Penelope Bamford

Metabolism of acetyl-L-carnitine for energy and neurotransmitter synthesis in the immature rat brain.

J. Neurochem. (2010) 114, 820–831.

Escherichia coli transcytosis in a Caco-2 cell model: Implications in neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a serious gastrointestinal disorder of preterm infants. Other than an association with prematurity and gastrointestinal feeding, no single factor or mechanism has been consistently linked to this disease. We have previously demonstrated that Escherichia coli isolates obtained from the stool of infants with NEC caused NEC-like injury in a weanling rabbit ileal loop model; this injury, in turn, could be blocked by coinfection with selected Gram(+) bacteria(Enterococcus faecium) isolated from asymptomatic controls. Using Caco-2 cells in a trans-well system, we now demonstrate that the same E. coli isolates can cross epithelial cell monolayers in the absence of ultrastructural change or damage. These results with E. coli contrast with those seen with Salmonella typhimurium, which passed through the monolayer at a higher rate and were associated with striking ultrastructural damage. Transcytosis of E. coli was reduced 3-5-fold in the presence of E. faecium previously shown to block NEC-like injury in the loop model. There was a mild increase in the rate of E. coli transcytosis when studies were conducted with younger, undifferentiated cells; these immature cells had no brush border, had decreased production of brush border-specific enzymes, but retained well defined tight junctions, as demonstrated by transepithelial electrical resistance and electron microscopy. A further reduction/complete blockage of E. coli transcytosis was observed when E. faecium was used as the coinfectant in studies with these undifferentiated cells. We hypothesize that the ability of E. coli to cross epithelial cell layer is a critical initial step in the cascade of events which lead ultimately to NEC; blockage or reduction in E. coli transcytosis in the presence of certain Gram(+) organisms may play a significant role in prevention of NEC.

CLC-2 single nucleotide polymorphisms (SNPs) as potential modifiers of cystic fibrosis disease severity

BackgroundCystic fibrosis (CF) lung disease manifest by impaired chloride secretion leads to eventual respiratory failure. Candidate genes that may modify CF lung disease severity include alternative chloride channels. The objectives of this study are to identify single nucleotide polymorphisms (SNPs) in the airway epithelial chloride channel, CLC-2, and correlate these polymorphisms with CF lung disease.MethodsThe CLC-2 promoter, intron 1 and exon 20 were examined for SNPs in adult CF dF508/dF508 homozygotes with mild and severe lung disease (forced expiratory volume at one second (FEV1) > 70% and < 40%).ResultsPCR amplification of genomic CLC-2 and sequence analysis revealed 1 polymorphism in the hClC -2 promoter, 4 in intron 1, and none in exon 20. Fishers analysis within this data set, did not demonstrate a significant relationship between the severity of lung disease and SNPs in the CLC-2 gene.ConclusionsCLC-2 is not a key modifier gene of CF lung phenotype. Further studies evaluating other phenotypes associated with CF may be useful in the future to assess the ability of CLC-2 to modify CF disease severity.

Role of Bacteria and Immunosuppressive Agent in a Weanling Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Role of Bacteria and Immunosuppressive Agent in a Weanling Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Differential Induction of Cytokine mRNA by Gram Negative and Gram Positive Bacterial Infections in a Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Differential Induction of Cytokine mRNA by Gram Negative and Gram Positive Bacterial Infections in a Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Cytokine mRNA Expression in the Rabbit Ileal Loop Model of Necrotizing Enterocolitis † 582

Mechanisms involved in the pathogenesis of necrotizing enterocolitis (NEC) are not well understood. Prematurity, ischemia, presence of bacteria and substrate in the intestine have all been implicated in this disease process. The role of bacteria in eliciting a proinflammatory cytokine response has been widely reported, and elevated levels of IL-1β, IL-8 and IL-6 have been demonstrated in blood and tissue samples from babies with NEC. In the current study, we examined the expression of cytokine mRNAs in response to defined bacterial insults in the rabbit gut. An E. coli strain was used to produce NEC-like injury in the weanling rabbit ileal loop model using our previously reported protocols (Pediatr Res 36:115-21, 1994). Control and diseased ileal tissue samples were obtained from multiple loops in 4 rabbits 16-24 hours after bacterial infection and quickly frozen in liquid nitrogen for RNA extraction and further analysis. Expression of cytokine mRNAs was examined by Reverse Transcriptase- Polymerase Chain Reaction using rabbit-specific primers and quantified in a fluorimager. Each cytokine was normalized against β-actin. There was a significant increase (p<0.05) in the expression of IL-8 in infected loops. The mean value of IL-8 in E. coli infected loops was 0.34 (SEM 0.05) compared to 0.19 (SEM 0.04) in controls [p < 0.05]. For IL-1β, the mean value was 0.21 (SEM 0.09) compared to 0.12 (SEM 0.05) in controls [p = NS]. In contrast to the increased expression of IL-8 and IL-1β, the infected loops had a value of 0.16 (SEM 0.04) compared to 0.19 (SEM 0.05) in uninfected controls for IL-6 [p= NS]. Although IL-6 has been implicated in inflammatory states, its inhibitory effect on TNF and IL-1β has also been reported (Aderka et al.J Immunol 143:3517-23, 1989; Barton et al. Infect Immun 60:749-53, 1992). Failure to upregulate IL-6 as observed in our system may potentiate proinflammatory cytokines and may contribute to the pathogenesis of NEC.

Expression of Surfactant-Associated Protein A in the Fetal Rat Gastrointestinal Tract |[dagger]| 562

Gastrointestinal epithelium produces surface-active material closely related to that produced by pulmonary alveolar type II epithelial cells. This material includes surfactant-associated protein A (SP-A) which may play a role in gut defense mechanisms. Rubio et al. (J Biol Chem 270:12162, 1995) first demonstrated SP-A in the large and small intestines but not in the stomach of the adult rat. To ascertain the ontogeny and localization of gastrointestinal SP-A we investigated the expression of mRNA in fetal rat gut using RT-PCR with nested primers. Amplification of the cDNA yielded appropriately sized SP-A bands (372 base pairs) in the 17-day fetal rat stomach and intestine; 18-day fetal rat stomach and intestine; 20- and 21-day fetal rat stomach, small intestine and large intestine. These findings were correlated with SP-A expression in the adult rat lung as a positive control and the lack of SP-A expression in the adult rat kidney as a negative control. This is the first documentation of SP-A expression by the fetal rat gastrointestinal tract. Expression of pulmonary SP-A by the fetal rat was reported at gestational day 15.5 by in situ hybridization(Meneghetti, et al. J Histochem Cytochem 44:1173,1996) and at gestational day 13 by RT-PCR (Wang, et al. Am J Respir Cell Mol Biol 10:222, 1994). Our on-going studies are aimed at quantitative analysis of the temporal and spatial profile of SP-A expression in the rat G.I. tract and its possible role in gut defenses.

ROLE OF GLUTAMINE IN BACTERIAL TRANSLOCATION AND EPITHELIAL INJURY IN CACO-2 CELL AND WEANLING RABBIT ILEAL LOOP MODELS. • 737

ROLE OF GLUTAMINE IN BACTERIAL TRANSLOCATION AND EPITHELIAL INJURY IN CACO-2 CELL AND WEANLING RABBIT ILEAL LOOP MODELS. • 737