Effect of recombinant platelet-activating factor acetylhydrolase on two models of experimental acute pancreatitis

BACKGROUND & AIMS: Recent reports suggest that platelet-activating factor (PAF) plays a role in pancreatitis and pancreatitis-associated lung injury. In this study, the effects on these processes of termination of PAF action by recombinant PAF-acetylhydrolase (rPAF-AH) were investigated. METHODS: Rats were given rPAF-AH and then infused with a supramaximally stimulating dose of cerulein to induce mild pancreatitis. Opossums underwent biliopancreatic duct ligation to induce severe pancreatitis, and rPAF-AH administration was begun 2 days later. RESULTS: In mild, secretagogue-induced pancreatitis, rPAF-AH given before the cerulein reduced hyperamylasemia, acinar cell vacuolization, and pancreatic inflammation but did not alter pancreatic edema or pulmonary microvascular permeability. In severe, biliopancreatic duct ligation-induced pancreatitis, rPAF-AH delayed and reduced the extent of inflammation and acinar cell injury/necrosis and completely prevented lung injury even though the rPAF-AH administration was begun after the onset of pancreatitis. CONCLUSIONS: PAF plays an important role in the regulation of pancreatic injury but not pancreatic edema or increased pulmonary microvascular permeability in mild, secretagogue-induced pancreatitis. PAF plays a critical role in the regulation of progression of pancreatic injury and mediation of pancreatitis-associated lung injury in severe biliary pancreatitis. Amelioration of pancreatitis and prevention of pancreatitis-associated lung injury can be achieved with rPAF-AH even if treatment is begun after pancreatitis is established.     

Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer

The platelet-activating factor PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid first messenger active in general cell activation, fertilization, inflammatory and allergic reactions, asthma, HIV pathogenesis, carcinogenesis, and apoptosis. There is substantial evidence that PAF is involved in intracellular signalling, but the pathways are poorly understood. Inactivation of PAF is carried out by specific intra- and extracellular acetylhydrolases (PAF-AHs), a subfamily of phospholipases A2 that remove the sn-2 acetyl group. Mammalian brain contains at least three intracellular isoforms, of which PAF-AH(Ib) is the best characterized. This isoform contains a heterodimer of two homologous catalytic subunits alpha1 and alpha2, each of relative molecular mass 26K, and a non-catalytic 45K beta-subunit, a homologue of the beta-subunit of trimeric G proteins. We now report the crystal structure of the bovine alpha1 subunit of PAF-AH(Ib) at 1.7 A resolution in complex with a reaction product, acetate. The tertiary fold of this protein is closely reminiscent of that found in p21(ras) and other GTPases. The active site is made up of a trypsin-like triad of Ser 47, His 195 and Asp 192. Thus, the intact PAF-AH(Ib) molecule is an unusual G-protein-like (alpha1/alpha2)beta trimer.     

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.     

Serum paraoxonase and platelet-activating factor acetylhydrolase in chronic renal failure

PURPOSE: Since clinically apparent varicoceles may affect testicular volume and sperm production, early repair has been advocated. However, repair of the pediatric varicocele with conventional nonmagnified techniques may result in persistence of the varicocele after up to 16% of these procedures. Also testicular artery injury and postoperative hydrocele formation can occur after nonmagnified repair. The microsurgical technique has been successfully completed in a large series of adults with a dramatic reduction in complication and recurrence rates. We report our experience with the microsurgical technique in boys. MATERIALS AND METHODS: A total of 30 boys (average age 15.9 years) underwent 42 microsurgical varicocelectomies (12 bilateral). All patients had a large left varicocele. Indications for repair included testicular atrophy (size difference between testicles of greater than 2 ml.) in 20 boys, pain in 5 and a large varicocele without pain or testicular atrophy in 5. Six boys were referred following failure of conventional nonmicrosurgical techniques. All boys were examined no sooner than 1 month postoperatively (mean followup 12). RESULTS: Preoperative volume of the affected testis averaged 13.0 ml., and an average size discrepancy between testicles of 2.8 ml. was noted before unilateral varicocelectomy. No cases of persistent or recurrent varicoceles were detected, and 1 postoperative hydrocele resolved spontaneously. After unilateral varicocelectomy the treated testes grew an average of 50.1%, while the contralateral testes grew only 23%. Overall, 89% of patients with testicular atrophy demonstrated reversal of testicular growth retardation after unilateral varicocelectomy. In contrast, both testes showed similar growth rates after bilateral varicocelectomy (45% left testis, 39% right testis). CONCLUSIONS: The meticulous dissection necessary to preserve arterial and lymphatic supply, and to ligate all spermatic veins in the pediatric patient is readily accomplished using a microsurgical approach, and results in low recurrence and complication rates. Rapid catch-up growth of the affected testis after microsurgical varicocelectomy suggests that intervention during adolescence is effective and warranted.     

Neonatal pneumatosis coli: a mild form of neonatal necrotizing enterocolitis

A distinctive form of necrotizing enterocolitis was observed in seven newborn infants in a 4 1/2-year period. Characteristically these patients had gross blood in the stools, but other local and systemic signs were mild or absent. Radiography mainly revealed submucosal intramural intestinal gas which was limited to the colon (pneumatosis coli). There was no evidence of small bowel involvement as judged by the absence of small intestinal pneumatosis or distension. This form of colonic disease should be recognized as a benign variety of neonatal necrotizing enterocolitis that carries a favorable prognosis and responds to medical management without sequelae.     

Role of bacterial colonization in neonatal necrotizing enterocolitis and its prevention

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. A major component of the pathophysiology of NEC is the nature of the interaction of bacteria with the premature gut. Intestine microflora are important to the host in resistance to bacterial infections. Diet and environmental conditions can influence this ecosystem. A breast-fed full-term infant has a preferred intestine microbiota in which bifidobacteria predominate over the potentially harmful bacteria, whereas in formula-fed infants coliforms, enterococci and bacteroides predominate. The pattern of bacterial colonization in the premature neonate gut is quite different from that in the gut of the healthy full-term infant. Those infants requiring intensive care acquire intestinal organisms slowly, and the establishment of bifidobacterial flora is retarded. A delayed bacterial colonization of the gut with a limited number of bacterial species tends to be virulent. Bacterial overgrowth is one of major factors promoting bacterial translocation. The aberrant colonization of the premature infant may contribute to the development of NEC. Breast feeding protects infants against NEC. Oligosaccharides and glycoconjugates, natural components in human milk, may prevent intestinal attachment of enteropathogens by acting as receptor homologues. Probiotics and prebiotics modulate the composition of human intestine microflora to the benefit of the host. The beneficial effects may result in the suppression of colonization of harmful microoganisms and/or the stimulation of bifidobacterial growth. In the future, control and manipulation of bacterial colonization in the neonate gut may be a new approach to the prevention and treatment of bacterial intestinal disease of various etiologies.     

Study of platelet-activating factor acetylhydrolase in the perioperative period of patients undergoing cardiac surgery

This study was conducted to determine whether brief, intermittent exposure to hypoxia with little change in nutrient intake would affect fetal growth. Pregnant rats were exposed to 1 or 2 h of hypoxia (FiO2 = 0.09-0.095) from days 15 to 19 of gestation. Exposure to 1 h of hypoxia decreased fetal body weight and length, liver weight and increased the brain/liver weight ratio (p < 0.05) as compared to controls. Two hours of hypoxia decreased fetal body weight and length, and heart, lung, kidney, gut, brain and liver weights (p < 0.01), but did not affect the brain/liver weight ratio. Two hours of hypoxia decreased maternal food intake and weight gain (p < 0.05), but fetal growth was not significantly altered in pair-fed controls. These data demonstrate that brief, intermittent periods of intrauterine hypoxia have significant effects on fetal growth.     

Reduction of microtubule catastrophe events by LIS1, platelet-activating factor acetylhydrolase subunit

Forming the structure of the human brain involves extensive neuronal migration, a process dependent on cytoskeletal rearrangement. Neuronal migration is believed to be disrupted in patients exhibiting the developmental brain malformation lissencephaly. Previous studies have shown that LIS1, the defective gene found in patients with lissencephaly, is a subunit of the platelet-activating factor acetylhydrolase. Our results indicated that LIS1 has an additional function. By interacting with tubulin it suppresses microtubule dynamics. We detected LIS1 interaction with microtubules by immunostaining and co-assembly. LIS1-tubulin interactions were assayed by co-immunoprecipitation and by surface plasmon resonance changes. Microtubule dynamic measurements in vitro indicated that physiological concentrations of LIS1 indeed reduced microtubule catastrophe events, thereby resulting in a net increase in the maximum length of the microtubules. Furthermore, the LIS1 protein concentration in the brain, measured by quantitative Western blots, is high and is approximately one-fifth of the concentration of brain tubulin. Our new findings show that LIS1 is a protein exhibiting several cellular interactions, and the interaction with the cytoskeleton may prove to be the mode of transducing a signal generated by platelet-activating factor. We postulate that the LIS1-cytoskeletal interaction is important for neuronal migration, a process that is defective in lissencephaly patients.     

Loss of activity of plasma platelet-activating factor acetylhydrolase due to a novel Gln281-->Arg mutation

The prevalence of plasma platelet-activating factor (PAF) acetylhydrolase deficiency was investigated in 477 healthy Japanese individuals and 985 patients with various cardiovascular diseases. The genotype for this enzyme with regard to a G994-->T mutation (MM, normal; Mm, heterozygote; mm, mutant homozygote) was determined by an allele-specific polymerase chain reaction in 80 subjects shown to have no or low plasma activity (<10 nmol/min/ml). In 72 subjects, the genotype was consistent with plasma enzyme activity; 44 individuals with no activity were mm, and 28 with low activity were Mm. However, eight subjects with the MM genotype showed plasma enzyme activities of <10 nmol/min/ml. Determination of the DNA sequence of exon 9 of the plasma PAF acetylhydrolase gene in these eight subjects revealed a previously unidentified A1001-->G missense mutation, resulting in a Gln281-->Arg substitution, in a 72-year-old woman with coronary artery disease, essential hypertension, and no plasma enzyme activity. Site-directed mutagenesis in vitro showed that the corresponding recombinant mutant protein lacked PAF acetylhydrolase activity. Thus, the Gln281-->Arg substitution appears responsible for the loss of plasma PAF acetylhydrolase activity.     

A mutation in plasma platelet-activating factor acetylhydrolase (Val279-->Phe) is a genetic risk factor for stroke

BACKGROUND AND PURPOSE: Platelet-activating factor (PAF) is a phospholipid with multiple actions that include thrombosis and inflammation. It is inactivated by a plasma enzyme, PAF acetylhydrolase. Deficiency of this enzyme in plasma is caused by a missense mutation in the gene (Val279-->Phe). We have studied a possible association of this mutation with the risk of stroke. SUBJECTS AND METHODS: We studied 120 consecutive patients with cerebral thrombosis. The control group consisted of 134 patients matched for age and sex with minor complaints but without stroke. Genomic DNA was analyzed for the mutant allele by a specific polymerase-chain reaction. Plasma PAF acetylhydrolase activity was determined by the method of Stafforini et al. RESULTS: The prevalence of the mutant gene was 43.4% in stroke patients (39.2% heterozygotes and 4.2% homozygotes), which was significantly higher than the 25.4% in control subjects (22.4% heterozygotes and 3.0% homozygotes) (chi 2 = 9.22, P < .01). The prevalence was slightly higher in stroke patients without hypertension than those with hypertension, but the difference was not significant. The patients with family histories of stroke had a slightly higher but not a significant prevalence of the mutant gene as compared with those without family histories of stroke. Plasma PAF acetylhydrolase activity was higher in patients than in control subjects, in normal subjects, or patients with a heterozygous genotype. CONCLUSIONS: These results suggest that plasma PAF acetylhydrolase deficiency may be a risk factor for stroke. This may explain the relatively high prevalence of stroke in Japan, as the mutation is more common among Japanese than Caucasians.     

Generation of reactive oxygen species and activity of platelet-activating factor acetylhydrolase in human monocyte-derived macrophages

Monocytes were prepared from healthy human volunteers and were allowed to differentiate into macrophages by adhesion to plastic surface and cultured over 7 days in presence of either 10% fetal calf serum (FCS), human control serum or serum from hyperlipaemic patients. Hyperlipaemic serum stimulated the differentiation (measured as an increase in cellular protein and DNA content) to a higher extent when compared to control serum and FCS. With all sera a marked increase of the cellular activity of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) and a tremendous decrease in the capacity of cells to generate reactive oxygen species (ROS) was observed. After seven days of culture the increase in PAH-AH activity was about 19-fold with hyperlipaemic serum, 11-fold with control serum and 6-fold with FCS. During the same period of time ROS generation measured as zymosan-induced chemiluminescence decreased by about 98% and no significant differences between the three types of serum were found. The results indicate that the activity of PAF-AH and the capacity of ROS generation which are both assumed to play an important role in the oxidation of low-density lipoproteins (LDL) and thus in the development of atherosclerosis, change in opposite direction during the differentiation of blood monocytes into macrophages, and that hyperlipaemic serum stimulates PAF-AH activity but not ROS generation.     

Effect of 17 beta-estradiol on secretion of platelet-activating factor acetylhydrolase by HepG2 cells

Estrogen has been shown to decrease plasma platelet-activating factor (PAF) acetylhydrolase activity, but the precise mechanisms are not known. We examined the effect of estradiol on the secretion of PAF acetylhydrolase by HepG2 cells. In our previous study, we demonstrated the production of this enzyme by HepG2 cells, which we used as an experimental model of normal hepatocytes. 17 beta-Estradiol mildly but consistently inhibited the secretion of PAF acetylhydrolase by HepG2 cells in a concentration-dependent manner. Under basal conditions, HepG2 cells secreted 42.3 pmol/mg cell protein/min PAF acetylhydrolase in 24 hours (mean of 8 dishes), and the presence of 10(-7) mol/L 17 beta-estradiol decreased the secretion to 77% +/- 10.3% of control values (mean +/- SD, n = 8, P < .02). 17 beta-Estradiol treatment affected neither the secretion of apolipoprotein (apo) A-I nor cell-associated PAF acetylhydrolase activity. Electrophoretic separation of [35S]methionine-labeled PAF acetylhydrolase revealed a single band whose molecular weight was approximately 43,000 d. We conclude that estrogen decreases the secretion of PAF acetylhydrolase by the liver, and it may explain, at least in part, the effect of estrogen on plasma PAF acetylhydrolase.     

Alterations of the enteric nervous system in neonatal necrotizing enterocolitis revealed by whole-mount immunohistochemistry

We report the results of a comprehensive search of Drosophila melanogaster DNA sequences in GenBank for di-, tri-, and tetranucleotide repeats of more than four repeat units, and a DNA library screen for dinucleotide repeats. Dinucleotide repeats are more abundant (66%) than tri- (30%) or tetranucleotide (4%) repeats. We estimate that 1917 dinucleotide repeats with 10 or more repeat units are present in the euchromatic D. melanogaster genome and, on average, they occur once every 60 kb. Relative to many other animals, dinucleotide repeats in D. melanogaster are short. Tri- and tetranucleotide repeats have even fewer repeat units on average than dinucleotide repeats. Our WorldWide Web site (http://www.bio.cornell.edu/genetics/aquadro/+ ++aquadro.html) posts the complete list of 1298 microsatellites (> or = five repeat units) identified from the GenBank search. We also summarize assay conditions for 70 D. melanogaster microsatellites characterized in previous studies and an additional 56 newly characterized markers.