical and trauma patients. Prevention of these intestinal events is a potential target for therapies aimed to control systemic inflammation. Previously, we demonstrated in a rodent shock model that lipid-rich enteral nutrition attenuated systemic inflammation and prevented organ damage through a cholecystokinin receptor-dependent vagal pathway. The influence of lipid-rich nutrition on very early intestinal compromise as seen after shock is investigated. Next, the involvement of cholecystokinin receptors on the nutritional modulation of immediate gut integrity loss and intestinal inflammation is studied. DESIGN: Randomized controlled in vivo study. SETTING: University research unit. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Liquid lipid-rich nutrition or control low-lipid feeding was administered per gavage before hemorrhagic shock. Cholecystokinin receptor antagonists were used to investigate involvement of the vagal antiinflammatory pathway. MEASUREMENTS AND MAIN RESULTS: Gut permeability to horseradish peroxidase increased as soon as 30 mins postshock and was prevented by lipid-rich nutrition compared with low-lipid (p<.01) and fasted controls (p<.001). Furthermore, lipid-rich nutrition reduced plasma levels of enterocyte damage marker ileal lipid binding protein at 60 mins (p<.05). Early gut barrier dysfunction correlated with rat mast cell protease plasma concentrations at 30 mins (rs=0.67; p<.001) and intestinal myeloperoxidase levels at 60 mins (rs=0.58; p<.05). Lipid-rich nutrition significantly reduced plasma rat mast cell protease (p<.01) and myeloperoxidase (p<.05) before systemic inflammation was detectable. Protective effects of lipid-rich nutrition were abrogated by cholecystokinin receptor antagonists (horseradish peroxidase; p<.05 and rat mast cell protease; p<.05). CONCLUSIONS: Lipid-rich enteral nutrition prevents early gut barrier loss, enterocyte damage, and local intestinal inflammation before systemic inflammation develops in a cholecystokinin receptor-dependent manner. This study identifies activation of the vagal antiinflammatory pathway with lipid-rich nutrition as a potential therapy in patients prone to develop a compromised gut.

Urine-based detection of intestinal tight junction loss.

BACKGROUND: Tight junction breakdown, with loss of the important sealing protein claudin-3, is an early event in the development of intestinal damage. Therefore, noninvasive analysis of intestinal tight junction status could be helpful in early detection of intestinal injury. AIM: To investigate the usefulness of urinary claudin-3 as marker for intestinal tight junction loss. METHODS: A rat hemorrhagic shock model and a human setting of known intestinal damage, that is, patients with relapsed inflammatory bowel disease (IBD), were used to investigate intestinal tight junction status by immunohistochemical staining and urinary claudin-3 levels by western blot. RESULTS: In rats claudin-3 urine levels increased rapidly after histologically proven intestinal tight junction loss, with significantly elevated levels at 90 minutes after shock compared with sham-operated animals [mean+/-SEM: 611+/-101 intensity (INT), n=6 vs. 232+/-30 INT, n=6; P<0.05]. Moreover, in colonic biopsies of patients with IBD relapse claudin-3 staining was reduced compared with biopsies of patients with IBD without signs of disease. Concomitantly, significantly increased claudin-3 urine levels were found in these patients (502+/-67 INT, n=10) compared with patients with IBD in remission (219+/-17 INT, n=10, P<0.001) and healthy volunteers (225+/-38 INT, n=10, P<0.001). CONCLUSION: Here we show for the first time in both an experimental and clinical setting a strong relation between intestinal tight junction loss and urinary claudin-3 levels. These findings suggest that measurement of urinary claudin-3 can be used as noninvasive marker for intestinal tight junction loss. This offers new opportunities for early diagnosis and follow-up of intestinal injury.

Endotoxin induced chorioamnionitis prevents intestinal development during gestation in fetal sheep.

Chorioamnionitis is the most significant source of prenatal inflammation and preterm delivery. Prematurity and prenatal inflammation are associated with compromised postnatal developmental outcomes, of the intestinal immune defence, gut barrier function and the vascular system. We developed a sheep model to study how the antenatal development of the gut was affected by gestation and/or by endotoxin induced chorioamnionitis.Chorioamnionitis was induced at different gestational ages (GA). Animals were sacrificed at low GA after 2d or 14d exposure to chorioamnionitis. Long term effects of 30d exposure to chorioamnionitis were studied in near term animals after induction of chorioamnionitis. The cellular distribution of tight junction protein ZO-1 was shown to be underdeveloped at low GA whereas endotoxin induced chorioamnionitis prevented the maturation of tight junctions during later gestation. Endotoxin induced chorioamnionitis did not induce an early (2d) inflammatory response in the gut in preterm animals. However, 14d after endotoxin administration preterm animals had increased numbers of T-lymphocytes, myeloperoxidase-positive cells and gammadelta T-cells which lasted till 30d after induction of chorioamnionitis in then near term animals. At early GA, low intestinal TLR-4 and MD-2 mRNA levels were detected which were further down regulated during endotoxin-induced chorioamnionitis. Predisposition to organ injury by ischemia was assessed by the vascular function of third-generation mesenteric arteries. Endotoxin-exposed animals of low GA had increased contractile response to the thromboxane A2 mimetic U46619 and reduced endothelium-dependent relaxation in responses to acetylcholine. The administration of a nitric oxide (NO) donor completely restored endothelial dysfunction suggesting reduced NO bioavailability which was not due to low expression of endothelial nitric oxide synthase.Our results indicate that the distribution of the tight junctional protein ZO-1, the immune defence and vascular function are immature at low GA and are further compromised by endotoxin-induced chorioamnionitis. This study suggests that both prematurity and inflammation in utero disturb fetal gut development, potentially predisposing to postnatal intestinal pathology.

Intestinal cytoskeleton degradation precedes tight junction loss following hemorrhagic shock.

Hemorrhagic shock (HS) leads to intestinal barrier loss, causing systemic inflammation, which in turn can ultimately lead to multiorgan dysfunction syndrome. Barrier function is based on tight junctions (TJs) between intact epithelial cells. These TJs are anchored in the cell via the filamentous actin (F-actin) cytoskeleton. We hypothesize that HS causes hypoperfusion, leading to loss of F-actin, via activation of actin-depolymerizing factor/cofilin (AC), and consequently TJ loss. This study is aimed at unraveling the changes in cytoskeleton and TJ integrity after HS in organs commonly affected in multiorgan dysfunction syndrome (liver, kidney, and intestine) and to elucidate the events preceding cytoskeleton loss. Adult rats were subjected to a nonlethal HS and sacrificed, along with unshocked controls, at 15, 30, 60, and 90 min after induction of shock. Cytoskeleton, TJ integrity loss, and its consequences were studied by assessment of globular actin, F-actin, AC, zonula occludens protein 1, claudin 3, and bacterial translocation. In the liver and kidney, TJ and the F-actin cytoskeleton remained intact at all time points studied. However, in the intestine, significant loss of F-actin and increase of globular actin was seen from 15 min after shock. This change preceded statistically significant loss of the TJ proteins claudin 3 and zonula occludens protein 1, which were observed starting at 60 min after induction of shock (P < 0.05 vs. controls). Early after induction of shock (15 and 30 min) the nonactive AC (phosphorylated AC) in the intestine was significantly decreased (by 21% and 27%, P < 0.05 vs. control), whereas total AC remained constant, reflecting an increase in activated AC in the intestine from 15 min after shock. Bacterial translocation to mesenteric lymph nodes, liver, and spleen was present from 30 min after shock. This study shows for the first time that HS results in AC activation, selective intestinal actin cytoskeleton disruption, and TJ loss very early after the onset of shock. Loss of this intestinal barrier results in translocation of toxins and bacteria, which enhances inflammation and leads to infections.

New Insight in Loss of Gut Barrier during Major Non-Abdominal Surgery.

BACKGROUND: Gut barrier loss has been implicated as a critical event in the occurrence of postoperative complications. We aimed to study the development of gut barrier loss in patients undergoing major non-abdominal surgery. METHODOLOGY/PRINCIPAL FINDINGS: Twenty consecutive children undergoing spinal fusion surgery were included. This kind of surgery is characterized by long operation time, significant blood loss, prolonged systemic hypotension, without directly leading to compromise of the intestines by intestinal manipulation or use of extracorporeal circulation. Blood was collected preoperatively, every two hours during surgery and 2, 4, 15 and 24 hours postoperatively. Gut mucosal barrier was assessed by plasma markers for enterocyte damage (I-FABP, I-BABP) and urinary presence of tight junction protein claudin-3. Intestinal mucosal perfusion was measured by gastric tonometry (P(r)CO2, P(r-a)CO2-gap). Plasma concentration of I-FABP, I-BABP and urinary expression of claudin-3 increased rapidly and significantly after the onset of surgery in most children. Postoperatively, all markers decreased promptly towards baseline values together with normalisation of MAP. Plasma levels of I-FABP, I-BABP were significantly negatively correlated with MAP at (1/2) hour before blood sampling (-0.726 (p<0.001), -0.483 (P<0.001), respectively). Furthermore, circulating I-FABP correlated with gastric mucosal P(r)CO2, P(r-a)CO2-gap measured at the same time points (0.553 (p = 0.040), 0.585 (p = 0.028), respectively). CONCLUSIONS/SIGNIFICANCE: This study shows the development of gut barrier loss in children undergoing major non-abdominal surgery, which is related to preceding hypotension and mesenterial hypoperfusion. These data shed new light on the potential role of peroperative circulatory perturbation and intestinal barrier loss.

Usefulness and applicability of femorofemoral crossover bypass grafting.

We examined the usefulness of femorofemoral crossover bypass grafting (FFC) and factors influencing its outcome by retrospectively analyzing all FFCs performed in our hospital over a 5-year period, focusing on both patency rates and clinical efficacy. For 95 patients Kaplan-Meier patency rates were calculated (follow-up 40.4 +/- 3.0 months). Clinical outcome was defined according to Rutherford's standardized categories. The influence of cardiovascular risk factors and technical characteristics on outcome was determined. Clinical status of the limb remained improved in 89%. One- and 5-year primary, primary assisted, and secondary patency rates were 88.2% and 57.3%, 90.6% and 62.4%, and 92.6% and 68.1%, respectively. Clinical outcome of the limb was better in patients with <50% stenosis in the femoral arteries preoperatively (p = 0.033). No predictors for patency rates were identified. FFCs are effective in the medium long term for patients in all age categories independently of cardiovascular risk factors. The best predictor of clinical outcome is the preoperative degree of stenosis, with a better outcome for patients affected by <50% stenosis. Success of FFC cannot be reliably measured by graft patency alone but should be assessed by combining patency rates and clinical outcome according to standardized categories.

The PPARgamma agonist rosiglitazone impairs colonic inflammation in mice with experimental colitis.

Various animal models showed that peroxisome proliferator-activated receptor (PPAR)gamma agonists, when given as a gavage shortly preceding colitis induction, protect against inflammatory bowel disease (IBD). We have examined the effects of 16 days rosiglitazone treatment via the diet prior to dextran sodium sulphate (DSS)-induced colitis in mice. After 7 days DSS in the drinking water, rosiglitazone-fed mice had lost significantly more weight than control mice. Rosiglitazone-treated mice had more diarrhea, weight of colon and spleen were increased, and length of colon was decreased. Histology showed that rosiglitazone-treated mice had more severe colitis, mainly caused by more ulceration, crypt loss, and edema. Immunofluorescence showed a loss of tight junction structure Zonula Occludens protein 1 (ZO-1) in colons of rosiglitazone-treated mice as compared to control mice. Also, serum amyloid P component (SAP) concentrations in plasma were increased. However, concentrations of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma in colon homogenates, and TNF-alpha in spleen homogenates were significantly decreased, whereas interleukin (IL)-10 in spleen homogenates was increased. Other cytokines (IL-2, IL-4, IL-6, IL-12p70 and monocyte chemotactic protein (MCP)-1) and myeloperoxidase (MPO) concentrations showed no differences. In conclusion, 16 days pretreatment with rosiglitazone impaired DSS-induced colitis in mice.