Michael Amer

Bifidobacterial supplementation reduces the incidence of necrotizing enterocolitis in a neonatal rat model

BACKGROUND & AIMS Neonatal necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of premature infants partly caused by intestinal bacterial proliferation. Because bifidobacteria are thought to reduce the risk for intestinal disturbances associated with pathogenic bacterial colonization, we hypothesized that exogenous bifidobacterial supplementation to newborn rats would result in intestinal colonization and a reduction in the incidence of neonatal NEC. METHODS Newborn rat pups were given Bifidobacterium infantis (10(9) organisms per animal daily), Escherichia coli, or saline control and exposed to the NEC protocol consisting of formula feeding (Esbilac; 200 cal. kg(-1). day(-1)) and asphyxia (100% N(2) for 50 seconds followed by cold exposure for 10 minutes). Outcome measures included stool and intestinal microbiological evaluation, gross and histological evidence of NEC, plasma endotoxin concentration, intestinal phospholipase A(2) expression, and estimation of intestinal mucosal permeability. RESULTS Bifidobacterial supplementation resulted in intestinal colonization by 24 hours and appearance in stool samples by 48 hours. Bifidobacteria-supplemented animals had a significant reduction in the incidence of NEC compared with controls and E. coli-treated animals (NEC, 7/24 B. infantis vs. 19/27 control vs. 16/23 E. coli; P < 0.01). Plasma endotoxin and intestinal phospholipase A(2) expression were lower in bifidobacteria-treated pups than in controls, supporting the role of bacterial translocation and activation of the inflammatory cascade in the pathophysiology of NEC. CONCLUSIONS Intestinal bifidobacterial colonization reduces the risk of NEC in newborn rats.

Effect of Polyunsaturated Fatty Acid (PUFA) Supplementation on Intestinal Inflammation and Necrotizing Enterocolitis (NEC) in a Neonatal Rat Model

Inasmuch as long-chain polyunsaturated fatty acids (PUFA, metabolites of the essential n-3 and n-6 fatty acids) are known to modulate inflammation, we hypothesized that supplementation of formula with these compounds would prevent necrotizing enterocolitis (NEC) and intestinal inflammation in our neonatal rat model. Newborn rats were stressed with asphyxia and formula feeding, and randomly assigned to control formula, control with PUFA supplementation, and PUFA with nucleotides. Animals were followed for 72–96 h and assessed for death, gross and histologic NEC, intestinal apoptosis, endotoxemia, and intestinal mRNA synthesis of phospholipase A2-II (rate-limiting enzyme for platelet activating factor production), platelet activating factor receptor, and inducible nitric oxide synthase. We found that PUFA reduced the incidence of death and NEC compared with the other groups (NEC 8 of 24 versus 17 of 24 control and 13 of 23 PUFA + nucleotides, p < 0.05). Furthermore, PUFA reduced plasma endotoxemia at 48 h (25 ± 4 EU/mL versus 276 ± 39 EU/mL in control and 170 ± 28 EU/mL in PUFA + nucleotide), intestinal phospholipase A2-II expression at 24 h, and platelet activating factor receptor expression at 48 h. Formula supplementation had no effect on apoptosis of intestinal epithelium or intestinal inducible nitric oxide synthase expression. Addition of nucleotides with PUFA abrogated the beneficial effects of PUFA on intestinal inflammation. We conclude that PUFA reduces the incidence of NEC and intestinal inflammation in a neonatal rat model.

Are the Duke Criteria Superior to the Beth Israel Criteria for the Diagnosis of Infective Endocarditis in Children

Accurate diagnosis of infective endocarditis may be difficult. The Beth Israel criteria and the newer Duke criteria assign probability to the diagnosis of infective endocarditis on the basis of the presence of common features and manifestations. We reviewed 111 cases of pediatric infective endocarditis diagnosed and treated over 19 years. Each case was classified by the two criteria, and the results were compared. Of 111 cases, 73 (66%) and 18 (16%) were classified as definite by the Duke criteria and the Beth Israel criteria, respectively. No cases were rejected by the Duke criteria, while 21 (19%) of 111 were rejected by the Beth Israel criteria. In 18 pathologically proven cases, reanalysis without pathological data showed that the Duke criteria had significantly greater sensitivity (83%) than the Beth Israel criteria (67%) (P < .03). Echocardiographic evidence was required in 22 cases for definite classification by the Duke criteria; none were rejected, however, when echocardiographic findings were ignored. Our results suggest that the Duke criteria are superior to the Beth Israel criteria for the diagnosis of pediatric infective endocarditis.

Platelet-Activating Factor Concentration in the Stool of Human Newborns: Effects of Enteral Feeding and Neonatal Necrotizing Enterocolitis

Epidemiologic studies have identified enteral feedings as a risk factor for necrotizing enterocolitis (NEC). Enteral feedings provide the substrate for colonization of the newborn gut with gram-negative bacteria with endotoxin production, which may trigger the production of endogenous inflammatory mediators, including platelet-activating factor (PAF). In this prospective study, we examined the effect of enteral feeding on PAF concentration in the stool of preterm and full-term human newborns. The concentration of PAF levels in stool was measured at the following times: at passage of first meconium, within 24 h prior to the onset of feedings, at the 3rd and 14th day of feeding and at any time confirmed NEC developed. Stool samples also were analyzed for levels of acetylhydrolase, the PAF breakdown enzyme. Stool PAF concentration rose significantly following the start of enteral feedings. The mean PAF concentration for day 14 samples was significantly higher than the mean concentration of meconium samples (4.90 ± 1.03 vs. 1.81 ± 0.38 ng/g, p < 0.05) and day 0 samples (4.90 ± 1.03 vs. 1.79 ± 0.39 ng/g, p < 0.05). For the 7 patients diagnosed with definite NEC, the mean stool PAF concentration was 12.42 ± 0.77 ng/g, significantly elevated compared to the mean PAF levels in stool from healthy infants at all sampling times (p < 0.01). There was no significant change in acetylhydrolase activity at any of the sampling times. Stool PAF concentration increases with the provision of enteral feedings and rises further with the development of NEC. Since stool acetylhydrolase activity remained unchanged, we speculate the increase of PAF in stool likely represents increased PAF production at the local level following the provision of enteral feedings or the development of neonatal necrotizing enterocolitis.

The Role of Human Milk in Necrotizing Enterocolitis

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants, and accounts for significant morbidity and mortality worldwide (Kliegman et al., 1984; Uauy et al., 1991). Although the etiology of intestinal inflammation and necrosis that characterizes this disease remains poorly understood, studies have suggested that human milk feedings reduce its’ incidence significantly (Lucas et al., 1990). As compared to neonatal formula, mother’s milk has a myriad of bioactive components that provide immunoprotection and host defense that presumably contribute to the beneficial effects (Goldman, 2000; Goldman et al., 1990; Hanson, 1999). One key component of breast milk may be PAF-acetylhydrolase (PAF-AH), a specific PAF degrading enzyme that is deficient in human newborns, and has been shown in animal models to markedly alter the development of neonatal NEC (Caplan et al., 1990a; Caplan et al., 1997b; Farr et al., 1983). Since data strongly support the role of endogenous PAF in the initiation of intestinal injury (Hsueh et al., 1994; Hsueh et al., 1987), the presence or absence of PAF-AH may be a critical factor in the pathophysiology of the neonatal disease.

Endotoxin Increases Type II-Phospholipase A 2 Gene Expression in Rat Intestinal Epithelial Cells

Endotoxin Increases Type II-Phospholipase A 2 Gene Expression in Rat Intestinal Epithelial Cells

Bifidobacteria Supplementation Prevents NEC in Newborn Rats by Modulation of the Inflammatory Cascade • 566

Bifidobacteria Supplementation Prevents NEC in Newborn Rats by Modulation of the Inflammatory Cascade • 566

Asphyxia and Formula Feeding Increase Platelet Activating Factor Receptor Gene Expression in the Intestine of Newborn Rats • 555

Asphyxia and Formula Feeding Increase Platelet Activating Factor Receptor Gene Expression in the Intestine of Newborn Rats • 555

Are the Duke Criteria Superior to the von Reyn (Beth Israel) Criteria for the Diagnosis of Infective Endocarditis in Children? |[bull]| 769

The diagnosis of pediatric infective endocarditis (IE) is often difficult. Strict diagnostic criteria were developed in 1981 by von Reyn and in 1994 at Duke University and have been evaluated thoroughly in adults. Both criteria consider microbiologic data, physical exam findings, and predisposing cardiac disease. The most striking differences are that the Duke Criteria (DC) allow use of pathologic or clinical features for definite IE and consider echocardiographic findings, whereas the von Reyn Criteria (VRC) require pathologic evidence to diagnose definite IE. Several adult IE series have demonstrated increased sensitivity of the DC compared to VRC. No North American series has evaluated these criteria for diagnosis of pediatric IE. We compared the DC to the VRC in 69 children identified by hospital discharge diagnosis who were treated for IE at two large pediatric centers from 1978 through 1990. All were evaluated echocardiographically. 60/69 patients had predisposing heart disease, most congenital. By VRC, 12/69 (17%) were definite IE, 22/69 (32%) were probable IE, 27/69 (39%) were possible IE, and 8/69 (12%) were rejected. By DC, 47/69 (68%) met criteria for definite IE, 22/69 (32%) were possible and none were rejected. All 22 cases classified as probable by VRC were definite by DC. Of cases rejected by VRC, 4/8 were definite and 4/8 were possible by DC. The 12 definite cases classified by VRC (i.e. with pathologic confirmation) were re-classified using clinical criteria only: 8 became probable, one possible, and 3 were rejected by VRC; 9/12 remained definite and 3 were re-classified as possible by DC. Among 44/47 definite cases by clinical DC (excluding 3 confirmed pathologically), 30/44 are probable, 12/44 are possible, and 2/44 are rejected by VRC when echocardiographic data are ignored. We conclude that application of DC to pediatric patients with suspected IE identifies many more patients with definite IE and rejects fewer patients than VRC and that echocardiographic findings add greatly to the increased sensitivity of DC.

ASPHYXIA AND FORMULA FEEDING INCREASE TYPE-II PHOSPHOLIPASE A2 ENZYME ACTIVITY AND mRNA LEVELS IN THE INTESTINE OF NEWBORN RATS. |[bull]| 797

Phospholipase A2 (PLA2) regulates the production of platelet-activating factor (PAF), a phospholipid inflammatory mediator which may be important in the pathogenesis of necrotizing enterocolitis (NEC). We have developed a newborn rat model of NEC induced by asphyxia (A) and formula feedings (F). In this model, the clinical signs and intestinal lesions are similar to those of human NEC. This experiment measured intestinal PLA2 enzyme activity and PLA2 gene transcription in the rat model of NEC. Intestinal PLA2 enzyme activity from 14 formula-fed and asphyxiated(F,A) and 13 non-asphyxiated, mother-fed control rat pups was assayed by measuring the conversion of a radiolabeled phosphatidylethanolamine substrate to free arachidonic acid. The mean PLA2 enzyme activity for the stressed animals compared to the control pups was 90.6 ± 10.6 vs 58.7± 7.7 pmol/gm protein/hr (p=0.02). Intestinal PLA2-II mRNA was quantified by competitive RT-PCR using a 401bp cRNA of rat PLA2-II(gift of Dr. W. Hsueh). Following the competitive RT-PCR amplification, reaction products were separated by electrophoresis and transcript and competitor cDNA bands quantified by phosphorimaging. Intestinal samples were studied from 5 starved (S), 6 formula-fed (F) and 14 formula-fed and asphyxiated (F,A) newborn rats. Mean PLA2 transcript levels (pg PLA2 transcript per ug total RNA) were similar between the starved and formula-fed groups (0.366 ± 0.259 vs 0.260 ± 0.052) but increased more than 5-fold in the formula-fed rat pups subjected to asphyxia(0.260 ± 0.052 vs 1.399 ± 0.427). These data suggest that in the newborn rat model of NEC these stresses increase PLA2 enzyme synthesis at the level of gene transcription and that the increased PLA2 enzyme activity may represent an increase in PLA2 -II gene activity. The increased PLA2 enzyme activity following exposure to risk factors for human NEC further supports the role of PAF in the pathogenesis of NEC. Supported by NIH grant HD 00999 and a grant from The March of Dimes.