The expression and localization of plasma platelet-activating factor acetylhydrolase in endotoxemic rats.

The production of platelet-activating factor (PAF) and PAF-like phospholipids that also bind the PAF receptor are implicated in numerous pathological situations including bacterial endotoxemia and injury-induced oxidative damage. PAF and PAF-like phospholipids are hydrolyzed and inactivated by the enzyme PAF acetylhydrolase. In the intact rat, infusion of lipopolysaccharide (LPS) into a mesenteric vein served as an acute, liver-focused model of endotoxemia. We determined that the liver responds to LPS exposure with the production of plasma-type PAF acetylhydrolase mRNA and protein expression specifically in the resident macrophages of the liver. Liver macrophages, defined immunohistochemically using antibodies against ED1, present in livers from saline-treated animals contained no detectable PAF acetylhydrolase. Twenty-four hours following in vivo LPS administration, immunohistochemistry detected a slight increase in the number of ED1 staining cells and the ED1-positive cells now contained an abundance of PAF acetylhydrolase. The systemic administration of LPS resulted in increased expression of PAF acetylhydrolase in several tissues. Of the tissues examined, the greatest increase in PAF acetylhydrolase expression was observed in lung followed by increases in spleen, liver, kidney, and thymus. Additionally, the expression of PAF acetylhydrolase mRNA increased in circulating leukocytes and in peritoneal macrophages in response to systemic exposure to LPS. We examined the regulation of PAF acetylhydrolase expression and demonstrated the administration of the PAF receptor antagonists, BN 50739 and WEB 2170, inhibited by 50% the increase in PAF acetylhydrolase expression in response to LPS. The up-regulation of the plasma-type PAF acetylhydrolase expression constitutes an important mechanism for elevating the local and systemic ability to inactivate PAF and oxidized phospholipids in order to minimize PAF-mediated pathophysiology consequent from exposure to endotoxin. The abundance of PAF acetylhydrolase production in the liver lobule likely limits endotoxin-mediated tissue damage due to PAF synthesis.

Outbreak of Pseudomonas aeruginosa ventriculitis among patients in a neurosurgical intensive care unit.

OBJECTIVE: To determine the cause of an outbreak of Pseudomonas aeruginosa cerebral ventriculitis among eight patients at a community hospital neurosurgical intensive care unit. All had percutaneous external ventricular catheters (EVCs) to monitor cerebrospinal fluid (CSF) pressure. METHODS: Cohort study of all patients who had EVCs placed during the epidemic period (August 8-October 22, 1997). A case-patient was any patient with P aeruginosa ventriculitis during the epidemic period. Pulsed-field gel electrophoresis (PFGE) was performed on all isolates. RESULTS: P aeruginosa was significantly more likely to be isolated from CSF per EVC placed in the epidemic than pre-epidemic (January 1-August 7, 1997) periods (8/61 [13%] vs 2/131 [1.5%], P=.002). During the epidemic period, ventriculitis was significantly more likely after EVC placement in the operating room than in other units (8/24 vs 0/22, P=.004). EVC placement technique differed for EVCs placed in the operating room (little hair was removed, preventing application of an occlusive dressing) versus other hospital units (more hair was removed, and an occlusive dressing was applied). Among patients who had operating room EVC placement, contact with one healthcare worker was statistically significant (7/13 vs 0/8, P=.02). Hand cultures of this worker were negative. All isolates had closely related PFGE patterns. CONCLUSIONS: These data suggest that a single healthcare worker may have contaminated EVC insertion sites, resulting in an outbreak of P aeruginosa ventriculitis. Affected patients were unlikely to have had an occlusive dressing at the EVC insertion site. Application of a sterile occlusive dressing may decrease the risk of ventriculitis in patients with EVCs.

Incidence and types of adverse events and negligent care in Utah and Colorado.

BACKGROUND: The ongoing debate on the incidence and types of iatrogenic injuries in American hospitals has been informed primarily by the Harvard Medical Practice Study, which analyzed hospitalizations in New York in 1984. The generalizability of these findings is unknown and has been questioned by other studies. OBJECTIVE: We used methods similar to the Harvard Medical Practice Study to estimate the incidence and types of adverse events and negligent adverse events in Utah and Colorado in 1992. DESIGN AND SUBJECTS: We selected a representative sample of hospitals from Utah and Colorado and then randomly sampled 15,000 nonpsychiatric 1992 discharges. Each record was screened by a trained nurse-reviewer for 1 of 18 criteria associated with adverse events. If > or =1 criteria were present, the record was reviewed by a trained physician to determine whether an adverse event or negligent adverse event occurred and to classify the type of adverse event. MEASURES: The measures were adverse events and negligent adverse events. RESULTS: Adverse events occurred in 2.9+/-0.2% (mean+/-SD) of hospitalizations in each state. In Utah, 32.6+/-4% of adverse events were due to negligence; in Colorado, 27.4+/-2.4%. Death occurred in 6.6+/-1.2% of adverse events and 8.8+/-2.5% of negligent adverse events. Operative adverse events comprised 44.9% of all adverse events; 16.9% were negligent, and 16.6% resulted in permanent disability. Adverse drug events were the leading cause of nonoperative adverse events (19.3% of all adverse events; 35.1% were negligent, and 9.7% caused permanent disability). Most adverse events were attributed to surgeons (46.1%, 22.3% negligent) and internists (23.2%, 44.9% negligent). CONCLUSIONS: The incidence and types of adverse events in Utah and Colorado in 1992 were similar to those in New York State in 1984. Iatrogenic injury continues to be a significant public health problem. Improving systems of surgical care and drug delivery could substantially reduce the burden of iatrogenic injury.

Costs of medical injuries in Utah and Colorado.

Patient injuries are thought to have a substantial financial impact on the health care system, but recent studies have been limited to estimating the costs of adverse drug events in teaching hospitals. This analysis estimated the costs of all types of patient injuries from a representative sample of hospitals in Utah and Colorado. We detected 459 adverse events (of which 265 were preventable) by reviewing the medical records of 14,732 randomly selected 1992 discharges from 28 hospitals. The total costs (all results are discounted 1996 dollars) were $661,889,000 for adverse events, and $308,382,000 for preventable adverse events. Health care costs totaled $348,081,000 for all adverse events and $159,245,000 for the preventable adverse events. Fifty-seven percent of the adverse event health care costs, and 46% of the preventable adverse event costs were attributed to outpatient medical care. Surgical complications, adverse drug events, and delayed or incorrect diagnoses and therapies were the most expensive types of adverse events. The costs of adverse events were similar to the national costs of caring for people with HIV/AIDS, and totaled 4.8% of per capita health care expenditures in these states.

Secretory PAF-acetylhydrolase of the rat hepatobiliary system: characterization and partial purification.

Hepatocytes and Kupffer cells in primary culture both secrete plasma-type platelet-activating factor-acetylhydrolase (pPAF-AH) into serum-free culture medium. The rate of secretion of pPAF-AH by Kupffer cells was 20 to 25 times higher than from hepatocytes, and Kupffer cells expressed a higher level of pPAF-AH mRNA than did hepatocytes. Purified liver cell-secreted pPAF-AH exhibited a major protein band of 65-67 kDa on SDS-PAGE; this was the band predominantly labeled when the enzyme catalytic center was reacted with [3H]diisopropylfluorophosphate ([3H]DFP). Rat bile collected from cannulated bile ducts contained significant PAF-AH activity, and bile samples possessed a prominent band at 30-32 kDa, which was the exclusive target for [3H]DFP. Experiments using tunicamycin, an inhibitor of N-linked glycosylation, and endoglycosidase H suggested that pPAF-AH secreted constitutively by cultured hepatocytes and Kupffer cells is glycosylated. The present study supports the notion that hepatic secretion of pPAF-AH into the blood contributes to the regulation of PAF and oxidized phospholipid levels in the circulation, whereas secretion of PAF-AH into the bile may allow hepatic control of these phospholipid signaling molecules in the gastrointestinal tract.

Cell-specific regulation of expression of plasma-type platelet-activating factor acetylhydrolase in the liver.

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that causes hypotension, increases vascular permeability, and has been implicated in anaphylaxis, septic shock and several other inflammatory responses. PAF is hydrolyzed and inactivated by the enzyme PAF-acetylhydrolase. In the intact rat, a mesenteric vein infusion of lipopolysaccharide (LPS) served as an acute, liver-focused model of endotoxemia. Plasma PAF-acetylhydrolase activity increased 2-fold by 24 h following LPS administration. Ribonuclease protection experiments demonstrated very low levels of plasma-type PAF-acetylhydrolase mRNA transcripts in the livers of saline-infused rats; however, 24 h following LPS exposure, a 20-fold induction of PAF-acetylhydrolase mRNA was detected. In cells isolated from endotoxin-exposed rat livers, Northern blot analyses demonstrated that Kupffer cells but not hepatocytes or endothelial cells were responsible for the increased PAF-acetylhydrolase mRNA levels. In Kupffer cells, plasma-type PAF-acetylhydrolase mRNA was induced by 12 h, peaked at 24 h, and remained substantially elevated at 48 h. Induction of neutropenia prior to LPS administration had no effect on the increase in PAF-acetylhydrolase mRNA seen at 24 h. Although freshly isolated Kupffer cells contain barely detectable levels of plasma-type PAF-acetylhydrolase mRNA, when Kupffer cells were established in culture, PAF-acetylhydrolase expression became constitutively activated concomitant with cell adherence to the culture plates. Alterations in plasma-type PAF-acetylhydrolase expression may constitute an important mechanism for elevating plasma PAF-acetylhydrolase levels and an important component in minimizing PAF-mediated pathophysiology in livers exposed to endotoxemia.

Improved manufacture and application of an agarose magnetizable solid-phase support.

A simple, semiautomated, nonhazardous procedure for the production of a magnetizable solid-phase support (MSPS) has been developed based on the extrusion of molten agarose-iron oxide mixtures, which enables manufacture of a range of differently sized spherical agarose-iron oxide beads. This system has enabled scale-up of an original manufacture procedure and reproducible preparation of kg quantities of MSPS suitable for biomolecular purifications. An improved protocol for the isolation of plasmid DNA directly from cell lysates using this MSPS, derivatized with diethylaminoethyl (DEAE) groups, is reported. This involves a modified alkaline lysis, followed by adsorption to and elution from the support, yielding plasmid DNA of a purity comparable with, or better than, other methods of plasmid isolation. Using the same procedure, plasmid DNA can be isolated from bacterial cell culture volumes of 1.5 mL and 100 mL with equal efficiency and purity.

Endothelin-1 production by hepatic endothelial cells: characterization and augmentation by endotoxin exposure.

Activation of endothelin (ET) receptors in the liver causes vasoconstriction, glucose production, and lipid and peptide mediator synthesis. In the intact rat, a bolus infusion of endotoxin into a mesenteric vein served as an acute exposure model of endotoxemia. In response to this challenge, a ninefold increase in hepatic ET-1 mRNA occurred within 3 h. The plasma level of immunoreactive ET-1 (irET-1) increased correspondingly by 8.5-fold within 6 h. ET-1 mRNA levels in liver endothelial cells (EC) isolated from livers of endotoxin-treated rats at various times after endotoxin challenge showed a more gradual increase. Northern blot analyses of the major liver cell types demonstrated that ET-1 mRNA was most abundant in the EC. The present results document a significant increase in the circulating level of irET-1 during episodes of endotoxemia. The increased hepatic ET-1 production in response to endotoxin infusion suggests that ET-1 produced in the liver could make a significant contribution to the plasma irET-1 and may be an important component in the hepatic responses to systemic trauma.

Platelet-activating factor augments lipopolysaccharide-induced nitric oxide formation by rat Kupffer cells.

Acute endotoxic shock is accompanied by an increase in the production of nitric oxide (NO) by several different hepatic cell types. Platelet-activating factor (PAF) is a potent proinflammatory mediator with many pathophysiological actions and, in fact, elevated plasma and tissue levels of PAF are observed in animal models of endotoxic shock. The current study demonstrates that PAF induced nitrite formation, the end product of nitric oxide synthesis, by Kupffer cells in a dose- and time-dependent manner. Moreover, PAF was seen to initiate NO synthase gene expression and protein synthesis. PAF augmented lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase messenger RNA (mRNA), protein, nitrite and cyclic guanosine monophosphate (cGMP) levels in Kupffer cells. Treatment of Kupffer cells with actinomycin D or cycloheximide inhibited PAF- and LPS-stimulated nitrite and nitric oxide synthase protein formation confirming that de novo synthesis of the enzyme occurred. In Kupffer cells, the presence of an arginine analog, NG-methyl-L-arginine, attenuated nitrite formation induced by PAF and LPS alone or in combination. L-arginine is the principal substrate for nitric oxide synthase. PAF and LPS individually and in combination induced a time-dependent uptake of L-[3H]-arginine into the Kupffer cell, and this response was sensitive to cycloheximide. The current study indicates that exogenous PAF contributes to the induction of nitric oxide synthase by LPS in cultured rat Kupffer cells.

Interaction of platelet-activating factor with rat hepatocytes: uptake, translocation, metabolism, and effects on PAF-acetylhydrolase secretion and protein tyrosine phosphorylation.

In the present study, the interaction of the phospholipid mediator platelet-activating factor (PAF) with rat hepatocytes in primary culture was examined. Following exposure to hepatocytes, exogenous [3H]alkyl-PAF was metabolized rapidly to [3H]lyso-PAF, the content of which was raised in the outer leaflet of the plasma membrane within the initial 5 min of incubation. Thereafter [3H]lyso-PAF was translocated into cells with concomitant reacylation to [3H]alkyl-acyl-glycerophosphocholine. A portion of untransformed [3H]PAF accumulated in the outer leaflet, and only a small amount of the [3H]PAF was translocated into the inner leaflet of the plasma membrane. Detectable levels of [3H]lyso-PAF were found in the medium of hepatocyte cultures at all times of incubation. These findings suggest that at least a portion of the cellular PAF-acetylhydrolase (PAF-AH) activity is located in the outer leaflet of the plasma membrane and can be secreted into the medium. Indeed, rat hepatocytes in culture released PAF-AH into the medium in a time-dependent fashion, Incubation of hepatocytes with exogenous PAF increased secretion of PAF-AH, whereas lyso-PAF and the nonhydrolyzable analog methylcarbamyl-PAF significantly reduced secretion. The structurally related PAF receptor antagonist CV 3988 markedly inhibited the activity of PAF-AH and also diminished its release by hepatocytes. In contrast, BN 50739 amd WEB 2170, thienotriazolodiazepine PAF receptor antagonists, did not affect the PAF-AH activity, but increased its secretion by the cells. A full-length 3.8-kb mRNA encoding the cell surface PAF receptor was absent in hepatocytes as indicated by Northern blot analysis using the rat PAF receptor cDNA, whereas PAF receptor mRNA was readily detected in Kupffer cells. Upon incubation with hepatocytes, PAF induced tyrosine phosphorylation of proteins with molecular masses of 120-130 and 160-180 kDa and dephosphorylation of 80-90-kDa proteins; these responses were not inhibited by WEB 2170 and BN 50739. The protein tyrosine kinase inhibitor genistein abolished the release of free arachidonic acid, suggesting a crucial role for tyrosine phosphorylation in PAF-induced phospholipase A2 activation in rat hepatocytes. Taken together, our data indicate that the interaction of PAF with rat hepatocytes is dependent upon its metabolism, involves protein tyrosine phosphorylation/dephosphorylation and arachidonic acid release, and does not involve the heteromeric G-protein-coupled PAF receptor which has been characterized in Kupffer cells. This metabolically regulated mechanism for PAF action on hepatocytes may be of potential biological importance in the liver under normal and pathological conditions.

Metacyclogenesis of Leishmania (Viannia) braziliensis in vitro: evidence that lentil lectin is a marker of complement resistance and enhanced infectivity.

Axenic culture in modified Grace's medium was used to induce metacyclogenesis of Leishmania (Viannia) braziliensis in vitro. Morphological characteristics, lectin agglutination profiles, susceptibility to complement lysis, and infectivity in vivo were compared between metacyclic promastigotes and promastigotes in mid-log phase growth. Short, arrow-like promastigotes and round, oval promastigotes were defined as putative metacyclic forms on the basis of being highly motile and free swimming, with a small cell body and long flagellum. These forms increased during metacyclogenesis to > 80% whereas long-bodied, slender promastigotes and intermediate slender promastigotes declined progressively. Lentil lectin selectively agglutinated L. braziliensis after the induction of metacyclogenesis, whereas concanavalin A, wheat germ agglutinin and peanut agglutinin similarly agglutinated metacyclic promastigotes and mid-log phase promastigotes. Metacyclic promastigotes survived in 7.5%-20% human serum whereas mid-log phase promastigotes did not. Five hundred metacyclic promastigotes were highly infective to hamsters whereas 500 mid-log phase promastigotes rarely caused any lesion. Specific agglutination by lentil lectin should allow purification of metacyclic organisms for standardization of immunoprotection and challenge experiments.