Multicenter Evaluation of BD Max Enteric Parasite Real-Time PCR Assay for Detection of Giardia duodenalis, Cryptosporidium hominis, Cryptosporidium parvum, and Entamoeba histolytica.

Common causes of chronic diarrhea among travelers worldwide include protozoan parasites. The majority of parasitic infections are caused by Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, and Cryptosporidium hominis Similarly, these species cause the majority of parasitic diarrhea acquired in the United States. Detection of parasites by gold standard microscopic methods is time-consuming and requires considerable expertise; enzyme immunoassays and direct fluorescent-antibody (DFA) stains have lowered hands-on time for testing, but improvements in sensitivity and technical time may be possible with a PCR assay. We performed a clinical evaluation of a multiplex PCR panel, the enteric parasite panel (EPP), for the detection of these common parasites using the BD Max instrument, which performs automated extraction and amplification. A total of 2,495 compliant specimens were enrolled, including 2,104 (84%) specimens collected prospectively and 391 (16%) specimens collected retrospectively. Approximately equal numbers were received in 10% formalin (1,273 specimens) and unpreserved (1,222 specimens). The results from the EPP were compared to those from alternate PCR and bidirectional sequencing (APCR), as well as DFA (G. duodenalis and C. parvum or C. hominis) or trichrome stain (E. histolytica). The sensitivity and specificity for prospective and retrospective specimens combined were 98.2% and 99.5% for G. duodenalis, 95.5% and 99.6 for C. parvum or C. hominis, and 100% and 100% for E. histolytica, respectively. The performance of the FDA-approved BD Max EPP compared well to the reference methods and may be an appropriate substitute for microscopic examination or immunoassays.

GLUT-5 expression in neonatal rats: crypt-villus location and age-dependent regulation.

The rat fructose transporter normally appears after completion of weaning but can be precociously induced by early feeding of a high-fructose diet. In this study, the crypt-villus site, the metabolic nature of the signal, and the age dependence of induction were determined. In weaning rats fed high-glucose pellets, GLUT-5 mRNA expression was modest, localized mainly in the upper three-fourths of the villus, and there was little expression in the villus base. When fed high-fructose pellets, GLUT-5 mRNA expression was two to three times greater in all regions except the villus base. Intestinal perfusion in vivo of a nonmetabolizable fructose analog, 3-O-methylfructose, tended to increase fructose uptake rate and moderately increased GLUT-5 mRNA abundance but had no effect on glucose uptake rates and SGLT1 mRNA abundance. Gavage feeding of high-fructose, but not high-glucose, solutions enhanced fructose uptake only in pups > or =14 days, suggesting that GLUT-5 regulation is markedly age dependent. Fructose or its metabolites upregulate GLUT-5 expression in all enterocytes, except those in the crypt and villus base and in pups <14 days old.

Stimulation of tyrosylprotein sulfotransferase activity by ethanol: role of increased enzyme level.

Tyrosylprotein sulfotransferase (TPST), an enzyme involved in the posttranslational modification of proteins, plays important role in the biological activity and secretion of proteins. Previously we have shown an increased activity of this enzyme in gastric mucosa of alcoholics. In the present study, effect of ethanol on TPST was examined in rat liver and gastric mucosa utilizing enzyme assays and Western blot analyses for TPST levels. Male Sprague-Dawley rats were pair-fed Leiber-DeCarli liquid diets for 10 days and controls received a liquid diet in which dextrose was isocalorically substituted for ethanol. After ethanol feeding, rats were sacrificed and liver and gastric mucosa were processed for Golgi membrane preparation. The TPST activity was measured using poly(Glu6, Ala3, Tyr1) as the sulfate acceptor and PAPS as sulfate donor. There was a threefold increase in TPST activity of gastric mucosa of animals subjected to chronic alcohol feeding. In the liver, the increase in tyrosine sulfating activity was also around threefold. The kinetic studies performed to understand the mechanism involved in ethanol stimulation of TPST activity showed no change in the Km values of the enzyme by ethanol. In control and ethanol-treated animals, the Km for EAY was 0.41-0.53 and 0.43-0.53 microM, and the Km for PAPS was 10-12.5 and 9-17 microM, respectively. The Vmax in ethanol-fed animals was increased by 1.5- to 2-fold. The increase in TPST activity in experimental rats was further tested by analyzing the Western blots by Imaging Densitometer for TPST levels. Analysis of TPST levels also showed over threefold increase in the stomach and liver of ethanol-fed rats. Our results indicate that stimulation of TPST by ethanol involves increased TPST level rather than change in affinity for its substrates.

Plasma nitric oxide in posttrauma critical illness: a function of "sepsis" and the physiologic state severity classification quantifying the probability of death.

The time course of nitric oxide (NO) production in posttrauma critical illness was monitored, and its relationship to posttrauma "sepsis/SIRS" and physiologic patterns was described. Eighty multiple trauma patients were studied (514 samples) during their course in the intensive care unit (Injury Severity Score 27.6; 36% deaths). Plasma NO was estimated from NO3 + NO2 by the Griess test and compared with that of 10 healthy controls (HC). At each sample period, the patient was categorized as having bacteremic sepsis (BAC), sepsis syndrome (SS), or systemic inflammatory response syndrome (SIRS), and classified by Physiologic State Severity Classification (PSSC) into normal stress response (A-State), metabolic insufficiency (B-State), or respiratory insufficiency (C2-State), each quantified by their physiologic "distance" from reference state of recovering trauma patients (R-State). A severity index (L2PDEATH), based on a logistic model of state distances from R-State, quantified probability of death. Deaths showed increased NO (p < .05) over survivors or HC by day 3 posttrauma. A fall in vascular tone in deaths was related to the increased NO (p < .0001). The level of NO was higher as sepsis worsened: BAC > SS > SIRS > HC (all simultaneous, p < .05). PSSC and L2PDEATH correlated with incidence > HC and level of NO. In conclusion, the severity of posttrauma critical illness was classified by PSSC and quantified by the L2PDEATH index. These reflect progressively increased NO levels and suggest worsening sepsis status. The reduced total peripheral resistance (TPR)-to-flow relationship (vascular tone) in deaths characteristic of the more severe septic PSSC states appeared related to the increased plasma NO.

Chronic ethanol feeding alters the structure and function of the epidermal growth factor receptor in rat stomach.

We investigated the effect of chronic ethanol feeding on the EGF receptor in rat stomach. Adult male rats were fed either an isocaloric control or ethanol (EtOH)-containing liquid diet (36% total calories as EtOH) for 4 weeks EtOH significantly reduced the specific binding of 125I-EGF to the gastric mucosal membrane (control vs. EtOH, 2.07 +/- 0.2 vs. 0.94 +/- 0.16 fmol/mg protein; p < 0.01). Scatchard analysis suggested that the lower binding might be due to the reduction of EGF receptor number, and/or the affinity of the high-affinity binding site. Western blot analysis, using anti-EGF receptor antibody, revealed four immunoreactive protein bands (180, 150, 60, and 50 kDa) in the lectin-purified gastric membrane prepared from both groups. However, the intensities of these protein bands in the EtOH-fed animals were 90% lower compared to the controls. In the EGF-responsive protein kinase assay, 32P-ATP was incubated with lectin-purified samples in the absence or presence of 1 microM EGF. EGF stimulated autophosphorylation of the EGF receptor (180 kDa) in stomach from the control groups, but not the EtOH-fed animals. This EtOH-related alteration of the gastric EGF receptor may be one of the mechanisms underlying the gastric pathology associated with alcohol abuse.

Physiologic state severity classification as an indicator of posttrauma cytokine response.

A Physiologic State Severity Classification (PSSC) derived from clustering of 17 cardiorespiratory variables was used to predict cytokine response in critically ill posttrauma patients. The PSSC defined physiologic states: A-State (A), normal stress response; B-State (B), metabolic insufficiency; C2-State (C), respiratory insufficiency. Bayesian analysis of these states defined a probability of death (Pdeath). 416 studies from 60 newly studied multiple trauma patients (70% males, Injury Severity Score = 27.5) were analyzed; 45 (75%) had sepsis (s), 28 (47%) had sepsis-adult respiratory distress syndrome (s-ARDS). Of 35 survivors (66% s, 37% s-ARDS, mean Pdeath = .42) 23% were predominantly A, 66% B, and 11% C. Of 25 deaths (88% s, 60% s-ARDS, mean Pdeath = .64) 0% were A, 44% B, and 56% C. PSSC States were correlated with incidence and mean plasma levels (pl) in picograms/mL of cytokines. 23 samples from recovering nonseptic trauma patients were used as controls.

Regulatory properties of brain glutamate decarboxylase (GAD): the apoenzyme of GAD is present principally as the smaller of two molecular forms of GAD in brain.

The apoenzyme of glutamate decarboxylase [enzyme without bound cofactor, pyridoxal 5'-phosphate (pyridoxal-P)] serves as a reservoir of inactive glutamate decarboxylase (GAD) that can be activated when additional GABA synthesis is required. We have investigated which of two molecular forms of GAD is present as apoenzyme in synaptosomes and in cortex, caudate nucleus, hippocampus, and cerebellum of rat brain. Endogenous glutamate apodecarboxylase (apoGAD) was labeled by incubating extracts of synaptosomes or punches of each region with 32P-pyridoxal-P, followed by reduction with NaBH4, to link covalently the 32P-pyridoxal-P to GAD. Proteins were separated by SDS-PAGE. Punches from all four brain regions and forebrain synaptosomes contained two forms of GAD with apparent Mrs of 63 and 65 kDa as identified by immunoblotting with four antiGAD sera. Punches and synaptosomes contained a major 32P-pyridoxal-P-labeled band with an apparent Mr of 63 kDa that was stained on immunoblots by the antiGAD serum 1440 and the monoclonal antibody GAD-6, and a minor labeled band at 65 kDa that was stained by the 1440, 6799, and K2 antisera. Synaptosomes contained remarkably few other strongly labeled proteins, but punches contained several other labeled bands. Three additional lines of evidence indicate that the labeled 63-kDa protein is apoGAD: (1) it was purified by immunoaffinity chromatography with the GAD-1 monoclonal antibody; (2) it yielded one major labeled peptide when digested with chymotrypsin, and that peptide appeared identical in peptide-mapping experiments to the labeled active-site peptide isolated from chromatographically prepared rat brain GAD; and (3) its labeling was selectively blocked by 4-deoxypyridoxine 5'-phosphate, a competitive inhibitor of the binding of pyridoxal-P to GAD.(ABSTRACT TRUNCATED AT 250 WORDS)

Cofactor interactions and the regulation of glutamate decarboxylase activity.

More than 50% of glutamate decarboxylase (GAD) in brain is present as apoenzyme. Recent work has opened the possibility that apoGAD can be studied in brain by labeling with radioactive cofactor. Such studies would be aided by a compound that inhibits specific binding. One possibility is 4-deoxy-pyridoxine 5'-phosphate, a close structural analog of the cofactor pyridoxal 5'-phosphate. The effects of deoxypyridoxine-P on the cyclic series of reactions that interconverts apo- and holoGAD was investigated and found to be consistent with simple competitive inhibition of the activation of apoGAD by pyridoxal-P. As expected from the cycle GAD was inactivated when incubated with glutamate and deoxypyridoxine-P even though cofactor was present, but no inactivation was observed with deoxypyridoxine-P in the absence of glutamate. Deoxypyridoxine-P also stabilized apoGAD against heat denaturation. These effects were quantitatively accounted for by a kinetic model of the apo-holoGAD cycle. Deoxypyridoxine-P inhibited the labeling by [32P]pyridoxal-P of GAD isolated from rat brain. Hippocampal extracts were labeled with [32P]pyridoxal-P and analyzed by SDS-polyacrylamide gel electrophoresis. Remarkably few bands were strongly labeled. The major labeled band (at 63 kDa) corresponded to one of the forms of GAD. Other strongly-labeled bands were observed at 65 kDa (corresponding to the higher molecular weight form of GAD) and at 69--72 kDa. Labeling of the 63- and 65-kDa bands was inhibited by deoxypyridoxine-P, but the 69-72 kDa bands were unaffected, suggesting that the latter were non-specifically labeled. The results suggest that the 63-kDa form of GAD makes up the majority of apoGAD in hippocampus.

In vitro and in vivo inhibitory effect of ethanol and acetaldehyde on O6-methylguanine transferase.

Human and rat O6-methylguanine transferase (O6MeGT) are inhibited in vitro by ethanol at concentrations of 10 to 50 mM and by acetaldehyde, the first metabolite of ethanol, at concentrations as low as 0.01 microM. Several other enzymes, including glyceraldehyde-3-phosphate dehydrogenase and yeast alcohol dehydrogenase, which like O6MeGT have cysteines in their active sites, were not inhibited by acetaldehyde at the levels that inhibited O6MeGT. Disulfiram, an acetaldehyde dehydrogenase inhibitor, enhanced the inhibitory effect of ethanol in vivo. These results indicate that the inhibitory effect of ethanol on O6MeGT activity is mediated primarily via its metabolite, acetaldehyde.

The effects of chronic ethanol consumption on carcinogen metabolism and on O6-methylguanine transferase-mediated repair of alkylated DNA.

This article presents a review and update of recent experiments conducted in collaboration with Dr. C. S. Lieber on mechanisms underlying the increased cancer risk associated with alcohol abuse. Ethanol has been found to be a potent inducer of microsomal enzymes involved in carcinogen metabolism in a variety of rat tissues including liver, esophagus, lungs, and intestines. In some of these tissues, ethanol's inductive effect on microsomal cytochrome P-450 enzyme activity may result in enhanced levels of electrophilic metabolites of procarcinogens which are not readily detoxified. In addition, chronic ethanol feeding has been found to depress the activity of O6-methylguanine transferase, an enzyme involved in the repair of carcinogen-induced DNA alkylation. The effects of ethanol on carcinogen metabolism and on DNA repair would be expected to enhance the initiation phase of chemically induced cancers.