HCV lookback in the United States: effectiveness of an extended lookback program.

BACKGROUND: In 1998, the FDA recommended look-back for HCV. The recommendation was initially limited, however, to donors who reacted on a multiantigen HCV screening test and to components collected since January 1, 1988. A lookback program was extended to include donors who reacted on the first-generation (single-antigen) HCV screening test and who were positive on a supplemental assay (RIBA-1 or -2) and all components for which transfusion records could be found (back to 1978). STUDY DESIGN AND METHODS: The yield of the incremental lookback programs was compared to that originally recommended by the FDA by comparing the number of newly identified HCV-positive recipients in each program. The results of lookbacks were reviewed on 385 blood components for which 314 transfusion recipients were identified. RESULTS: Of the 135 recipients in the FDA program, 70 percent were dead, 28 percent were living and notified, and 2 percent could not be located. In the incremental programs, there were 179 recipients, of whom 80.4 percent were dead, 16.2 percent were living and notified, and 3.4 percent could not be located. Most adult recipients were dead (81%), but the majority of pediatric recipients were alive (57%); 76 percent of tested recipients were HCV seropositive, with no significant difference between programs. One-half of test-positive recipients in each program were newly identified through the lookback program. Seven of the 20 newly identified HCV-positive recipients were found through the incremental programs. The yield, defined as newly detected HCV cases per total number of recipients, was 9.6 percent for the FDA and 3.9 percent for the incremental programs. This difference was significant (p = 0.04). CONCLUSION: The yield of both programs was limited by the high percentage of recipients who had died. Pediatric recipients were more likely to be living at the time of notification. The incremental program was less efficacious than the FDA program in identifying newly HCV-positive recipients, but one-third of the newly detected HCV cases were identified through the incremental program.

Efficacy of preoperative donation of blood for autologous use in radical prostatectomy.

To determine the amount of blood lost, the number of transfusions, and the effectiveness of preoperative autologous blood donation in radical prostatectomy, 163 patients' records from 1987 to 1991 were reviewed at four university hospitals and three community hospitals. Calculated red cell volume lost was 1003 +/- 535 mL (mean +/- SD), which corresponds to 44 +/- 18 percent (mean +/- SD) of total red cell volume. Preoperative donation of blood for autologous use reduced the rate of transfusion of allogeneic blood from 66 to 20 percent (p < 0.001). Of the patients who donated 1 to 2 units, 32 percent received allogeneic blood; 14 percent of those who donated 3 units received allogeneic blood. Donation of 4 units reduced the allogeneic transfusion rate to 11 percent. However, as the number of units donated increased (1-3 units), the units not transfused also increased (0-21%). Ninety-one (56%) of 163 patients donated fewer than 3 units. Autologous blood donation is effective in minimizing the transfusion of allogeneic blood to radical prostatectomy patients, but many patients do not donate enough blood (< 3 units). The donation of 3 units of blood for autologous use is recommended for patients who undergo radical prostatectomy.

Occupation of low-affinity cholecystokinin (CCK) receptors by CCK activates signal transduction and stimulates amylase secretion in pancreatic acinar cells.

Based on the effects of monensin on binding of 125I-CCK-8 and its lack of effect on CCK-8-stimulated amylase secretion we previously proposed that pancreatic acinar cells possess three classes of CCK receptors: high-affinity receptors, low-affinity receptors and very low-affinity receptors [1]. In the present study we treated pancreatic acini with carbachol to induce a complete loss of high-affinity CCK receptors and then examined the action of CCK-8 on inositol trisphosphate IP3(1,4,5), cytosolic calcium and amylase secretion in an effort to confirm and extend our previous hypothesis. We found that first incubating pancreatic acini with 10 mM carbachol decreased binding of 125I-CCK-8 measured during a second incubation by causing a complete loss of high-affinity CCK receptors with no change in the low-affinity CCK receptors. Carbachol treatment of acini, however, did not alter the action of CCK-8 on IP3(1,4,5), cytosolic calcium or amylase secretion or the action of CCK-JMV-180 on amylase secretion or on the supramaximal inhibition of amylase secretion caused by CCK-8. The present findings support our previous hypothesis that pancreatic acinar cells possess three classes of CCK receptors and suggest that high-affinity CCK receptors do not mediate the action of CCK-8 on enzyme secretion, that low-affinity CCK receptors may mediate the action of CCK on cytosolic calcium that does not involve IP3(1,4,5) and produce the upstroke of the dose-response curve for CCK-8-stimulated amylase secretion and that very low-affinity CCK receptors mediate the actions of CCK on IP3(1,4,5) and cytosolic calcium and produce the downstroke of the dose-response curve for CCK-8-stimulated amylase secretion. Moreover, CCK-JMV-180 is a full agonist for stimulating amylase secretion by acting at low-affinity CCK receptors and is an antagonist at very low-affinity CCK receptors.

Neutral endopeptidase (EC 3.4.24.11) modulates the contractile effects of neuropeptides on muscle cells from the guinea-pig stomach.

The objectives of this investigation were to characterize neuropeptide-degrading enzymes on the surface of gastric muscle cells and to determine their physiological function. Neutral endopeptidase (NEP, EC 3.4.24.11) activity was measured using the fluorogenic substrate glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine. The NEP inhibitors phosphoramidon and DL-thiorphan (1 microM) inhibited degradation of the substrate by gastric muscle membranes by 100% and by freshly dispersed gastric muscle cells by 55-60%. The phosphoramidon or DL-thiorphan-inhibitable activity, attributed to NEP, of membranes was 112 +/- 4.0 pmol h-1 (micrograms protein)-1 and of cells was 4.2 +/- 0.8 nmol h-1 (10(6) cells)-1. This activity was associated with membranes prepared from cells and was not detected in the cytoplasm or in the cell incubation solution. Gastric muscle membranes were fractionated by electrophoresis and analysed by Western blotting using two NEP antisera. Both antisera recognized a protein in membranes with an electrophoretic mobility identical to that of recombinant human NEP and an apparent molecular mass of approximately 95 kDa. Neuropeptides were degraded by membranes with specific activities in the order of [Leu5]enkephalin > [Met5]enkephalin > gastrin-releasing peptide-10 (GRP-10) > [D-Ala2][Leu5]enkephalin > somatostatin-14. Phosphoramidon and DL-thiorphan similarly inhibited the degradation of GRP-10 (mean of 35% inhibition), somatostatin-14 (57%) and the aminopeptidase-resistant analogue, [D-Ala2][Leu5]enkephalin (75%). When aminopeptidases were inhibited with amastatin (10 microM) phosphoramidon inhibited degradation of [Leu5]enkephalin (54%) and [Met5]enkephalin (100%). Phosphoramidon increased the potency of the contractile effects of neuropeptides on muscle cells by > 280-fold for somatostatin-14, 17-fold for GRP-10, 18-fold for [Met5]enkephalin and 14-fold for [Leu5]enkephalin. The results show that an NEP-like enzyme on the surface of gastric muscle cells degrades and inactivates enkephalins, GRP-10 and somatostatin-14 and acts in a manner analogous to that of acetylcholinesterase in the neuromuscular junction of skeletal muscle.

Identification of neuropeptide-degrading enzymes in the pancreas.

Neutral endopeptidase (NEP) and aminopeptidase M (APM) were identified in the pancreas by enzymatic assays and Western blotting. The NEP activity, assessed by the phosphoramidon- and DL-thiorphan-inhibitable degradation of glutaryl-Ala-Ala-Phe-4-methoxy-2-naphthylamine, was 28.8 pmol/h/micrograms of pancreatic membrane protein and 124 pmol/h/10(6) pancreatic acinar cells. The APM enzymatic activity, assessed by the actinonin- and amastatin-inhibitable degradation of Ala-4-methoxy-2-naphthylamine, was 633 pmol/h/micrograms pancreatic membrane protein and 17.4 nmol/h/10(6) pancreatic acinar cells. Proteins corresponding to NEP (95 kDa) and APM (140 kDa) were identified in membranes by Western blotting. Both NEP and APM on acinar cells may degrade neuropeptides and regulate their effects on exocrine secretion.

Inhibition of peptidases potentiates enkephalin-stimulated contraction of gastric muscle cells.

Cell surface peptidases degrade enkephalins and thereby restrict the number of molecules available to activate receptors. The effects of peptidase inhibitors on degradation of enkephalins and on enkephalin-stimulated contraction of gastric smooth muscle cells were examined. Muscle cells dispersed from the guinea pig stomach degraded [Tyr1-3H] [Leu5]enkephalin (41.6 +/- 9.0% degradation at 60 min incubation, mean +/- SD, n = 4 animals). Amastatin (10 microM, an aminopeptidase inhibitor) inhibited degradation by 72.1 +/- 1.5% The residual peptidase activity was inhibited by phosphoramidon (1 microM, an endopeptidase EC 3.4.24.11 inhibitor) by 58.0 +/- 11.0%. [Tyr1-125I] [Met5]enkephalin was similarly degraded. Phosphoramidon (1 microM) inhibited the degradation of the aminopeptidase-resistant peptide [Tyr1-3H] [D-Ala2]-[Leu5]enkephalin by greater than 95%. [Met5]enkephalin, incubated with cells for 30 s, stimulated contraction [50% maximal contraction (EC50) 120 +/- 50 nM, n = 6]. Pretreatment of cells with phosphoramidon alone, amastatin alone, or phosphoramidon plus amastatin, caused 20-fold (EC50 6.5 +/- 1.1 nM), 2-fold (EC50 63 +/- 23 nM), and 100-fold (EC50 1.1 +/- 0.3 nM) increase in potency of [Met5]enkephalin, respectively. The results show that endopeptidase EC 3.4.24.11 and aminopeptidases contribute to degradation of enkephalins by gastric muscle cells. The rapidity and magnitude of the potentiating effects of the inhibitors on enkephalin-stimulated contraction suggest a close physical relationship between the peptidases and the enkephalin receptors.

Down-regulation and recycling of high affinity cholecystokinin receptors on pancreatic acinar cells.

First incubating dispersed acini from rat pancreas with monensin, a cation ionophore that can inhibit recycling of receptors, inhibited binding of 125I-cholecystokinin 8 (125I-CCK-8) measured during a second incubation by as much as 50%. A maximal effect of monensin required 90 min of first incubation. Detectable inhibition of binding of 125I-CCK-8 occurred with 300 nM monensin, and inhibition increased progressively with concentrations of monensin up to 25 microM. Pancreatic acini possess two classes of receptors that bind 125I-CCK-8. One class has a high affinity (Kd = 461 pM) and a low capacity for CCK (512 fmol/mg DNA); the other class has a low affinity (Kd = 47 nM) and a high capacity for CCK (18 pmol/mg DNA). First incubating acini with monensin caused an 84% decrease in the number of high affinity CCK receptors with no change in the number of low affinity CCK receptors or the values of Kd for either class of receptors indicating that there is recycling of high affinity CCK receptors but not low affinity CCK receptors. First incubating acini with monensin did not alter CCK-stimulated amylase secretion indicating that in contrast to previous conclusions, occupation of low affinity CCK receptors mediates CCK-stimulated enzyme secretion. Moreover, the biphasic dose-response curve for CCK-stimulated enzyme secretion from monensin-treated acini suggests that pancreatic acini also possess a third, previously unrecognized class of very low affinity CCK receptors.

Properties of receptors for gastrin and CCK on gastric smooth muscle cells.

Previous studies have demonstrated that cholecystokinin (CCK), gastrin, and structurally related peptides can interact with various types of receptors that can be distinguished by their relative affinities for agonists and antagonists. In the present study we examined the effect of gastrin, the COOH-terminal octapeptide of CCK (CCK-8), and the tetrapeptide of CCK (CG-4) on contraction of dispersed gastric smooth muscle cells from guinea pig and tested the ability of various CCK receptor antagonists to affect agonist-induced muscle cell contraction. For purposes of comparison we tested the effect of each antagonist on CCK-stimulated amylase secretion by pancreatic acini from guinea pig. On gastric smooth muscle cells, CCK-8, gastrin, and CG-4 were all full agonists. CCK-8 and gastrin were equipotent and CG-4 was 6,000-fold less potent. Each antagonist caused inhibition of CCK-stimulated contraction with relative potencies (IC50): L364,718 (4 microM) = CBZ-CCK-(27-32)-NH2 (3 microM) greater than proglumide analogue 10 (90 microM). Inhibition by each of the antagonists was competitive in nature, specific for CCK peptides, and each had the same IC50 whether contraction was stimulated by CCK-8, gastrin, or CG-4. Relative potencies (IC50) of the three antagonists for inhibiting CCK-stimulated amylase release from pancreatic acini were L364,718 (3 nM) greater than proglumide analogue 10 (200 nM) greater than CBZ-CCK-(27-32)-NH2 (3 microM). These results demonstrate that gastric smooth muscle cells possess receptors that differ from CCK receptors on pancreatic acini in terms of affinities for both agonists and certain antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholecystokinin (CCK)-induced desensitization of pancreatic enzyme secretion is mediated by low affinity CCK receptors.

CCK-8-induced desensitization of carbachol-stimulated amylase secretion occurred over a range of concentrations of CCK-8 that occupy low affinity CCK receptors. CCK-JMV-180 [BOC-Tyr(SO3)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester] at concentrations up to 1 microM did not cause desensitization of enzyme secretion; however, the peptide was able to inhibit CCK-8-induced desensitization. Analysis of the relationship between receptor occupation and CCK-8-induced desensitization caused by CCK-8 and CCK-JMV-180 in combination also indicated that CCK-8-induced desensitization of carbachol-stimulated amylase secretion is caused by occupation of low affinity CCK receptors.

Cyclic GMP does not inhibit protein kinase C-mediated enzyme secretion in rat pancreatic acini.

In pancreatic acini, cGMP can be increased by secretagogues such as cholecystokinin (CCK), cholinergic agents, and bombesin, whose actions on enzyme secretion are believed to be mediated by protein kinase C. However, the role of cGMP in acinar cell function has been unclear. A recent paper by Rogers et al. (Rogers, J., Hughes, R.G., and Matthews, E. K. (1988) J. Biol. Chem. 263, 3713-3719) reported that two analogues of cGMP, N2,O2-dibutyl guanosine 3':5'-monophosphate (Bt2cGMP) and 8-bromoguanosine 3':5'-monophosphate (8Br-cGMP), at concentrations in the nanomolar range, inhibited the stimulation of amylase secretion caused by CCK-8, bethanechol, bombesin, and 12-O-tetradecanoylphorbol-13-acetate (TPA). Rogers et al. also reported that sodium nitroprusside inhibited the stimulation of enzyme secretion caused by CCK-8 or TPA. These authors concluded that cGMP inhibits protein kinase C-mediated secretion in pancreatic acini. In the present study we attempted to confirm the findings of Rogers et al., We found, however, that Bt2cGMP inhibited CCK-8-stimulated amylase release only at concentrations of the nucleotide above 10 microM. Moreover, there was a close correlation between the ability of Bt2cGMP to inhibit CCK-8-stimulated amylase release and its ability to inhibit binding of 125I-CCK-8. Bt2cGMP, at concentrations as high as 3 mM, did not alter the stimulation of amylase release caused by carbachol, bombesin, TPA, or A23187. 8Br-cGMP, at concentrations up to 1 mM, did not inhibit the stimulation of amylase release caused by CCK-8 or TPA. At concentrations above 0.1 mM, 8Br-cGMP augmented the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Sodium nitroprusside, at a concentration that causes a 60-fold increase in cGMP, did not inhibit the stimulation of amylase release caused by CCK-8, carbachol, bombesin, or TPA. Our results do not confirm the findings of Rogers et al. and indicate that cGMP does not inhibit protein kinase C-mediated secretion in pancreatic acini.

In vivo evaluation of cholesterol 7 alpha-hydroxylation in humans: effect of disease and drug treatment.

7 alpha-Hydroxylation of cholesterol is a stereospecific reaction consisting of the replacement of the 7 alpha-hydrogen with a hydroxyl group. When cholesterol labeled with tritium at the 7 alpha position is administered, the hydroxylation of the substrate will result in the loss of tritium which in turn will label the body water. The rate of tritium enrichment of the body water could thus give a quantitative estimate of the hydroxylation rate. This study describes the validation of the procedure with some 21 studies performed on 15 subjects in different conditions. [7 alpha-3H]cholesterol was administered intravenously in 50 ml of plasma and thereafter blood was sampled at timed intervals for 4 to 5 days. The rate of the hydroxylation of cholesterol was calculated from the time course of the specific activities of plasma cholesterol and body water after tracer administration and was expressed as 7 alpha-hydroxycholesterol formed/24 hr. Calculated values of hydroxylation in three control subjects (493 +/- 206), five patients with hyperlipoproteinemia (539 +/- 168), and seven cirrhotic patients (153 +/- 136) are in good agreement with figures reported for bile acid synthesis determined with other techniques. Cholesterol 7 alpha-hydroxylation rate is reduced in patients with cirrhosis, the impairment being related to the severity of the disease. Cholestyramine administered to one subject for 4 weeks produced a threefold increase of the hydroxylation. Administration of chenodeoxycholic acid resulted in a 50% decrease, whereas that of ursodeoxycholic did not produce consistent changes of the hydroxylation rate. The results support the current view that 7 alpha-hydroxylation of cholesterol is rate-limiting in the synthesis of bile acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ursocholic acid on bile lipid secretion and composition.

To further clarify the relationship between physical-chemical characteristics of bile acids and biliary lipid secretion, we investigated the effect of ursocholic acid, the 7 beta-hydroxyepimer of cholic acid, on bile lipid secretion and composition. The study included acute duodenal infusion (1 g/h for 5 h) of ursocholic acid contrasted with a less hydrophilic bile acid, ursodeoxycholic acid, in 3 T-tube patients and short-term oral administration (2 wk) of ursocholic acid (10-15 mg/kg X day) to 10 gallstone patients. Following acute infusion, ursocholic acid, similarly to ursodeoxycholic acid, accounted for greater than 80% of the biliary bile acids. However, ursocholic acid induced (per micromole of secreted bile acid) a significantly lower (p less than 0.01) secretion of cholesterol (0.013 mumol) and phospholipids (0.054 mumol) than that induced by ursodeoxycholic acid (0.034 mumol of cholesterol and 0.138 mumol of phospholipids). Biliary alkaline phosphatase activity during ursocholic acid administration was significantly lower (p less than 0.01) than during ursodeoxycholic acid administration. After short-term oral administration, ursocholic acid, undetectable before treatment, constituted 20.50% +/- 8.60% of the biliary bile acids. The percentage of deoxycholic acid increased from 32.35% +/- 18.79% to 47.53% +/- 16.19% (p less than 0.05). Mean saturation index decreased from a pretreatment value of 1.23 +/- 0.22 to 0.99 +/- 0.17 (p less than 0.05), but only in 4 of 10 subjects did bile become undersaturated. It is concluded that ursocholic acid, due to its higher hydrophilicity, stimulates a lower cholesterol and phospholipid output than ursodeoxycholic acid. Consequently, despite the low enrichment of the biliary bile acids with ursocholic acid, oral administration of ursocholic acid induces a reduction of bile cholesterol saturation.

Effects of acute changes of bile acid pool composition on biliary lipid secretion.

To elucidate the mechanism responsible for the bile acid-induced changes of biliary lipid secretion, we evaluated bile flow and biliary output of bile acids, cholesterol, phospholipids, and alkaline phosphatase activity in seven cholecystectomized subjects with a balloon occludable T-tube during two experimental periods: (a) depletion of the endogenous bile acid pool and (b) replacement of the pool by means of duodenal infusion with individual bile acids, such as deoxycholic (DCA), chenodeoxycholic (CDCA), cholic (CA), and ursodeoxycholic (UDCA) acids. Bile flow, cholesterol, and phospholipid output were linearly related to bile acid secretion in all experimental periods. During the replacement periods, the amount of cholesterol and phospholipids coupled to bile acids was significantly different (at 1% level at least) for each individual bile acid secreted; it was the highest during DCA secretion (slope value: 0.209 for cholesterol and 0.434 for phospholipids) followed, in the order, by CDCA (0.078 and 1.794), CA (0.044 and 0.127), and UDCA (0.030 and 0.122). The phospholipid to cholesterol ratio was higher during secretion of CA and UDCA as compared with DCA and CDCA. The secretion of CA seemed to stimulate a greater bile flow than the other bile acids did. The infusion of all bile acids, except UDCA, induced an increase of biliary alkaline phosphatase activity as compared with the values of the depletion period. The mean highest increase (13-fold the pretreatment value) was observed during DCA secretion followed by CDCA (fivefold) and CA (1.5-fold). These results would suggest that the physical chemical properties, namely the lipid-solubilizing capacity, of bile acids could directly contribute to the regulation of biliary lipid secretion. The observed changes in biliary alkaline phosphatase activity lend support to the view that bile acid-induced lipid secretion may be, at least in part, contributed by membrane solubilization.