Intestinal alkaline phosphatase is protective to the preterm rat pup intestine

Background

Necrotizing enterocolitis (NEC) is the most common surgical emergency in neonates, with a mortality rate between 10 and 50%. The onset of necrotizing enterocolitis is highly variable and associated with numerous risk factors. Prior research has shown that enteral supplementation with intestinal alkaline phosphatase (IAP) decreases the severity of NEC. The aim of this study is to investigate whether IAP is protective to the preterm intestine in the presence of formula feeding and in the absence of NEC.

Methods

Preterm rat pups were fed formula with or without supplementation with IAP, and intestine was obtained on day of life 3 for analysis of IAP activity, mRNA expression of TNFα, IL-6 and iNOS and permeability and cytokine expression after LPS exposure.

Results

There was no difference in the absolute and intestine specific alkaline phosphatase activity in both groups. Rat pups fed IAP had decreased mRNA expression of the inflammatory cytokines TNFα, IL-6 and iNOS. Pups supplemented with IAP had decreased permeability and inflammatory cytokine expression after exposure to LPS ex vivo when compared to formula fed controls.

Conclusions

Our results support that IAP is beneficial to preterm intestine and decreases intestinal injury and inflammation caused by LPS.     

Serum levels of interleukin-8 and gut-associated biomarkers in diagnosing necrotizing enterocolitis in preterm infants

Background

In recent years several potential biochemical markers have been evaluated to facilitate a reliable diagnosis of necrotizing enterocolitis (NEC), but none have made progress to clinical routine. We performed a comparative assessment in premature infants to evaluate the diagnostic value of the routinely available cytokine interleukin (IL)-8, and two promising experimental biomarkers, the gut barrier proteins liver fatty acid binding protein (L-FABP) and intestinal fatty acid binding protein (I-FABP), respectively, for the diagnosis of NEC.

Methods

IL-8, L-FABP, and I-FABP concentrations were analyzed in the serum of 15 infants with NEC and compared with 14 gestational age-matched infants serving as a control group.

Results

Serum concentrations of I-FABP, L-FABP and IL-8 were significantly higher in infants with NEC compared with controls. IL-8 showed the highest diagnostic value with an area under the curve of 0.99, followed by L-FABP and I-FABP. In addition we found a significant correlation between IL-8 and both FABPs in infants with NEC.

Conclusion

Our results further advocate the possible role of IL-8 as a specific marker for NEC. The diagnostic value of IL-8 seems to be superior to I-FABP, and similar to L-FABP. The routinely availability facilitates IL-8 as a possible candidate for further clinical investigations.     

Interleukin-8 predicts 60-day mortality in premature infants with necrotizing enterocolitis

Objective

The purpose of this study was to evaluate the predictiveness of circulating interleukin (IL)-8 for 60-day mortality in premature infants with necrotizing enterocolitis (NEC).

Background

NEC affects up to 5% of premature infants and remains a leading cause of mortality among neonates.

Methods

A total of 113 infants with surgically (n = 50) or medically (n = 63) treated NEC were retrospectively analyzed. Laboratory parameters including serum IL-8 were assessed at the diagnosis of NEC and during the preoperative workup.

Results

The 60-day mortality was 19% (22/113), 10% (6/63) in medical and 33% (16/50) in surgical NEC. IL-8 levels significantly correlated with 60-day mortality (odds ratio: 1.38; CI 1.14–1.67; p = 0.001). Median IL-8 levels at diagnosis were significantly higher in neonates who were later treated surgically (median = 2625 pg/ml; range: 27–7500) compared with those treated medically (median = 156 pg/ml; range: 5–7500; p < 0.001). The AUC to discriminate between medical and surgical NEC was 0.82 (CI, 0.74–0.90), and an exploratory IL-8 cutoff point could be established at 1783 pg/ml (sensitivity of 90.5%; specificity of 59.2%).

Conclusions

Our findings that serum IL-8 (i) correlates directly with 60-day mortality and (ii) differs significantly between medically and surgically treated infants may change the process of therapeutic decision making in NEC.     

Glutamine downregulates TLR-2 and TLR-4 expression and protects intestinal tract in preterm neonatal rats with necrotizing enterocolitis

Background/Purpose

Toll-like receptor (TLR)-4 and TLR-2 play an essential role in the pathogenesis of necrotizing enterocolitis (NEC). In this study, we investigated the protective effect of glutamine (Gln) in an NEC neonatal rat model, and the potential association with TLR-4 and TLR-2 expression in local intestinal tissues.

Methods

Preterm neonatal rats were randomly divided into 3 groups: normal control; NEC model; and NEC plus Gln intervention. NEC was induced by feeding with artificial milk substitutes, plus exposure to hypoxia and cold stress. All preterm rats were sacrificed at 3 days after birth. The intestinal tissues were taken for pathological analysis. Protein and mRNA expression of TLR-2, TLR-4, and caspase-3 was examined by immunohistochemistry and real-time RT-PCR, respectively.

Results

Compared with the normal control, the NEC neonatal rats showed mucosal injury and upregulated mRNA and protein expression of TLR-2, TLR-4, and caspase-3 in ileum and colon. Gln intervention significantly reduced the mucosal injury and suppressed the upregulated expression of TLR-2, TLR-4, and caspace-3 in the ileum and colon of NEC neonatal rats.

Conclusions

Gln protects the intestinal tract of NEC neonatal rats, which may be associated with the reduction of TLR-2 and TLR-4 expression in intestines     

Oral relaxin maintains intestinal blood flow in a rat model of NEC

Purpose

Intestinal vasoconstriction is a critical step in development of necrotizing enterocolitis (NEC). Relaxin (RLXN), a hormone found in breast milk but absent from formula, is a potent vasodilator. We hypothesized that relaxin-supplemented feeds with an NEC protocol would decrease NEC severity and increase intestinal blood flow.

Methods

Timed-pregnant Sprague–Dawley rats were randomly assigned to CONTROL, NEC, NEC + 1xRLXN, or NEC + All Feeds RLXN, and all but CONTROL underwent NEC protocol. NEC + 1xRLXN and NEC + All Feeds RLXN groups were fed relaxin-supplemented formula with the last feed or every feed. At 48 h of life, intestinal blood flow was measured at baseline and after application of 2.5% Delflex® solution.

Results

The addition of relaxin to NEC group feeds (1x or All Feeds) improved the degree of ileal injury. Ileal blood flow was decreased in the NEC pups compared to the CONTROLS, but the addition of relaxin to one feed increased baseline ileal blood flow in the NEC group compared to NEC alone. Furthermore, the addition of relaxin to ALL feeds significantly increased baseline ileal blood flow.

Conclusion

Pups who received relaxin with all feeds had substantially increased ileal perfusion compared to control pups. Our data suggest that relaxin supplementation maintains intestinal blood flow and results in less histologic NEC.     

Soybean-derived recombinant human epidermal growth factor protects against experimental necrotizing enterocolitis

Background

Epidermal Growth Factor (EGF) reduces necrotizing enterocolitis (NEC). However, its high cost virtually prohibits clinical use. To reduce cost, soybean expressing human EGF was developed. Here we report effectiveness of soybean-derived EGF in experimental NEC.

The effect of supplemental parenteral nutrition on outcomes of necrotizing enterocolitis in premature,low birth weight neonates

Background

We hypothesized that supplemental parenteral nutrition (PN) decreases the need for surgery and mortality associated with necrotizing enterocolitis (NEC).

Methods

Single institution retrospective review of all premature, low birth weight infants with NEC from January 2006 to December 2013 was conducted.

Results

NEC was identified in 114 premature, low birth weight infants, 59 (51.8%) of which required surgical management. Surgical NEC infants were born younger (25.8 ± 4.0 vs 27.8 ± 3.3 weeks, P = .005) and weighed less at birth (829 ± 281 vs 938 ± 271 g, P = .038) than those managed medically. There was no difference in the use of PN (37.7% vs 31.4%, P = .541) between surgical and medical NEC patients. There was no statistically significant difference in mortality at discharge between patients who had PN at NEC onset and those who did not (31.4% vs 42.6%, P = .294)

Conclusion

In this single-center study, supplemental PN at NEC onset does not appear to significantly improve outcomes as demonstrated by rates of surgical intervention and in-hospital mortality.     

A novel scoring system to predict the development of necrotizing enterocolitis totalis in premature infants

Background/Purpose

Necrotizing enterocolitis totalis (NEC-totalis) is the severest form of NEC, with mortality rate of almost 100% even in the busiest neonatal centers. Despite such a prognosis, its risk factors remain elusive. We seek to identify clinical and laboratory parameters that differentiate NEC-totalis from NEC, and to use these factors to develop a scoring system to identify patients at risk for NEC-totalis upon presentation.

Method

NEC patients were identified from our electronic medical record using ICD9 code. Diagnosis of NEC-totalis was based on operative and autopsy reports. Patients were divided into 2 groups: NEC-but-no-totalis and NEC-totalis. Clinical/laboratory data were obtained for each group. T-test, multivariate logistic regression and backward stepwise regression analysis were performed to identify risk factors for NEC-totalis and these risk factors were formulated into a “Totalis Score.”

Result

Among 157 NEC patients, 13 had NEC-totalis. NEC-totalis patients, compared to NEC alone, had fewer platelets, older age at diagnosis of NEC and greater phosphorus and creatinine levels. A 0-5 point scale “Totalis Score” based on these risk factors had sensitivity of 92% and a specificity of 78% for the diagnosis of NEC-totalis.

Conclusion

Low platelet, high phosphorus, high creatinine and older age at diagnosis of NEC were associated with a greater risk of developing NEC-totalis.     

Ductus venosus closure results in transient portal hypertension—Is this the silent trigger for necrotizing enterocolitis?

Introduction

The etiology of necrotizing enterocolitis (NEC) remains elusive and no definite trigger has been identified. There are no studies to date examining the potential role of closure of the ductus venosus (DV), its effect on increasing portal venous pressure (PVP) and its association to mesenteric venous ischemia in the development of NEC. Our aim was to develop an animal model to examine this physiology.

Methods

Fifteen near-term lambs were used. The DV was occluded in experimental animals by a balloon tip catheter, while the sham controls underwent catheterization without DV occlusion. Vital signs and PVP were monitored for 4 h, followed by intestinal biopsy.

Results

The experimental group (n = 5) demonstrated a significant increase in PVP following DV occlusion (11.87 mm Hg [95% CI: 11.40–12.34]), compared to controls (8.95 mm Hg [95% CI: 8.34–9.56]) (F = 12.16, p = 0.001). Histology of the terminal ileum showed vacuolar degeneration, indicative of reversible cellular damage in the experimental group.

Conclusions

We demonstrate that DV closure in the neonatal lamb leads to transient portal hypertension which is associated with cellular damage and inflammatory changes of the intestinal mucosa. Additional studies will be necessary to determine if the transient portal hypertension following DV closure leads to clinically apparent intestinal ischemia and NEC.     

Overexpression of ELAV-like protein HuR is associated with increased COX-2 expression in atrophy, high-grade prostatic intraepithelial neoplasia, and incidental prostate cancer in cystoprostatectomies

Background

The human ELAV-like protein HuR regulates the stability of several mRNA targets, including that of cyclooygenase-2 (COX-2). Their expression in prostatic carcinogenesis is uncertain.

Objective

To analyze HuR and COX-2 expression in cystoprostatectomies (CyPs) with incidental prostate cancer and compare their expression with those in radical prostatectomies (RPs) with clinically detected cancer.

Design, setting, and participants

HuR and COX-2 were immunohistochemically evaluated in normal-looking epithelium (NEp), atrophy, high-grade prostatic intraepithelial neoplasia (HGPIN), and prostate carcinoma (PCa) in 20 CyPs and 20 RPs, both types of specimens with pT2a Gleason score 6 PCa.

Measurements

At least 1000 cells were counted in contiguous 400X microscopic fields in each case, separately for NEp, atrophy, HGPIN, and PCa.

Results and limitations

There was an increase in the percentage of secretory cells with cytoplasmic HuR staining from NEp to atrophy, HGPIN, and PCa. The mean percentages in NEp, atrophy, and HGPIN adjacent to PCa were greater than away from cancer, both in the CyP and RPs. There was a trend towards a reduced nuclear HuR expression in atrophy, HGPIN, and PCa, compared to NEp. COX-2 staining was seen in the cytoplasm of the basal and secretory cells. There was a reduction in the mean proportion of positive basal cells and progressive increase in the percentage of positive secretory cells from atrophy to HGPIN and PCa, compared to NEp. Cytoplasmic HuR overexpression was correlated with COX-2 expression. There was no difference in HuR and COX-2 expression between cancers with tumour volume <0.5 ccm or >0.5 ccm.The limitations of this study were the small number of cases investigated and lack of a control group without cancer.

Conclusions

The secretory cells showed shift in HuR staining from nuclear in NEp to cytoplasmic in PCa. This is associated with a parallel shift in COX-2 expression from basal to secretory cells.     

Captopril reduces the severity of bowel damage in a neonatal rat model of necrotizing enterocolitis

Background/Purpose

Selective mesenteric ischemia may result from activation of the renin-angiotensin system during periods of shock and is implicated in the pathogenesis of neonatal necrotizing enterocolitis (NEC). We investigated the effectiveness of captopril, an angiotensin-converting enzyme inhibitor, in reducing the severity of bowel damage in a neonatal rat model of NEC.

Methods

Necrotizing enterocolitis was induced by a combination of gavage feeding of hypertonic formula, hypoxia, and oral lipopolysaccharide (LPS). Rats were randomly divided into 3 groups: group A, control (breast fed; n = 20); group B, NEC (gavage/hypoxia/LPS; n = 31); group C, NEC with captopril 20 mg/kg per dose with the formula for 4 days (gavage/hypoxia/LPS/captopril; n = 35). Pups were killed after 4 days. Incidence of NEC was evaluated microscopically.

Results

Severity of bowel damage was higher in the NEC group compared to controls and was reduced by administration of captopril. Dilatation of the intestinal vasculature was observed in the captopril group. There were no cases of NEC in the controls; the incidence increased to 55% in NEC group and reduced to 29% by captopril.

Conclusions

In this model of neonatal NEC, captopril supplementation of formula reduces the severity of intestinal damage and the incidence of NEC, presumably by affecting mesenteric blood flow.     

Predictive utility of cyclo-oxygenase-2 expression by colon and rectal cancer

Background

Cyclo-oxygenase-2 (COX-2), an inducible enzyme expressed in areas of inflammation, is a target of interest for colorectal cancer therapy. Currently, the predictive significance of COX-2 in colorectal cancer remains unclear.

Methods

Tissue microarrays were constructed using 118 colon cancer and 85 rectal cancer specimens; 44 synchronous metastatic colon cancer and 22 rectal cancer lymph nodes were also evaluated. COX-2 expression was assessed by immunohistochemistry. Univariate analysis was used to determine the predictive significance of clinicopathologic variables. Overall survival, disease-specific survival, and disease-free survival were the main outcomes examined.

Results

COX-2 was found to be expressed in 93% of colon cancers and 87% of rectal cancers. Decreased COX-2 expression was related to decreased disease-specific survival (P = .016) and decreased disease-free survival (P = .019) in the rectal cancer cohort but not in the colon cancer cohort.

Conclusions

COX-2 expression has predictive utility for management of rectal but not colon cancer.     

Intraperitoneal 1.5% Delflex improves intestinal blood flow in necrotizing enterocolitis

Background

Necrotizing enterocolitis (NEC) alters intestinal microvascular control mechanisms causing significant vasoconstriction. Our prior work with intraperitoneal 2.5% dextrose solution demonstrated increased intestinal perfusion in experimentally induced NEC. In the current study, we examine whether a buffered solution with lower glucose and osmolar loads similarly increases intestinal blood flow. We hypothesized that buffered 1.5% dextrose solution would increase ileal blood flow compared with baseline in NEC.

Methods

We randomly assigned pregnant Sprague-Dawley rats to control (n = 103) or NEC (n = 123) groups, by litter. We induced NEC by previously published methods. Control pups were vaginally delivered and dam-fed. We used laser Doppler flowmetry to evaluate perfusion in the terminal ileum at 12, 24, 48, 72, or 96 h after delivery at baseline and after application of topical 1.5% dextrose solution. We evaluated differences between groups and time points by analysis of variance and Tukey post hoc test.

Results

Baseline blood flow in the terminal ileum increased with gestational age in both groups (P < 0.05). Control groups had significantly greater baseline blood flow than NEC groups (P < 0.05), and topical application of buffered 1.5% dextrose solution increased blood flow compared with baseline in both groups at all time points (P < 0.05).

Conclusions

Topical 1.5% dextrose solution significantly enhanced blood flow in the terminal ileum to the same degree as 2.5% dextrose solution. Thus, the use of buffered 1.5% dextrose solution might be more beneficial in treating clinical NEC, because it places a lower glucose and osmotic load on NEC-injured intestine.     

Late onset of necrotizing enterocolitis in the full-term infant is associated with increased mortality: Results from a two-center analysis

Purpose

The effect of timing of onset of necrotizing enterocolitis (NEC) on outcomes has not been determined for the full-term infant. In this study we aimed to characterize the full-term NEC population and to evaluate onset of NEC.

Methods

We performed a two-center retrospective review of all full-term infants (≥ 37 weeks) with a diagnosis of NEC between 1990 and 2012. Patients were identified by ICD-9 and age. Early onset for NEC was ≤ 7 days and late onset after 7 days of life. Demographics, comorbidities, maternal factors, clinical factors, surgical intervention, complications, and mortality were evaluated. Wilcoxon’s test was performed on continuous variables and Fisher’s exact test on categorical data. A p-value < 0.05 was considered significant. Univariate outcomes with a p-value < 0.1 were selected for multivariable analysis.

Results

Thirty-nine patients (24 boys, 15 girls) with median EGA of 39 weeks were identified. Overall mortality was 18%. Univariate predictors of mortality included congenital heart disease and placement of an umbilical artery (UA) catheter. Multivariate analysis revealed late onset of NEC to be an independent predictor of mortality (OR 90.8, 95% CI 2.6–3121).

Conclusion

Full-term infants who develop NEC after 7 days of life, have congenital heart disease, and/or need UA catheterization have increased mortality.     

Protective effect of tumor necrosis factor alpha antibody on experimental necrotizing enterocolitis in the rat

Background

Necrotizing enterocolitis (NEC) is a common and devastating disorder of premature infants. Elevated proinflammatory cytokines, especially tumor necrosis factor α (TNF-α), have been implicated in the pathogenesis of NEC. The aim of this study was to evaluate the effects of TNF-α on the inflammatory response in NEC by immunoneutralizing TNF-α with a selective antibody.

Methods

Neonatal Sprague-Dawley rats were divided in 3 groups: group 1 (n = 20), a NEC-like enterocolitis was induced by formula feeding, asphyxia, and cold exposure; group 2 (n = 9), animals were treated like in group 1 and additionally received TNF-α antibody intraperitoneally; and group 3 (n = 17), animals were dam-fed (controls). Animals were killed in case of imminent death or after 96 hours. Specimens from small bowel were processed for blinded histologic (H&E) and immunhistologic (myeloperoxidase [MPO]) analysis.

Results

In group 1, animals developed severe NEC (mean NEC score, 3.28 ± 0.32; mean MPO, 65.85 ± 9.46). In group 2, animals developed mild NEC (mean NEC score, 1.72 ± 0.41; mean MPO, 34.33 ± 9.69; P < .05). In group 3, no NEC was induced (mean NEC score, 0.0 ± 0; mean MPO, 6 ± 1.32; P < .05).

Conclusion

Tumor necrosis factor α antibody may have an attenuating effect on experimental NEC in rats.     

Effects of puerarin on the inflammatory role of burn-related procedural pain mediated by P2X7 receptors

Background

Burn injury can induce an inflammatory response in the blood and wound of patients. Procedural activities in burn patients are particularly problematic in burn care due to their high intensity and frequency; hence, procedural pain evoked by burn dressing changes is a common severe issue. Previous studies demonstrated that purinergic signalling is one of the major pathways involved in the initiation, progression and down-regulation of the inflammatory response. Adenosine 5′-triphosphate (ATP) contributes to inflammation, and increased extracellular ATP levels amplify inflammation in vivo via the P2X₇ receptor. In the present study, the effect of puerarin, an active ingredient extracted from Chinese herbal medicine Ge Gen, on pain relief of burn patients during dressing change and the mechanism related to the regulation of the purinergic signalling pathway were investigated.

Methods

Burn patients were randomly divided into the normal saline group (NS-treated burn patients) and the puerarin-treated group (PUE-treated burn patients), and healthy volunteers were recruited as a control group. The visual Analogue Scale (VAS) scores, heart rate (HR) and respiratory rate (RR) of NS- and PUE-treated burn patients were observed. In addition, interleukin (IL)-1 and IL-4 levels in blood samples, as well as expression of P2X₇ receptor messenger RNA (mRNA) and protein in peripheral blood mononuclear cells (PBMCs) were determined.

Results

The IL-1 levels in the PUE-treated burn patients at post-dressing changes were significantly decreased in comparison with those in NS-treated burn patients; in contrast, the IL-4 levels in PUE-treated burn patients were increased. The expression levels of P2X₇ protein and mRNA in PBMCs of PUE-treated burn patients were significantly decreased in comparison with those in NS-treated burn patients.

Conclusions

The inflammation and associated pain involved in dressing changes of burn patients were relieved by puerarin treatment. The effects were correlated with the decreased expression level of P2X₇ receptor mRNA and protein in PBMCs of burn patients.     

Hyperbaric oxygen therapy reduces the severity of necrotizing enterocolitis in a neonatal rat model

Introduction

Hyperbaric oxygen (HBO) therapy is known to increase oxygen concentration in tissues leading to induction of an adaptive increase in antioxidants, stimulation of angiogenesis, improvement of white blood cell action, and regulation of inflammatory process. Therefore, we tested the potential beneficial effect of HBO in neonatal rat model of necrotizing enterocolitis (NEC).

Materials and Methods

Thirty newborn Sprague-Dawley rats, provided by the Experimental Research Council, Gulhane Military Medical Academy, Ankara,Turkey, were randomly divided into 3 groups as follows: NEC, NEC + HBO, and control. Necrotizing enterocolitis was induced by enteral formula feeding and exposure to hypoxia after cold stress at 4°C and oxygen. The NEC + HBO group received HBO at 2.8 atmosphere absolute (ATA) for 90 minutes daily for 3 days. The pups were killed on the fourth day, and their intestinal tissues were harvested for biochemical and histopathologic analysis. Blood samples were also obtained from the pups.

Results

The mortality rate was highest in the NEC group (3 pups in the NEC group vs 1 pup in the NEC + HBO group). Malondialdehyde and protein carbonyl content were significantly increased, whereas superoxide dismutase and glutathione peroxidase were significantly decreased in the NEC group. All these changes were similar to control levels in the NEC group by HBO treatment. Nitrate plus nitrite (NO_x) levels and serum tumor necrosis factor α were increased in the NEC group and histopathologic injury score and apoptosis index in the NEC group were significantly higher than in the NEC + HBO group.

Conclusion

Hyperbaric oxygen significantly reduced the severity of NEC in our study.     

Mortality associated with laparotomy-confirmed neonatal spontaneous intestinal perforation: A prospective 5-year multicenter analysis

Background

Spontaneous intestinal perforation (SIP) has been recognized as a distinct disease entity. This study sought to quantify mortality associated with laparotomy-confirmed SIP and to compare it to mortality of laparotomy-confirmed necrotizing enterocolitis (NEC).

Methods

Data were prospectively collected on 177,618 very-low-birth-weight (VLBW, 401–1500 g) neonates born between January 2006 and December 2010 admitted to US hospitals participating in the Vermont Oxford Network (VON). SIP was defined at laparotomy as a focal perforation of the intestine without features suggestive of NEC or other intestinal abnormalities. The primary outcome was in-hospital mortality.

Results

At laparotomy, 2036 (1.1%) neonates were diagnosed with SIP and 4076 (2.3%) with NEC. Neonates with laparotomy-confirmed SIP had higher mortality (19%) than infants without NEC or SIP (5%, P = 0.003). However, laparotomy-confirmed SIP patients had significantly lower mortality than those with confirmed NEC (38%, P < 0.0001). Mortality in both NEC and SIP groups decreased with increasing birth weight and mortality was significantly higher for NEC than SIP in each birth weight category. Indomethacin and steroid exposure were more frequent in the SIP cohort than the other two groups (P < 0.001).

Conclusions

In VLBW infants, the presence of laparotomy-confirmed SIP increases mortality significantly. However, laparotomy-confirmed NEC mortality was double that of SIP. This relationship is evident regardless of birth weight. The variant mortality of laparotomy-confirmed SIP versus laparotomy-confirmed NEC highlights the importance of differentiating between these two diseases both for clinical and research purposes.