Evaluation of unfractionated heparin versus low-molecular-weight heparin and fondaparinux for pharmacologic venous thromboembolic prophylaxis in critically ill patients with cancer.

Essentials Critically ill cancer patients require pharmacologic prophylaxis for venous thromboembolism (VTE). Patients from 566 hospitals in the United States between 2010 and 2014 were included. Low-molecular-weight heparin (LMWH) prophylaxis was not associated in a reduction of VTE rates. LMWH prophylaxis was associated with a reduction in bleeding and heparin induced thrombocytopenia. SUMMARY: Background Critically ill patients with cancer are at increased risk of venous thromboembolism (VTE) from physical and cellular factors, requiring pharmacologic prophylaxis to reduce the risk of VTE. Objectives To assess whether low-molecular-weight heparin (LMWH) prophylaxis reduces in-hospital rates of VTE or improves clinical outcomes compared with unfractionated heparin (UFH) prophylaxis in critically ill patients with cancer. Methods We used a propensity-matched comparative-effectiveness cohort from the Premier Database. Patients aged 18 years or older with a primary diagnosis of cancer, intensive care unit admission and VTE prophylaxis within 2 days of admission between 1 January 2010 and 31 December 2014 were included. Patients were divided into LMWH or UFH prophylaxis groups. Results A total of 103 798 patients were included; 75 321 (72.6%) patients received LMWH and 28 477 (27.4%) patients received UFH. Propensity analysis matched (2 : 1) 42 343 LMWH patients and 21 218 UFH patients. Overall, LMWH was not associated with a decreased incidence of VTE (5.32% vs. 5.50%). LMWH prophylaxis was associated with a reduction in pulmonary embolism (0.70% vs. 0.99%), significant bleeding (13.3% vs. 14.8%) and heparin-induced thrombocytopenia (HIT) (0.06% vs. 0.19%). In non-metastatic solid disease, LMWH was associated with decreased VTE (4.27% vs. 4.84%) and PE (0.47% vs. 0.95%). Conclusions The use of an LMWH for VTE prophylaxis was not associated with a reduction in the incidence of in-hospital VTE as compared with UFH, but was associated with significant reductions in PE, clinically important bleeding events, and incidence of HIT in critically ill patients with cancer.

Resistance to Phytophthora citrophthora and P. parasitica and Nursery Characteristics of Several Citrus Rootstocks.

Studies were conducted to compare existing and potential citrus rootstocks with respect to resistance to root rot and gummosis caused by Phytophthora citrophthora and P. parasitica in greenhouse and growth chamber experiments and horticultural performance under simulated nursery conditions. Depending upon rootstock and experiment, mean root weights resulting from inoculation with P. citrophthora were 27 to 96% lower than the comparable controls. In similar experiments with the same rootstocks, inoculation with P. parasitica resulted in root weights that were 38 to 95% less than weights of the noninoculated controls. During 1994 or 1995, mean root weight reduction compared with noninoculated plants among Citrus macrophylla, rough lemon, C. volkameriana, and Sunki mandarin × Flying Dragon trifoliate (62-109-19) attributable to P. citrophthora and mean root weight reduction among C. macrophylla, C. volkameriana, rough lemon, Sacaton citrumelo, Sunki mandarin × Flying Dragon trifoliate (62-109-19), African shaddock × Rubidoux trifoliate, and Shekwasha mandarin × English trifoliate attributable to P. parasitica were significantly less than those recorded for all other tested rootstocks. Rootstocks that sustained a low percentage of root weight reduction generally experienced a low percentage of shoot weight reduction and survived longer as well. In evaluation of resistance to gummosis, depending on rootstock and experiment, the mean length of stem lesions caused by P. citrophthora on rootstocks ranged from 0.2 to 25.0 mm, whereas values for P. parasitica ranged from 0.2 to 18.5 mm. Stem lesions smaller than 5 mm in length were recorded for 21 and 14 of 36 different rootstocks inoculated with P. citrophthora and P. parasitica, respectively. On the other hand, P. citrophthora and P. parasitica caused stem lesions of at least 10 mm in length on 8 and 16 citrus rootstocks, respectively. Desirable nursery characteristics, including vigorous growth, minimal branching, and high leaf chlorophyll content, were demonstrated most prominently by Gomiri rough lemon, C. volkameriana, and Benton citrange, and to a lesser degree by some other rootstocks. Possible factors that could account for inconsistent classification of some citrus rootstocks as susceptible or resistant to Phytophthora root rot and gummosis are discussed.

Comparative analyses of the latency-associated transcript promoters from herpes simplex virus type 1 strains H129, +GC and KOS-63.

We have analyzed the activity of a specific portion of the latency-associated transcript (LAT) promoter of three strains of herpes simplex virus type 1 (HSV-1) in neuronal and non-neuronal cell types. Restriction fragments containing the LAT promoter sequences and the 5'-end of the LATs were isolated from HSV-1 strains H129, +GC and KOS-63, sequenced and cloned into a chloramphenicol transferase (CAT) plasmid vector. These vectors were separately assayed for CAT production in human (SknSH) and mouse (C-1300) neuroblastoma cell lines and a human continuous cell line (HeLa). Strain KOS-63 contained a C to T base substitution within the LAT promoter binding factor element upstream of the cAMP response element binding sequence. In replicate experiments, in which the construct DNA was used for transfection, the CAT constructs from strains H129 and +GC functioned equally well in all three cell lines. In contrast, the strain KOS-63 CAT construct functioned significantly better in HeLa cells than in neuroblastoma cell lines and better than the identical CAT constructs from strains H129 and +GC. In addition, the construct from strain KOS-63 functioned less well in the human neuroblastoma cell line than in HeLa or C-1300 neuroblastoma cells. When LAT expression was examined directly in vivo by in situ hybridization, strain KOS-63 produced slightly less LAT RNA than strain H129 within trigeminal ganglionic neurons of latently infected rabbits. However, utilizing competitive gel-shift assays, DNA fragments containing the LAT promoter binding element from all three strains bound equivalent amounts of HeLa cell nuclear proteins. Together, these results suggest that the activity expressed by the strain KOS-63 LAT promoter in vivo and in vitro may relate to positive or negative effects of DNA binding proteins on LAT transcription, and that these effects are cell-type dependent.

22-Oxacalcitriol: dissection of 1,25(OH)2D3 receptor-mediated and Ca2+ entry-stimulating pathways.

22-Oxa-1,25-dihydroxyvitamin D3 (oxacalcitriol, or OCT) is a bioactive analogue of 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] with lower calcemic activity than the parent compound. We investigated the ability of OCT to stimulate 1) genomic pathways mediated by nuclear receptors for 1,25(OH)2D3 versus 2) nongenomic pathways mediated by voltage-sensitive Ca2+ channels in growth phase rat osteosarcoma cells (ROS 17/2.8) and in chick intestine. Effects on nuclear receptor-mediated pathways were evaluated by measuring the ability of OCT to compete with [3H]1,25(OH)2D3 for soluble receptors. We also measured the ability of OCT to increase mRNA encoding osteoblast marker proteins osteopontin (OPN) and osteocalcin (OCN), which are both increased by 1,25(OH)2D3. Effects on Ca2+ entry into osteoblasts were measured using 45Ca2+ influx assays. The rapid stimulation of calcium absorption (transcaltachia) in chick intestine treated with OCT also was measured. We found that OCT bound to the nuclear receptor with lower binding affinity [relative competitive index (RCI) = 48.1 for ROS 17/2.8; RCI = 14.8 for chick intestine] than 1,25(OH)2D3 (RCI = 100). Like 1,25(OH)2D3, OCT increased mRNA levels of OPN and OCN in ROS 17/2.8 cells over a 48-h period. In contrast, OCT had no effect on transmembrane influx of 45Ca2+ across ROS cell membranes, whereas uptake was stimulated within 1 min by 1 nM 1,25(OH)2D3. In transcaltachia assays in perfused duodenum, OCT stimulated absorption with a maximum response at 6.5 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Irish setter dogs affected with rod/cone dysplasia contain a nonsense mutation in the rod cGMP phosphodiesterase beta-subunit gene.

Irish setter dogs affected with a rod/cone dysplasia (locus designation, rcd1) display markedly elevated levels of retinal cGMP during postnatal development. The photoreceptor degeneration commences approximately 25 days after birth and culminates at about 1 year when the population of rods and cones is depleted. A histone-sensitive retinal cGMP phosphodiesterase (PDE; EC 3.1.4.35) activity, a marker for photoreceptor PDEs, was shown previously to be present in retinal homogenates of immature, affected Irish setters. Here we report that, as judged by HPLC separation, this activity originates exclusively from cone photoreceptors, whereas rod PDE activity is absent. An immunoreactive product the size of the PDE alpha subunit, but none the size of the beta subunit, can be detected on immunoblots of retinal extracts of affected dogs, suggesting a null mutation in the PDE beta-subunit gene. Using PCR amplification of Irish setter retinal cDNA, we determined the complete coding sequence of the PDE beta subunit in heterozygous and affected animals. The affected PDE beta-subunit mRNA contained a nonsense amber mutation at codon 807 (a G-->A transition converting TGG to TAG), which was confirmed to be present in putative exon 21 of the affected beta-subunit gene. The premature stop codon truncates the beta subunit by 49 residues, thus removing the C-terminal domain that is required for posttranslational processing and membrane association. These results suggest that the rcd1 gene encodes the rod photoreceptor PDE beta subunit and that a nonsense mutation in this gene is responsible for the production of a nonfunctional rod PDE and the photoreceptor degeneration in the rcd1/rcd1 Irish setter dogs.

Improved binding of acidic bone matrix proteins to cationized filters during solid phase assays.

A number of commercially available matrix filter supports have been designed for the immobilization of proteins following either electrotransfer from sodium dodecyl sulfate (SDS) polyacrylamide gels or direct application during dot blotting assays. These matrices differ with respect to chemical composition, charge, pore size, and degree of hydrophobicity. It follows that the properties of the protein(s) of interest will greatly influence the degree to which they interact with and ultimately bind to various filters. Acidic bone proteins contain diverse post-translational modifications that influence their interactions with solid phase matrices such as those used in immunoblotting (Western or dot blotting) or ion binding (overlay) procedures. This communication describes the results of a study comparing binding of various mixtures of non-collagenous acidic bone matrix phosphoproteins as well as purified osteopontin and osteocalcin to various filters including nitrocellulose and cationized paper or nylon. Based on our findings, we recommend the use of cationized filters for solid phase assays requiring the binding of these acidic macromolecules to background supports.

Bacterial phospholipid hydrolysis enhances the destruction of Escherichia coli ingested by rabbit neutrophils. Role of cellular and extracellular phospholipases.

Escherichia coli ingested by PMN are promptly growth arrested but undergo limited destruction. We have studied bacterial phospholipid hydrolysis as a possible limiting factor in the disassembly of ingested E. coli, comparing the fates, during phagocytosis by rabbit peritoneal exudate PMN, of three isogenic strains, differing in their content of the pldA gene encoding the principal E. coli phospholipase A (PLA), i.e., pldA-, pldA+, pldA (the latter strain bearing the pldA gene in a multicopy plasmid resulting in a 20-fold increase in PLA content). Ingestion and growth inhibition (greater than 99% within 15 min) were the same for the three strains, but phospholipid degradation differed according to bacterial PLA content: pldA up to 60%, pldA+ up to 30%, and pldA- up to 20%. Since the pldA- strain has no activatable PLA, phospholipid degradation in this strain demonstrates the action of a PMN PLA. Added PLA2-rich ascitic fluid (AF) or purified AF PLA2 increased the rate and extent of degradation of the pldA- strain, provided the enzyme was added before ingestion was complete. 125I-AF-PLA2 binds to both E. coli and PMN and thus can enter the vacuole during phagocytosis. Although up to 50-fold more AF-PLA2 than the PLA2 content of the PMN could be loaded into the PMN in this way, degradation of pldA- E. coli did not exceed 30%. Increased phospholipid degradation had no effect on the degradation of bacterial macromolecules. In contrast, bacterial disassembly manifest as structural disorganization, release of bacterial protein derived material, and inhibition of protein synthesis were markedly enhanced when greater than 50% of prelabelled bacterial phospholipids were degraded. These findings reveal a link between envelope phospholipid degradation and overall bacterial destruction, suggesting therefore that factors limiting PLA action limit the destruction of E. coli ingested by PMN.

A calcium-binding, asparagine-linked oligosaccharide is involved in skeleton formation in the sea urchin embryo.

We have previously identified a 130-kD cell surface protein that is involved in calcium uptake and skeleton formation by gastrula stage embryos of the sea urchin Strongylocentrotus purpuratus (Carson et al., 1985. Cell. 41:639-648). A monoclonal antibody designated mAb 1223 specifically recognizes the 130-kD protein and inhibits Ca+2 uptake and growth of the CaCO3 spicules produced by embryonic primary mesenchyme cells cultured in vitro. In this report, we demonstrate that the epitope recognized by mAb 1223 is located on an anionic, asparagine-linked oligosaccharide chain on the 130-kD protein. Combined enzymatic and chemical treatments indicate that the 1223 oligosaccharide contains fucose and sialic acid that is likely to be O-acetylated. Moreover, we show that the oligosaccharide chain containing the 1223 epitope specifically binds divalent cations, including Ca+2. We propose that one function of this negatively charged oligosaccharide moiety on the surfaces of primary mesenchyme cells is to facilitate binding and sequestration of Ca+2 ions from the blastocoelic fluid before internalization and subsequent deposition into the growing CaCO3 skeleton.

Stimulation of matrix formation in rabbit chondrocyte cultures by ascorbate. 1. Effect of ascorbate analogs and beta-aminopropionitrile.

The most consistent effects of 0.2 mM L-ascorbate on monolayer cultures of rabbit articular chondrocytes were a diversion of incorporated radiosulfate into a pericellular matrix and enhancement of cell proliferation. Only with certain batches of fetal bovine serum (FBS) was there a cell-for-cell increase of proteoglycan synthesis. These actions increased as the cell inoculum rose from 0.5 to 2 x 10(5) cells/T25 flask. Maximal effects of ascorbate and D-isoascorbate were found over a range of 0.05-0.2 mM. L-Dehydroascorbic acid was less effective than either, and no stimulatory action was exerted by L-cysteine, glutathione, dithiothreitol, methylene blue, or phenazine methosulfate. Ascorbate increased the hypro:pro ratio of newly synthesized proteins. beta-Aminopropionitrile (1 mM) reduced the proportion of [3H]hydroxyproline and [35S]O4-proteoglycans in the ascorbate-supplemented matrix 31 and 7%, respectively. In corresponding electronmicrographs, the number of pericellular filaments was reduced. We conclude: (a) Ascorbate has a general anabolic effect on chondrocytes in culture and enhances matrix assembly through mechanisms other than its redox function; (b) deposition of proteoglycans in the matrix is not simply the result of mechanical entrapment by allysine- or hydroxyallysine-derived cross-linking of collagen; and (c) contradictory reports on the subject result from variations in the serum employed, inoculum density, and concentration of ascorbate.

Effect of fusarochromanone and T-2 toxin on articular chondrocytes in monolayer culture.

The effect of fusarochromanone and T-2 toxin on DNA synthesis and radio-sulfate incorporation by rabbit articular chondrocytes was studied in monolayer culture. T-2 toxin reduced DNA more than 50% at 5 x 10(-9) M; fusarochromanone caused small but progressive decrements over a range of 5 x 10(-8) to 10(-6) M. These actions are not specific for chondrocytes. The findings lend no support to the hypothesis that fusarochromanone, at least in unmodified form, is the etiologic agent in Kashin-Beck disease.

Mseleni disease serum is not harmful to cultured chondrocytes.

The origins of Mseleni disease, an acquired polyarticular degenerative joint disease, are unknown. We examined sera from 12 patients with the disease, 5 unaffected Mseleni residents, and 5 Durban residents. The effects of sera from the 12 patients with Mseleni disease on DNA or sulfated proteoglycan synthesis by cultured rabbit or human infant articular chondrocytes were no different from those of control sera. Cells derived from 2 children contained no stainable DR antigens; coculture had no impact on either of the above measures of cell function.

Angiotensin I converting enzyme inhibitors containing unnatural alpha-amino acid analogues of phenylalanine.

The activity of three angiotensin I converting enzyme (ACE) inhibitors with unique related structures was assessed in vitro and in vivo. The three compounds were (S)(-)-1,2,3,4-tetrahydro-2-(3-mercapto-1-oxopropyl)-3-isoquinoline carboxylic acid (EU-4865), 1,2,3,4-tetrahydro-2-(3-mercapto-1-oxopropyl)-1- isoquinolinecarboxylic acid (EU-4881), and (S)(-)-1,2,3,4-tetrahydro-1-(3-mercapto-1-oxopropyl)-2- quinolinecarboxylic acid (EU-5031). In vitro EU-4881 was a competitive inhibitor that lacked potency (IC50 = 1980 nM) against purified ACE. The other two compounds were equipotent (IC50 = 41 nM) against purified ACE but differed in their inhibition kinetics. EU-4865 (Ki = 38 nM) was a noncompetitive inhibitor, and EU-5031 (Ki = 6.9 nM) was a competitive inhibitor. Against caveolae membrane-bound ACE EU-4881 also lacked potency (IC50 = 2852 nM). In vivo in the conscious acute aortic coarctate (AAC) rat it also lacked potency, having an ED30 (oral dose decreasing blood pressure 30 mmHg) greater than 100 mg/kg. The activity of EU-4865 and EU-5031 in the caveolae membrane-bound ACE and AAC rat reflected their different Ki values rather than their similar IC50 values. In vitro, EU-4865 and EU-5031 had IC50 values of 19 and 6.7 nM, respectively, and in vivo, they had ED30 values of 52 and 1.1 mg/kg, respectively. These results suggest that ACE has a binding requirement for a carboxy-terminus, hydrophobic amino acid that is important for in vivo activity.

The effect of sodium selenite on chondrocytes in monolayer culture.

The effect of sodium selenite on DNA and sulfated proteoglycan synthesis by cultured rabbit articular and growth plate chondrocytes was studied as an in vitro model for Kashin-Beck disease. The selenium content of a defined medium (DMEM, fibroblast growth factor, insulin, and dexamethasone) was below the limit of detection by isotope dilution mass spectrometry. The chondrocytes were viable in the Se-free basal medium. Selenite over a range of 5 X 10(-9) M to 5 X 10(-7) M had no stimulatory effect on DNA or sulfated proteoglycan synthesis by either type of chondrocyte or skin fibroblasts. Proliferation of bovine endothelial cells was enhanced by 5 X 10(-7) M Se. At Se concentrations of greater than or equal to 10(-6) M, there was progressive inhibition of cell growth and radiosulfate incorporation of the connective tissue cells; bovine endothelial cells were more resistant. Twice equimolar concentrations of vitamins C and E exerted no protective effect against the cytotoxicity of higher concentrations of Se. Se supplementation also failed to stimulate growth of human infant chondrocytes. The model enabled simulation of conditions of hyposelenosis below those encountered in nature. The data provide no evidence that chondrocytes have idiosyncratic requirements for Se, and do not support the hypothesis that Se deficiency is a major etiologic factor in Kashin-Beck disease.

Induction of bone xenografts of rabbit growth plate chondrocytes in the nude mouse.

Subcutaneous transplantation of growth plate chondrocytes isolated enzymatically from the proximal tibia of 6-week-old rabbits into athymic (nu/nu) mice resulted in the formation of cartilaginous nodules. Calcification of the matrix was first seen after 48 hrs, and endochondral ossification at 12 days. The mineral first occurred about hypertrophic cells. Histochemical alkaline phosphatase activity was concentrated in pericellular collars at the same location. Immunofluorescence examination with rabbit anti-mouse lymphocyte serum disclosed that the bulk of the osteoblasts was derived from the mouse. A small quantity of mouse antigen was present in the cartilage matrix at its junction with bone. It presumably diffused into the cartilaginous interface from the host, but the possibility that some chondrocytes were of murine origin has not been excluded. Five of six grafts of cells grown to confluence in monolayer culture for 10 to 14 days became ossified. The ability to induce mineralization declined in subculture. Chondrocytes killed by heating to 56 degrees did not induce calcified cartilage or bone.

2,3-dihydro-4H-1-benzopyran-4-one O-carbamoyloximes, a series of gastric antisecretory agents.

A series of 2,3-dihydro-4H-1-benzopyran-4-one O-carbamoyloximes were synthesized and evaluated for gastric antisecretory activity in a pylorus-ligated rat model. Various substituents in the 6-position did not afford any compounds more active than I.

5-phenyl-2-furamidines: a new chemical class of potential antidepressants.

A series of 5-phenyl-2-furamidines has been synthesized and evaluated for antidepressant activities. Substitution in the phenyl ring with a nitro (4) or an amino (12) group in the ortho-position resulted in an increase in antidepressant activity. Both 4 and 12 antagonized tetrabenazine-induced ptosis in rodents and inhibited norepinephrine (noradrenaline) uptake into crude synaptosomes of whole mouse brain at doses or concentrations comparable to those of the tricyclic antidepressants. However, these compounds did not possess the anticholinergic and antihistaminic activities common to tricyclic antidepressants. In addition, they lacked monoamine oxidase inhibitory activity. The 5-phenyl-2-furamidines represent a new chemical class of antidepressants and may be useful for depressive patients who cannot tolerate the compromising side effects of the tricyclic antidepressants and monoamine oxidase inhibitors.

Rotavirus infection in Otago: a serological study.

A method for measuring rotavirus antibody in human sera has been established using enzyme-linked immunosorbent assay (ELISA). A Simian strain of rotavirus (SA11) was used as the antigen. Serum eluted from dried blood spots on good quality chromatography paper was found suitable for analysis. Paired serum samples from children with gastroenteritis have shown a brisk antibody response in association with the presence of rotavirus in the faeces. Community studies indicate that although all older children and adults tested have detectable antibodies to rotavirus, there is a significant rise in the number of individuals with high titre antibody in the child bearing age group, after which the levels diminish. This finding suggests that repeated infections occur throughout childhood and early adult life.

Isolation, identification, and synthesis of 4-amino-6,7-dimethoxy-3-quinolinol, the major metabolite of amiquinsin hydrochloride in rats and humans.

The metabolism of amiquinsin hydrochloride (4-amino-6,7-dimethoxyquinoline hydrochloride monohydrate, I) was studied in rats and humans. The major metabolite isolated from human urine was identified through synthesis as 4-amino-6,7-dimethoxy-3-quinolinol hydrochloride hydrate (II). Acid hydrolysis of the major metabolite from rat urine also gave II. The structure of the rat metabolite subsequently was tentatively identified as the potassium salt of the 3-O-sulfate of II. The pharmacological and toxicological properties of I and II are discussed.

Synthesis and skeletal muscle relaxant activity of quaternary ammonium salts of dantrolene and clodanolene.

A series of quaternary ammonium salts of dantrolene and clodanolene was prepared and evaluated for skeletal muscle relaxant activity. The quaternary ammonium salts exhibit greater aqueous solubility and, therefore, facilitate intravenous administration. One member of this series, although less effective orally, exhibited greater aqueous solubility than the sodium salt. When administered intravenously, it was a more potent antagonist of skeletal muscle contraction and yielded comparable therapeutic and muscle relaxant efficacy indexes.