Patrick N. Nation

Immunoprophylactic Potential of Cloned Shiga Toxin 2 B Subunit

The Shiga toxins Stx1 and Stx2 contribute to the development of enterohemorrhagic O157:H7 Escherichia coli-mediated colitis and hemolytic-uremic syndrome in humans. The Stx2 B subunit, which binds to globotriaosylceramide (GB3) receptors on target cells, was cloned. This involved replacing the Stx2 B subunit leader peptide nucleotide sequences with those from the Stx1 B subunit. The construct was expressed in the TOPP3 E. coli strain. The Stx2 B subunits from this strain assembled into a pentamer and bound to a GB3 receptor analogue. The cloned Stx2 B subunit was not cytotoxic to Vero cells or apoptogenic in Burkitts lymphoma cells. Although their immune response to the Stx2 B subunit was variable, rabbits that developed Stx2 B subunit-specific antibodies, as determined by immunoblot and in vitro cytotoxicity neutralization assays, survived a challenge with Stx2 holotoxin. This is thought to be the first demonstration of the immunoprophylactic potential of the Stx2 B subunit.

Administration of Tyrosyl Radical–Oxidized HDL Inhibits the Development of Atherosclerosis in Apolipoprotein E–Deficient Mice

Objective—Tyrosyl radical–oxidized HDL (tyrHDL) increases the ability of cells to donate cholesterol to apolipoprotein (apo) A-I for HDL particle formation. We tested whether treatment with tyrHDL raises endogenous HDL cholesterol levels and decreases atherosclerosis development in apoE-deficient mice. Methods and Results—Tyrosyl radical oxidation of mouse HDL induced formation of apoAI-AII heterodimers and enhanced the ability of mouse HDL to deplete cultured fibroblasts of their regulatory pool of cholesterol. 125I-labeled HDL and tyrHDL delivered intraperitoneally were cleared at similar rates from plasma of chow-fed apoE-deficient mice. ApoE-deficient mice injected intraperitoneally twice weekly with 150 &mgr;g tyrHDL from age 10 to 18 weeks showed a maximum 2.3-fold increase in endogenous HDL cholesterol levels, which fell toward the end of the treatment period. tyrHDL treatment resulted in 37% less aortic lesion development than in control HDL-treated mice (P <0.001) and 67% less than in saline-injected animals (P <0.001). Conclusions—Administration of tyrHDL for 8 weeks resulted in significantly less atherosclerosis development in apoE-deficient mice than injection of HDL or saline. Molecules increasing mobilization of cellular cholesterol to apoAI for HDL particle formation would be expected to decrease atherosclerosis without necessarily causing sustained increases in circulating HDL cholesterol levels.

Novel Neonatal Piglet Models of Surgical Short Bowel Syndrome With Intestinal Failure

Objectives: Short bowel syndrome occurring after surgery for acquired or congenital intestinal abnormalities causes considerable neonatal morbidity and mortality. Animal models are a valuable research tool for this problem; however, few successful neonatal models have been developed and most do not include distal intestinal resection as seen commonly in human babies. We report novel piglet models addressing these gaps. Subjects and Methods: Neonatal piglets (1–6 days) underwent venous and gastric catheter insertion and 75% intestinal resection. Group 1 (n = 6) had midintestinal resection with jejunoileal anastomosis; group 2 (n = 5) had distal intestinal resection with jejunocolic anastomosis; group 3 (n = 5) were sham controls; and group 4 (n = 5) were sow reared. Postoperatively, groups 1 to 3 piglets commenced parenteral nutrition (PN), and enteral nutrition was introduced and advanced using a standard regimen. Data collection included days on PN, weight gain, fat absorption, small intestine lengthening, and bowel/liver histology. Results: Group 2 piglets had more days on PN (P = 0.008), less weight gain (P = 0.027), and greater malabsorption (P = 0.012). They did not show small intestine lengthening and had more cholestatic liver disease. Group 1 piglets had histological evident intestinal adaptation and 1.5-fold intestinal lengthening (P = 0.001). Conclusions: These novel piglet models of short bowel syndrome are the first to represent the full clinical spectrum of intestinal failure as observed in human neonates. By considering the impact of different short bowel anatomy on potential for adaptation and growth, these animal models are a significant advance. They permit evaluation of new therapies to promote intestinal adaptation and reduce complications, such as cholestasis.

Effects of chronic glucagon-like peptide-2 therapy during weaning in neonatal pigs☆

BACKGROUND The enteroendocrine hormone glucagon like peptide-2 (GLP-2) and its ligands are under development as therapeutic agents for a variety of intestinal pathologies. A number of these conditions occur in neonates and infants, and thus a detailed understanding of the effects of GLP-2 during the phase of rapid growth during infancy is required to guide the development of therapeutic applications. We studied the effects of GLP-2 in the neonatal pig to determine the potential effects of exogenous administration. METHODS Two day old newborn domestic piglets were treated with GLP-2 (1-33) at 40 μg/kg/day or control drug vehicle (saline), by subcutaneous injection, given in two doses per day, (n=6/group) for 42 days. Animals were weaned normally, over days 21-25. In the fifth week of life, they underwent neuro-developmental testing, and a pharmacokinetic study. On day 42, they were euthanized, and a complete necropsy performed, with histological assessment of tissues from all major organs. RESULTS GLP-2 treatment was well tolerated, one control animal died from unrelated causes. There were no effects of GLP-2 on weight gain, feed intake, or behavior. In the treated animals, GLP-2 levels were significantly elevated at 2400±600 pM while at necropsy, organ weights and histology were not affected except in the intestine, where the villus height in the small intestine and the crypt depth, throughout the small intestine and colon, were increased. Similarly, the rate of crypt cell proliferation (Ki-67 staining) was increased in the GLP-2 treated animals and the rate of apoptosis (Caspase-3) was decreased, the depth of the microvilli was increased and the expression of the mRNA for the GLP-2 receptor was decreased throughout the small and large intestine. CONCLUSIONS In these growing animals, exogenous GLP-2 at pharmacologic doses was well tolerated, with effects confined to the gastrointestinal tract.

Exogenous glucagon-like peptide-2 improves outcomes of intestinal adaptation in a distal-intestinal resection neonatal piglet model of short bowel syndrome

Background:Endogenous glucagon-like peptide-2 (GLP-2) levels and intestinal adaptation are reduced in distal-intestinal resection animal models of short bowel syndrome (SBS) that lack remnant ileum. We hypothesized that exogenous GLP-2 would improve intestinal adaptation in a distal-intestinal resection neonatal piglet model of SBS.Methods:In all, 35 piglets were randomized to 2 treatment and 3 surgical groups: control (sham), 75% mid-intestinal resection (JI), and 75% distal-intestinal resection (JC). Parenteral nutrition (PN) commenced on day 1 and was weaned as enteral nutrition (EN) advanced. IV GLP-2 (11 nmol/kg/d) or saline was initiated on day 2. Piglets were maintained for 14 d. Clinical, functional, morphological, and histological outcomes were obtained.Results:JC-GLP-2 piglets had fewer days on PN (10.0 ± 0.6 vs. 13.8 ± 0.2), more days on EN (4.0 ± 0.6 vs. 0.2 ± 0.2), a higher percentage of EN at termination (92 ± 5 vs. 52 ± 10%), fewer days of diarrhea (8.0 ± 0.7 vs. 12.3 ± 0.4), increased intestinal length (19 ± 4 vs. −5 ± 3%), and deeper jejunal crypts (248 ± 21 vs. 172 ± 12 μm), compared with saline piglets.Conclusion:GLP-2 therapy improves clinical, morphological, and histological outcomes of intestinal adaptation in a distal-intestinal resection model of SBS. Since this anatomical subtype represents the majority of clinical cases of neonatal SBS, these results support a potential role for GLP-2 therapy in pediatric SBS.

High plasma cholesterol in drug-induced cholestasis is associated with enhanced hepatic cholesterol synthesis.

In α-naphthylisothiocyanate-treated mice, plasma phospholipid (PL) levels were elevated 10- and 13-fold at 48 and 168 h, respectively, whereas free cholesterol (FC) levels increased between 48 h (17-fold) and 168 h (39-fold). Nearly all of these lipids were localized to lipoprotein X-like particles in the low-density lipoprotein density range. The PL fatty acyl composition was indicative of biliary origin. Liver cholesterol and PL content were near normal at all time points. Hepatic hydroxymethylglutaryl CoA reductase activity was increased sixfold at 48 h, and cholesterol 7α-hydroxylase activity was decreased by ∼70% between 24 and 72 h. These findings suggest a metabolic basis for the appearance of abnormal plasma lipoproteins during cholestasis. Initially, PL and bile acids appear in plasma where they serve to promote the efflux of cholesterol from hepatic cell membranes. Hepatic cholesterol synthesis is then likely stimulated in the response to the depletion of hepatic cell membranes of cholesterol. We speculate that the enhanced synthesis of cholesterol and impaired conversion to bile acids, particularly during the early phase of drug response, contribute to the accumulation of FC in the plasma.In alpha-naphthylisothiocyanate-treated mice, plasma phospholipid (PL) levels were elevated 10- and 13-fold at 48 and 168 h, respectively, whereas free cholesterol (FC) levels increased between 48 h (17-fold) and 168 h (39-fold). Nearly all of these lipids were localized to lipoprotein X-like particles in the low-density lipoprotein density range. The PL fatty acyl composition was indicative of biliary origin. Liver cholesterol and PL content were near normal at all time points. Hepatic hydroxymethylglutaryl CoA reductase activity was increased sixfold at 48 h, and cholesterol 7alpha-hydroxylase activity was decreased by approximately 70% between 24 and 72 h. These findings suggest a metabolic basis for the appearance of abnormal plasma lipoproteins during cholestasis. Initially, PL and bile acids appear in plasma where they serve to promote the efflux of cholesterol from hepatic cell membranes. Hepatic cholesterol synthesis is then likely stimulated in the response to the depletion of hepatic cell membranes of cholesterol. We speculate that the enhanced synthesis of cholesterol and impaired conversion to bile acids, particularly during the early phase of drug response, contribute to the accumulation of FC in the plasma.

Parenteral Soy Oil and Fish Oil Emulsions: Impact of Dose Restriction on Bile Flow and Brain Size of Parenteral Nutrition-Fed Neonatal Piglets.

BACKGROUND Parenteral nutrition (PN)-associated liver disease (PNALD) remains a significant cause of morbidity and mortality for neonates dependent on PN. Total fat emulsion dose and composition, particularly the large amount of ω-6 long-chain polyunsaturated fatty acids in plant oils, have been proposed as risk factors for PNALD. We hypothesized restriction of the dose of emulsion would prevent PNALD, regardless of the composition, but growth could be compromised. METHODS Using a neonatal piglet model, we compared conventional soy oil emulsion (Intralipid), dosed high (SO10, n = 8: 10 g/kg/d) and low (SO5, n = 6: 5 g/kg/d), with fish oil (Omegaven), dosed low (FO5, n = 8: 5 g/kg/d). Piglets were given isonitrogenous PN for 14 days. The normal range for all parameters was determined by measurement in equivalent aged sow-reared piglets. RESULTS Bile flow was lower with high-dose Intralipid, outside the normal range, while higher for the other groups (SO10, 5.4 µg/g; SO5, 8.6 µg/g; FO5, 13.4 µg/g; P = .010; normal range, 6.5-12.2 µg/g). Total body weight was low in all treatment groups (SO10, 4.4 kg; SO5, 4.5 kg; FO5, 5.0 kg; P = .038; normal range, 5.2-7.3 kg). Brain weight was not different between groups (SO10, 40.3 g; SO5, 36.0 g; FO5, 36.6 g; P = .122; normal range, 41.8-51.4 g). Corrected for body weight, brain weight was lowest in the fish oil group (SO10, 9.3 g/kg; SO5, 8.0 g/kg; FO5, 7.3 g/kg; P < .001; normal range, 5.9-9.0 g/kg). CONCLUSION Low-dose fat emulsions reduce the risk of developing PNALD. Further investigation of the risk to brain development in neonates exposed to dose restriction, particularly with fish oil, is required.

Effects of Polymeric Formula vs Elemental Formula in Neonatal Piglets With Short Bowel Syndrome

BACKGROUND Intestinal adaptation is important for recovery in short bowel syndrome (SBS). This process is dependent on the presence of enteral nutrition (EN) and trophic factors, such as glucagon-like peptide-2 (GLP-2). In clinical practice, elemental formula is often used to feed neonates with SBS, whereas animal studies suggest polymeric formula promotes better intestinal adaptation. In neonatal piglet models of SBS, with or without ileum, we compared the elemental with the polymeric formula, including the effect on endogenous GLP-2. MATERIALS AND METHODS Forty-eight piglets underwent 75% mid-intestinal resection with jejunoileal anastomosis, 75% distal-intestinal resection with jejunocolic anastomosis (JC), or sham without resection. Parenteral nutrition (PN) started postoperatively, tapering as EN was increased, according to clinical criteria, based on diarrhea and weight. Within groups, piglets were randomized to an isocaloric/isonitrogenous elemental (amino acid) or polymeric (intact protein) diet. Plasma GLP-2 and histology for adaptation were measured at 14 days. RESULTS Within both SBS and control groups, no difference in adaptation was observed according to diet. A difference was observed only within the JC piglet group with regard to clinical outcomes. In these piglets, compared with elemental formula, the polymeric formula was associated with more diarrhea ( P = .023) and longer duration of PN support (P = .047). CONCLUSION An overall benefit of the polymeric formula over the elemental formula on gut adaptation was not observed. Furthermore, SBS piglets without ileum had less ability to tolerate polymeric formula, contributing to more days of PN support.

Role of glucagon-like peptide-2 deficiency in neonatal short-bowel syndrome using neonatal piglets

Background:Short-bowel syndrome (SBS) is the most common cause of neonatal intestinal failure. Recovery requires intestinal adaptation, dependent on enteral nutrition (EN) and growth factors such as glucagon-like peptide–2 (GLP-2), which is secreted from L cells in the ileum. Neonatal SBS often results in loss of ileum; therefore, we hypothesized that without ileum, endogenous GLP-2 production would be inadequate to promote adaptation. We compared endogenous GLP-2 production and adaptation in neonatal animals with SBS, with and without ileum.Methods:Neonatal piglets (4–6 d) were randomized to 75% mid-intestinal resection, 75% distal-intestinal resection, or sham control without resection. Postoperatively, all piglets commenced parenteral nutrition (PN), tapering as EN was increased to maintain specific growth.Results:The resected SBS piglets developed intestinal failure, requiring a longer duration of PN support and experiencing fat malabsorption. The piglets without ileum were not able to wean from PN during the study and did not show adaptation, specifically growth in intestinal length or crypt hyperplasia on histology of the jejunum. Adaptation was observed in the resected SBS piglets with ileum, and these piglets also had an increased plasma GLP-2 level that was not observed in piglets without ileum.Conclusion:SBS piglets with ileum undergo adaptation associated with increased endogenous GLP-2 production. SBS piglets without ileum undergo limited adaptation and severe intestinal failure, requiring prolonged PN support. This appears to be related to a deficiency in endogenous GLP-2 production.

Hepatic ultrastructure in a neonatal piglet model of intestinal failure-associated liver disease (IFALD)

This study was designed to evaluate liver disease in neonatal piglets with surgical short bowel syndrome causing intestinal failure with partial parenteral nutrition dependence. The short bowel piglets had 75% surgical resection of distal small intestine, including all ileum and cecum, and were compared with sham controls, without resection, and to healthy sow-reared controls. After 18 days of combined parenteral and enteral nutrition in short bowel and sham piglets, liver tissue was collected for quantitative and semi-quantitative histological and ultrastructural evaluation. The short bowel piglets developed biochemical and histological cholestasis, not observed in sham and control piglets. Ultrastructural examination revealed bile canaliculus dilation with bile plugging, microvillus flattening and disappearance, but without abnormalities of the pericanalicular zone. Interestingly, these data are similar to bile canaliculus changes seen in human neonates with IFALD supporting an initial consideration of this model to elucidate the pathogenesis of IFALD.