Randomized, cross-over, controlled comparison of two inactivated hepatitis A vaccines.

The immunogenicity, tolerability and interchangeability of two hepatitis A vaccines, Vaqta (Merck and Co.) and Havrix (SmithKline) were studied in a randomized, crossover, controlled clinical trial. Vaccine was administered to 201 volunteers at 0 and 26 weeks in one of four vaccine regimens: Havrix-Havrix; Havrix-Vaqta; Vaqta-Havrix or Vaqta-Vaqta. Seroconversion rates (>/=10 mIU/ml) for those whose first dose was Vaqta or Havrix, respectively, were: 41/96 (43%) versus 30/95 (32%) (P=0.15) at 2 weeks and 91/98 (93%) versus 84/97 (87%) (P=0.43) at 4 weeks, and 100% at 26 weeks. Geometric mean concentrations (GMC) of total antibody to hepatitis A virus (anti-HAV) for Vaqta and Havrix were 189 and 114 mIU/ml (P=0.011) at 4 weeks and 234 and 136 mIU/ml (P<0.001) at 26 weeks. At 30 weeks, the GMC after two doses of Havrix was 2612 mIU/ml compared with 5497 after two doses of Vaqta (P<0.001). The GMC in the Havrix-Vaqta group was 5672 mIU/ml compared with 3077 mIU/ml in the Vaqta-Havrix group (P<0.001). Less than half of vaccine recipients reported tenderness or pain. In this study, seroconversion rates of the two vaccines were similar. Vaqta produces significantly higher anti-HAV antibody than Havrix. Crossover immunization is well tolerated and results in high antibody concentrations, especially when Vaqta is the booster dose. The significance of higher anti-HAV antibody concentrations in terms of long-term protection is unknown.

Permanent tracheostomy in standing horses: technique and results.

Permanent tracheal stomas were created in seven sedated, standing horses with severe upper airway obstruction. After local anesthesia, a 3-cm by 6-cm rectangle of skin was removed from the ventral surface of the neck, 3 cm distal to the cricoid cartilage. The sternothyrohyoideus muscles were clamped proximally and distally, then transected to expose the tracheal rings. The ventral third of four tracheal rings was dissected from the tracheal mucosa that was then incised in a double "Y." Two layers of suture were used to achieve mucocutaneous closure. Stomas healed without serious complications; two mares subsequently foaled, and three horses were used for riding.

Treatment of olecranon bursitis in horses: 10 cases (1986-1993).

Medical records of 10 horses with olecranon bursitis were reviewed to examine treatments, evaluate a technique for en bloc resection of the bursa in standing horses, and determine outcome of the horses after treatment. Before admission, 6 horses had been treated by needle aspiration of fluid from the mass, followed by injection of corticosteroids. Subsequent treatment for 2 of these 6 horses included open drainage and packing of the cavity with gauze soaked in 7% iodine solution. None resolved after these treatments. After admission to the hospital, 5 horses were treated medically and 5 were treated by en bloc resection of the bursa. One horse that had received intralesional injection of a radionuclide was lost to follow-up evaluation. One horse treated conservatively by open drainage and packing and 1 treated by injection of a radionuclide had resolution of the olecranon bursitis. Only 1 of these 2 horses had a cosmetic result. The acquired bursae decreased in size in 2 horses (1 treated with a corticosteroid and 1 with orgotein), but were still visible 7 and 46 months after treatment, respectively. The surgery site of 4 horses that were treated by en bloc resection healed by primary intention, and the owners of these horses were pleased with the cosmetic results. The suture line of 1 horse dehisced 5 days after surgery. Proliferative granulation tissue was removed on 2 occasions, and the site healed by second intention after 2 months. A small knot and some white hair remained at the surgery site.

Arthroscopic approach and intra-articular anatomy of the plantar pouch of the equine tarsocrural joint.

Arthroscopic examination of structures within the plantar pouch of the tarsocrural joint was accomplished via portals in both the plantaromedial and plantarolateral aspects of the joint. Flexion and extension of the tarsus while examining the joint through either portal allowed observation of the proximal and plantar aspects of the lateral and medial trochlear ridges, the trochlear groove, the caudal aspect of the distal tibia, and the deep digital flexor tendon (DDFT) in its sheath. From a plantarolateral portal, the plantar talocalcaneal ligament and the plantar aspect of the lateral malleolus could be observed. The caudal aspect of the medial malleolus could not be observed with flexion or extension of the joint from a plantaromedial portal, but in some horses, the caudal aspect of the lateral malleolus could be observed. The dorsolateral and dorsomedial aspects of the plantar pouch were best examined from a portal on the ipsilateral side of the joint. An instrument portal opposite either arthroscope portal allowed access to most regions of the joint except the abaxial surface of the trochlear ridge opposite the instrument.

An in vitro model of ovarian epithelial carcinogenesis: changes in cell-cell communication and adhesion occurring during neoplastic progression.

To investigate the cellular mechanisms of ovarian epithelial carcinogenesis, a series of progressively transformed rat ovarian surface epithelial (ROSE) cell lines were developed and studied. Transfection of primary ROSE cells and an immortalized ROSE line (ROSE 199) with the pSV3neo plasmid (SV40 T-antigen) yielded transformed lines which retained epithelial morphology. In vivo selection of these pSV3neo cell populations resulted in further phenotypic transformation. Transfection of ROSE 199 with pSV2neo/c-H-rasEJ (rasEJp21) resulted in a malignant line which appeared fibroblast-like and formed invasive sarcomas both in athymic mice and in immunocompetent rats. Gap junctional intercellular communication (GJIC) and cell-cell adhesion were studied in this series of ROSE lines. Both c-H-rasEJ-transformation and in vivo selection resulted in a significant reduction of GJIC between adjoining cells and a transition of in vitro migration as continuous epithelial sheets to the dissociation of individual cells. This apparent shift in cell adhesiveness was associated with reduced expression of the E-cadherin adhesion molecule. Our data suggest that neoplastic progression of the ovarian surface epithelium may be associated with concomitant reductions in GJIC, E-cadherin expression and functional adhesiveness.

Reduction of pulmonary surfactant in patients with human immunodeficiency virus infection and Pneumocystis carinii pneumonia.

We assessed qualitative and quantitative differences in surfactant lipid composition of bronchoalveolar lavage (BAL) fluid in patients with acquired immune deficiency syndrome (AIDS) and Pneumocystis carinii (PC) pneumonia. Five normal volunteers and 27 patients with human immunodeficiency virus (HIV) infection underwent BAL for evaluation of possible pulmonary infection. Bronchoalveolar lavage studies in eight patients were negative for PC organisms, and 19 were positive. Pneumocystis carinii pneumonia was graded (mild vs moderate to severe) by initial alveolar-arterial oxygen gradient. Bronchoalveolar lavage fluid was centrifuged, the lipids were extracted from the supernatant, and total lipid profiles of dephosphorylated glycerolipids were analyzed as trimethylsilylether derivatives by high temperature gas-liquid chromatography. Phospholipase A2 levels were determined using a radiolabeled E coli membrane method. Compared to the normal volunteers (109 +/- 13 micrograms/5 ml) and the PC negative group (107 +/- 13 micrograms/5 ml), total BAL lipid was reduced for both the mild PC pneumonia group (73 +/- 10 micrograms/5 ml) and the moderate to severe PC pneumonia group (46 +/- 4 micrograms/5 ml). There was a parallel reduction of diacylglycerol lipids: normal volunteers, 52 +/- 7 micrograms/5 ml; PC negative, 52 +/- 9 micrograms/5 ml; mild PC pneumonia, 35 +/- 7 micrograms/5 ml; and moderate to severe PC pneumonia, 15 +/- 2 micrograms/5 ml. Phospholipase A2 activity in moderate to severe PC pneumonia was twice that of the PC negative patients, and 30 times that for normals. The data demonstrate a marked diminution in surfactant glycerophospholipid in patients with AIDS and PC pneumonia and suggest a potential role for surfactant abnormality in the pathophysiology of this disease.

Hyperphospholipasemia A2 in human volunteers challenged with intravenous endotoxin.

Phospholipase A2 (PLA2) activity was measured in the serum of 23 individuals infused intravenously with endotoxin (EN) at a dose of 4 ng/kg body weight. A marked increase in PLA2 was noted 3 h after EN challenge (mean 828 +/- 513 units/ml), reached its maximum at 24 h after the challenge (mean 2667 +/- 2442 units/ml), and was still evident at 48 h (mean 763 +/- 366 units/ml). In contrast, TNF levels were maximal (mean 712 +/- 375 pg/ml) 90 min after the EN challenge and subsided to very low values (5 +/- 5 pg/ml) 5 h after the challenge. There was a positive correlation between the maximum response of TNF and that of PLA2 (r = 0.82, P < 0.01). Administration of ibuprofen or pentoxifylline did not alter the PLA2 response. EN challenge did not affect serum pancreatic PLA2 concentration or that of the lysosomal cationic enzyme, lysozyme. Neutralizing antibody against human recombinant (synovial type) PLA2 completely abolished PLA2 activity in the sera tested. We conclude that EN infusions cause marked intravascular release of nonpancreatic secretory PLA2 and that the magnitude of this response seems to be related to the prior generation of TNF.

An examination of the stereochemical course of acylation of 2-monoacylglycerols by rat intestinal villus cells using [2H3]palmitic acid.

The stereochemical course of acylation of 2-monoacylglycerols by rat intestinal mucosa was investigated using isolated villus cells with 2-lauroylglycerol and [2H3]palmitic acid as substrates. The newly synthesized X-1,2-diacylglycerols and triacylglycerols were recognized on the basis of the content, positional distribution and molecular association of the fatty acids by gas-liquid chromatography/mass spectrometry in combination with sterospecific analysis. It was found that the free X-1,2-[2H3]palmitoyllauroylglycerols contained 74% sn-1,2- and 26% sn-2,3-enantiomers, which were utilized for triacylglycerol formation in the same proportion. Some of the 2-monolauroylglycerol became hydrolyzed and the released lauric acid was utilized along with [2H3]palmitic acid for diacylglycerol and triacylglycerol formation via both the phosphatidic acid and the 2-monoacylglycerol pathway. The contribution of the phosphatidic acid pathway (12-16%) was determined by estimating the relative proportion of the newly synthesized triacylglycerols containing [2H3]palmitic acid in the sn-2 position. After correcting for the contribution of the phosphatidic acid pathway, the sn-1,2-/sn-2,3-enantiomer ratio in the X-1,2-[2H3]palmitoyllauroylglycerol was estimated to be 71/29. These results are consistent with those previously obtained with glycerol-labelled 2-monoacylglycerols and intact tissue or homogenates of rat intestinal mucosa. It is suggested that the 2-mono-acylglycerol transacylase, if a single enzyme, possesses a low stereospecificity, or that two enzymes exist of unequal concentration and/or activity.

Relative acylglycerol acyltransferase activities in homogenates of enzymically dispersed rat jejunal villus and crypt cells.

The relative acyltransferase activities were compared in homogenates of rat jejunal villus and crypt cells isolated by differential scraping and hyaluronidase dispersion. The contributions of the monoacylglycerol and phosphatidic acid pathways to the higher acylglycerol and phospholipid biosynthesis were assessed using 2-oleoyl-sn-[3H] glycerol and [I-14C] palmitic acid as tracers. The stereochemical course of the diacylglycerol biosynthesis was determined by stereospecific analysis. Using 2-oleoyl-sn-glycerol as a tracer, the villus cells exhibited for times higher diacylglycerol and 19 times higher triacylglycerol biosynthesis than crypt cells on an equivalent protein basis. Furthermore, while the villus cell homogenates yielded a preponderance (75%) of the 1, 2-diacyl-sn-glycerols, the crypt cell homogenates formed essentially racemic proportions of 1, 2- and 2,3-diacyl-sn-glycerols. Both villus and crypt cell homogenates exhibited comparable acyl acceptor and acyl donor concentration dependence and the same cofactor requirements. It is unlikely that these acyltransferase activities in the crypt cell preparation are due to contamination with the villus cells, because then more comparable proportions of the enantiomeric diacylglycerols and triacylglycerols would have been anticipated. It is concluded that the crypt cells possess intrinsic monoacylglycerol and to a much lesser extent diacylglycerol acyltransferase activities, which are acquired prior to the development of a distinct brush border and which probably do not require dietary stimulus for induction.

Synthesis and release of lipids and lipoproteins by isolated rat jejunal enterocytes in the presence of sodium taurocholate.

Isolated rat jejunal villus and crypt cells prepared by differential scraping and hyaluronidase dispersion were used in the presence of 8 mM sodium taurocholate to study the incorporation of sn-[3H]glycerol-2-monooleate, [1-14C]palmitate, [1-14C]acetate, L-[4,5(n)-3H]leucine and D-[1-14C]glucosamine into cellular and medium lipids and proteins, respectively. The villus cells were capable of an apparently normal biosynthesis of triacylglycerols and phospholipids, as well as of proteins and glycoproteins despite an altered dye permeability and increased release of cytosolic and membrane enzymes. About 20-30% of the newly formed triacylglycerols and about 35% of the newly formed phospholipids were secreted into the medium and were recovered as triacylglycerol-rich particles. Labelled proteins and glycoproteins were also recovered from this fraction. The crypt cells synthesized about one-half as much triacylglycerol and phospholipid as did the villus cells, but secreted little or no labelled lipid into the postincubation medium. The release into the medium of triacylglycerols synthesized by the villus cells was blocked by a pretreatment of the isolated cells with the microtubule disruptors, nocodazole, colchicine and colcemid; by the amino sugar, D-galactosamine; by the inhibitors of protein synthesis, puromycin and cycloheximide, and by the inhibitor of the biosynthesis of phosphatidylcholine, chlorocholine. These results indicate that the secretion of labelled lipids, proteins and glycoproteins by the upper villus enterocytes in the presence of sodium taurocholate is not entirely due to cell breakage and spillage of contents. It is concluded that incubations of isolated villus cells of rat jejunum with mixed micellar solutions containing 8 mM taurocholate are compatible with an apparently normal biosynthesis and secretion of triacylglycerol-rich particles.

Culture of presumptive epithelial cells from jejunal mucosa of axenic rats.

Strains of presumptive epithelial and fibroblast cells were prepared from the jejunal mucosa of axenic rats. Cells were cultured on collagen gels, in highly enriched media supplemented with homologous sera and hormones, and were maintained for more than 7 weeks.

Lipid absorption and metabolism.

Metabolic processes occurring within the mucosal cell are critical in determining results of interactions between environmental agents and the alimentary tract. The absorption, metabolism, and transport of lipids affects most those agents which are lipid soluble. The understanding of the process involved in lipid absorption and transport is therefore important for both appreciation of the mechanism of uptake of these toxins and for an effective interference with it. Most of the detailed mechanisms of lipid absorption and transport have been proposed from in vitro studies with soluble cell-free systems. The present review integrates these results with recent in vivo and in vitro findings with intact animal tissues and isolated mucosal cells. While there is much general agreement occasional startling differences are also observed, which may have a bearing on the mechanism of normal fat absorption and on the understanding of the transport of the fat-soluble toxins across the mucosal villus cell.

End product specificity of triacylglycerol lipases from intestine, fat body, muscle and haemolymph of the American cockroach, Periplaneta americana L.

The triacylglycerol-hydrolyzing capacity of tissue homogenates has been investigate for midgut, fat body, thoracic musculature and haemolymph of the American cockroach, Periplaneta americana. The greatest lipolytic activity was demonstrated in midgut homogenates with decreasing levels of activity present in fat body, muscle and haemolymph. Comparison of the lipolytic products resulting from triacylglycerol hydrolysis indicates that midgut homogenates effect the production of sn-2-monoacylglycerols and free fatty acids, whereas the other tissues that were examined favor the accumulation of diacylglycerols. Stereospecific analysis of the diacylglycerol products of triacylglycerol hydrolysis demonstrated that the lipolytic activities of midgut and muscle homogenates result in the production of a racemic mixture of the sn-1,2- and sn-2,3-enantiomers, but the fat body and haemolymph show a preference for the accumulation of the sn-1,2-isomer.

Improved isolation of villus and crypt cells from rat small intestinal mucosa.

An enzymic method is described which allows the isolation under comparable conditions of crypt and villus cells from rat jejunum with normal morphologic appearance and high metabolic activity when compared with previous preparations. The method is based on a differential scraping of short lengths of everted small intestine to yield two villus cell fractions and a gut wall residue. The scrapings and the gut tube are incubated for the same length of time in a HEPES-buffered modified Hanks' balanced salt solution containing hyaluronidase, DNase, and soybean trypsin inhibitor. The cells of the crypt region are recovered by a further scraping of the digested gut wall. Cells from all fractions are dispersed by gentle agitation, washed, and harvested by centrifugation. The final crypt and villus cells are 95--99% viable by dye exclusion and exhibit 5--20% cross-contamination on the basis of differential marker enzymes. The isolated crypt and villus cells prepared by the new procedure are suitable for comparative studies of metabolic activity in the absence of chelation-induced structural and metabolic abnormalities.

Comparative determination of plasma cholesterol and triacylglycerol levels by automated gas--liquid chromatographic and autoanalyzer methods.

Plasma samples obtained during a prevalence study of hyperlipemia in a free living urban population were analyzed for total cholesterol and triacylglycerol content by automated high-temperature gas--liquid chromatographic (GLC) and automated colorimetric (Auto-Analyzer, AA11) methods. The analyses were done over a three-year period. The methods gave excellent overall correlation for both total cholesterol (r = 0.9811) and total triacylglycerols (r = 0.9739). Detailed comparisons of the results obtained by the two methods with natural samples over the entire concentration range, indicated that the GLC method gave cholesterol values 5--10 mg% lower and triacylglycerol values 10--20 mg% lower than the corresponding AA11 determinations. The differences between the two methods are attributed to an overestimation of the cholesterol and triacylglycerol levels by the AA11 method due to presence of variable amounts of interfering chromogens in the plasma extracts. The between-method relative error ranged from 3 to 5% for cholesterol and from 5 to 10% for triacylglycerols. The within-day standard deviation of GLC averaged 2.3 mg% for cholesterol and 3.5 mg% for triacylglycerols. The between-day standard deviation of the GLC method averaged about 6 mg% for both cholesterol and triacylglycerols. The within-day, within GLC, relative error averaged 1.12% for cholesterol and 2.66% for triacylglycerols. The apparent high precision and high accuracy of the GLC method recommend it as an alternative to the indirect methods of plasma cholesterol and triacylglycerol analysis, especially where a smaller throughput of samples is not a limitation and where both total amount and composition of the lipids is of interest.

Preferential in vivo accumulation of sn-2,3-diacylglycerols in postheparin plasma of rats.

The stereochemical course of in vivo hydrolysis of triacylglycerols by lipoprotein lipase was investigated by determining the structure of diacylglycerol intermediates in postheparin plasma of rats which had been fed [3H]glycerol-labeled Intralipid 2 h before an injection of heparin or had been given an injection of a mixture of [3H]glycerol-Intralipid and heparin. During the clearance of both the natural chylomicrons and the artificial emulsion, sn-2,3-diacylglycerols (60-80%) were found to be the dominant enantiomers. Similar results were obtained when the contribution of the hepatic lipase was altered, either by tying off the mesentery artery and portal vein before injection of heparin, or by injection of heparin directly into the portal vein. These findings are consistent with a preferential release of the acyl group from the sn-1 position of the triacylglycerol molecule as demonstrated previously in vitro. A preferential orientation of the substrate in the enzyme-substrate complex or at the oil-water interface is discussed as a possible basis for these effects.