Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor.

Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI-1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin-coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor.

The effect of local anesthetics on bacterial proliferation: TAC versus lidocaine.

We compared the effect of topical 0.5% tetracaine, 1:2,000 epinephrine, and 11.8% cocaine (TAC) with 1% lidocaine infiltration on bacterial proliferation in experimental lacerations. Forty-eight lacerations were made on the backs of Hampshire pigs, inoculated by injection with infectious doses of Staphylococcus aureus and randomly anesthetized with either topical TAC or lidocaine infiltration. Wounds were sutured, and quantitative cultures were obtained by excision after 48 hours. The mean log10 bacteria per gram of tissue for wounds anesthetized with TAC was 6.818 (95% confidence interval [CI], 6.07 to 7.54) compared with 6.820 (95% CI, 5.91 to 7.75) for those treated with lidocaine; this difference was not significant (P less than .05 by paired two-tailed t test). The probability of failing to detect an intergroup difference of 0.5 log10 bacteria per gram was less than .0001. TAC does not increase bacterial proliferation more than lidocaine infiltration in contaminated experimental porcine lacerations.

Effects of substrata on the polarization of bovine endometrial epithelial cells in vitro.

Epithelial-cell function requires cellular polarity in which apical membrane surfaces have unique characteristics and cellular organelles are stratified. Physiological investigations of endometrial epithelial cells would be enhanced greatly by the ability of a method to polarize cells in culture. This study investigates the effects of different substrata on polarization of cultured bovine endometrial epithelial cells. Fetal bovine endometrial epithelial-cell lines were developed from explant outgrowth. Epithelial monolayers were subcultured onto amniotic membranes, Millicell-HA membranes, or Millicell-CM membranes coated with rat-tail collagen, Matrigel, laminin, Vitrogen, or fibronectin. Cultures on these substrata were maintained at the air/liquid interface. Cells grown on either collagen-coated or uncoated Millicell membranes also were maintained submerged in medium. Excellent polarized morphology was attained in cultures grown at the air/liquid interface on amniotic membranes and rat-tail collagen-coated membranes. Lectin-binding patterns to apical membranes of polarized epithelial cell cultures paralleled patterns of binding to bovine endometrial surfaces in vivo. Cultures on rat-tail collagen were maintained for several weeks. These methods provide a valuable system for studying the endometrium in vitro.

On the use of T61 for euthanasia of domestic and laboratory animals; an ethical evaluation.

A number of experiments was carried out to determine the sequence of events leading to death following administration of the euthanizing agent T61. Simultaneous recordings of the EMG, EEG, ECG and end-tidal CO2 (dogs only) were obtained in acutely instrumented rabbits and dogs. Results show that following T61 administration the loss of consciousness and loss of muscle activity occurred simultaneously. Vocalization and increased muscle movement occurred in the initial phase of the injection in 3 of 8 dogs, injected with T61 or butyramide. From this study it was concluded that the presence of the muscle relaxant does not pose an ethical problem for the use of T61 as an euthanizing agent, but our results suggest that the use of T61 may have some emotionally unpleasant side-effects.

Effects of a potassium-sparing/thiazide diuretic combination on cardiovascular reactivity to vasopressor agents.

The vascular effects of a diuretic combination (spironolactone/altizide) were studied in 5 anuric patients undergoing dialysis by measuring changes in cardiovascular reactivity to norepinephrine (NE) and angiotensin II (AII) after infusion of incremental doses of these 2 vasopressor agents. There was a marked dose-response relation between NE and AII administration and mean (NE) or diastolic (AII) blood pressure (BP). Diuretic treatment moderated the increase in mean or diastolic BP induced by NE or AII. In addition, the pressor doses of NE and AII, which are also parameters of vascular reactivity, were significantly increased after diuretic treatment (NE, +101%; AII, +163%). Right atrial and pulmonary capillary wedge pressures increased along with BP in response to NE. Diuretic treatment also moderated the increase in right atrial and pulmonary capillary wedge pressures induced by NE. These results suggest that the antihypertensive action of spironolactone and altizide in combination is mainly due to a direct effect on resistance and capacitance vessels. The mechanisms by which diuretics modify the cardioreactivity remain poorly understood; however, they may modify electrolyte transport (sodium and calcium) across vascular smooth muscle cell membranes or stimulate prostaglandin release.

[Meaningful combinations of chemotherapeutic agents]. / Sinnvolle Kombinationen von Chemotherapeutika.

It is frequently assumed that, in general, combinations of antibiotics and similar chemotherapeutic agents are more efficient than the single chemotherapeutic, and that they can be combined without disadvantages for the activity of the single component. However, the specific properties of the combined substances, such as their antimicrobial spectrum and resistance, their pharmacokinetics and side effects, as well as their physical properties and formulation, have an enormous impact on the activity of combined chemotherapeutics. They may lead to an advantageous, but also to an indifferent or diminished activity of the combination in patients. Some of the important requirements for an advantageous combination of antibiotics and similarly functioning compounds will be reviewed and ways of avoiding mistakes are described.

Regulation of ornithine decarboxylase activity in LoVo cells.

The role of Na+ and Na(+)-H+ exchange in the stimulation of ornithine decarboxylase (ODC) activity has been investigated in a human colon adenocarcinoma cell line, LoVo. Asparagine (Asn; 10 mM) or 10% fetal bovine serum (FBS) increased ODC activity from undetectable levels to greater than 500 pmol CO2.mg protein-1.h-1 in 4 h. This increase could be reduced 50% by concentrations of Na(+)-H+ exchange inhibitors that did not reduce protein synthesis. (approximately 0.2 mM for amiloride and 0.05 mM for hexamethyleneamiloride). Asn was able to double the uptake of 22Na+, whether an ionic (choline chloride) or nonionic (D-mannitol) substance was substituted for Na+, and the substitution of these compounds as well as N-methyl-glucamine for Na+ largely prevented the stimulation of ODC by Asn. Another factor influencing ODC activity was extracellular pH (pHo). When pHo was lowered, intracellular pH (pHi) also fell, and ODC activity was reduced. When pHo was raised, pHi also rose, and ODC activity increased. The well-known correlation between increased pHi and Na+ uptake with the stimulation of growth may be due to their influence on ODC activity.

Advantages of a new anticoagulant in routine hematology on the Coulter Counter S-Plus STKR analyzer.

The authors report the advantages of a new anticoagulant-antiaggregant mixture that avoids the deleterious effects of ethylenediaminotetraacetic acid (EDTA) on mean platelet volume and also prevents EDTA-induced platelet clumping. It is suitable for routine cell counting and sizing with the Coulter Counter S-Plus STKR. The values of the common hematologic parameters agree well with those from EDTA-treated samples and are stable for at least eight hours after sampling.

Functional and metabolic responses of isolated hearts to acidosis: effects of sodium bicarbonate and Carbicarb.

To investigate the mechanisms by which acidosis depresses cardiac function, a Langendorff isolated, perfused rat heart preparation was studied using 31P-magnetic resonance spectroscopy. Isolated hearts were subjected to normal perfusion conditions or moderate or severe metabolic acidosis. Acidosis resulted in dose-dependent decreases in O2 consumption (VO2), rate of developed ventricular pressure (dP/dt), intracellular pH (pHi), and the tissue concentration of creatine phosphate (PCr) after 30 min. Acidosis did not affect ATP concentration but resulted in significant increases in the concentrations of total and monovalent inorganic phosphate (Pi and Pim). When metabolic determinants of the change in dP/dt with acidosis were examined, changes in pHi, extracellular pH, and tissue concentrations of Pi and Pim all correlated significantly with the change in dP/dt. In a second set of experiments using the severe acidosis model, normal saline, 1 M sodium bicarbonate, and the experimental buffer Carbicarb (1 ml/kg body wt) were infused in a crossover manner at 20-min intervals. None had significant effects on tissue concentrations of ATP, PCr, Pi, or PCr/Pi. Administration of bicarbonate resulted in an increase in VO2 without significant effects on pHi or dP/dt. In contrast, Carbicarb effected little change in VO2 but resulted in sustained increases in pHi and decreases in Pim and marked increases in dP/dt. These data suggest that the decrease in cardiac function that occurs during acidosis may be related to the metabolic consequences of reductions in pHi and that a buffer that causes intracellular alkalinization may cause functional improvement in this model.

Hippocampal cell death following ischemia: effects of brain temperature and anesthesia.

The effect of brain temperature and anesthesia on ischemic neuronal damage was studied in the hippocampal formation using the four vessel occlusion model in awake and anesthetized rats. Neuronal damage was assessed by immunocytochemistry and silver impregnation of tissue sections. The degree of ischemia was monitored by recording spontaneous and evoked electrical activity from the hippocampus and dentate gyrus in all animals. In addition, the hippocampal temperature and oxygen tension were also recorded using a chamber-type thin-film microelectrode in the anesthetized animals. Fifteen minutes ischemia in the awake animals caused greater neuronal damage and mortality of animals than 30 min ischemia in anesthetized rats. The temperature of the brain was found to drop by 4-6 degrees C during complete forebrain ischemia in the latter group. Neuronal damage was observed infrequently in the hippocampus of these animals. When the brain temperature was kept constant at the preischemic level during 30 min occlusion, all animals died within a day, while after 15 min occlusion the majority showed an almost complete degeneration of CA1 pyramidal cells and hilar somatostatin immunoreactive neurons. Following 15 min ischemia, the awake animals showed a similar cell loss in the CA1 region and the hilus. It is concluded that, in the anesthetized animals prepared for acute recording, the decreased temperature of the brain during ischemia is a major factor in protecting neurons from damage, but that Equithesin anesthesia also has a significant protective effect. Consistent ischemic degeneration occurs in awake animals by four vessel occlusion, if the brain temperature is controlled and the completeness of ischemia is monitored by recording spontaneous and evoked electrical activity with chronic electrodes.

Relationship of anabolic status and phase and rate of growth to priorities for protein and fat deposition in steers.

The effects of anabolic implants, growth phase (growing vs finishing) and rate of growth on the priorities for protein and fat deposition were determined in yearling cattle. Santa Gertrudis crossbred yearling steers weighing 290 kg were individually fed diets varying in forage and grain content and either not implanted (n = 16) or implanted (90-d intervals) with Ralgro (n = 13) or Synovex-S (n = 12) implants. The cattle were fed toward a similar expected final empty BW (455 kg). Initial and interim empty body composition was measured via deuterium oxide dilution; final composition was determined by carcass specific gravity. During a 100-d growing phase, rates of protein gain were increased (P less than .12) to 118 and 131 g/d from 98 g/d for Ralgro and Synovex vs nonimplanted cattle, respectively. Concurrently, the fraction of protein in empty body growth was increased (P less than .09) from 17.5% for controls to 23.8 and 19.7% for Ralgro- and Synovex-implanted steers, respectively. This change in protein growth occurred concomitant with mobilization of fat and a reduction (P less than .04) in fat gain with Ralgro and Synovex implants. During the 136-d finishing phase, protein accretion was 115 and 132 vs 93 g/d for Ralgro- and Synovex-implanted cattle vs nonimplanted cattle; this represented a 24 and 42% increase (P less than .03) with Ralgro and Synovex, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The promotional effect of bone wax on experimental Staphylococcus aureus osteomyelitis.

The consequences of using surgical bone wax are not well studied. We evaluated the infection-promoting potential of sterile bone wax in a rat model of chronic Staphylococcus aureus osteomyelitis. The addition of bone wax greatly reduced the quantitative bacterial inoculum (log colony-forming units) required to establish chronic osteomyelitis in 50% and 100% of challenged animals. The 50% infection rate was reduced from log 6.9 to 2.6 and the 100% infection rate from 8.2 to 4.4, respectively (p less than 0.015, t test for parallelism). Separate experiments were done 10 to 30 minutes after inoculation with only log 6.4 staphylococci. Tibiae of animals that received bone wax yielded more organisms than those that did not (log 2.76 +/- 0.68 versus 1.72 +/- 0.94, p less than 0.01). At 24 hours quantitative colony counts were not significantly different whether animals received wax or not (log 5.02 +/- 0.42 versus 4.43 +/- 0.65, p greater than 0.09). These studies suggest that the routine surgical use of bone wax should be reassessed.

Addition of a hypoxic cell selective cytotoxic agent (mitomycin C or porfiromycin) to Fluosol-DA/carbogen/radiation.

In an effort to develop effective combination treatments for use with radiation against solid tumors, the cytotoxic effects of the addition of mitomycin C or porfiromycin on treatment with Fluosol-DA/carbogen (95% O2/5% CO2) breathing and radiation in the FSaIIC tumor system were studied. In vitro mitomycin C and porfiromycin were both preferentially cytotoxic toward hypoxic FSaIIC cells. After in vivo exposure, however, the cytotoxicity of mitomycin C toward single cell tumor suspensions obtained from whole tumors was exponential over the dose range studied, but for porfiromycin a plateau in cell killing was observed. With Fluosol-DA/carbogen breathing and single dose radiation, addition of either mitomycin C or porfiromycin increased the tumor cell kill achieved at 5 Gy by approximately 1.2 and 1.0 logs, respectively. Less effect was seen with addition of the drugs at the 10 and 15 Gy radiation doses. In tumor growth delay experiments, the addition of either mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation resulted in primarily an additive increase in tumor growth delay. The survival of Hoechst 33342 dye-selected tumor cell subpopulations indicated that Fluosol-DA/carbogen breathing increased the cytotoxicity of radiation (10 Gy) more in the bright cell subpopulation (4-fold) than in the dim cell subpopulation (2-fold) resulting in an overall 4-fold sparing of the dim subpopulation. Mitomycin C and porfiromycin were both more toxic toward the dim cell subpopulations. Addition of mitomycin C or porfiromycin to Fluosol-DA/carbogen breathing and radiation (10 Gy) resulted in a primarily additive effect of the drugs and radiation killing in both tumor cell subpopulations. Thus, with mitomycin C/Fluosol-DA/carbogen and radiation there was a 2-fold sparing of dim cells and with porfiromycin in the combined treatment a 1.6-fold sparing of the dim cell population. Our results indicate that treatment strategies directed against both oxic and hypoxic tumor subpopulations can markedly increase the tumor cell kill achieved by radiation.

Efficacy of vaginal spaying and anabolic implants on growth and carcass characteristics in beef heifers.

Ninety crossbred beef heifers averaging 260 kg were blocked by weight and allotted randomly to 15 pens of six heifers each, with three replicates per treatment. The treatments were spayed heifers (S); intact heifers (I); S + Synovex-H7 (SH); I + Synovex-H (IH); and S + Synovex-S7 (SS). The heifers were spayed vaginally with a Willis instrument; intact heifers were rectally palpated for reproductive soundness. Heifers were fed a growing diet (55 d), re-implanted and subsequently fed a finishing ration (73 or 101 d). All heifers were observed for estrus daily at approximately 0600 and 2000 until d 69. A jugular vein blood sample was obtained from each heifer on d 48, 55, 62 and 69 for blood progesterone analysis. Results of blood progesterone analysis and reproductive tract examination indicated that two spayed heifers were incompletely spayed, a 96% success rate. The SH and SS heifers continued to exhibit estrous behavior despite being successfully spayed. Heifers implanted with Synovex-H had greater (P less than .01) ADG and were more efficient (P less than .03) than nonimplanted heifers. The SS heifers had greater ADG (P less than .05) during the finishing phase than SH heifers, but no advantage of SS over SH was apparent during the growing phase. The overall ADG response to implantation was fourfold greater (P less than .07) in the spayed heifers than in the intact heifers (32 vs 8%). Heifers implanted with Synovex-H had greater adjusted hot carcass weight (P less than .02) and ribeye area (P less than .002) than nonimplanted heifers. For adjusted live weight and ribeye area, the response to implantation was approximately threefold greater in the spayed heifer than the intact heifer. These results demonstrate that spaying and implanting heifers can increase rates and efficiency of gain even though behavioral estrus is not eliminated.

Time course of cycloplegia induced by a new phenylephrine-tropicamide combination drug.

A motorized and computer-interfaced phoropter was used to track the development of cycloplegia and recovery of accommodation over a 60-min period, after the topical application of a phenylephrine 5%-tropicamide 0.8% drug combination (Phenyltrope). Phenyltrope was introduced into the Canadian market about 2 years ago (Compendium of Pharmaceuticals and Specialties, 1987), and advertised as a fast acting cycloplegic and mydriatic drug. Here we report the results of our investigation of the depth of action and the temporal aspects of cycloplegia for this drug combination as a function of iris color. We also compare the action spectrum of Phenyltrope to that of tropicamide 1% under similar test conditions. Our results indicate that the latency for cycloplegia was shorter for tropicamide, the maximum rate of accommodative loss similar for both drugs, and the resultant cycloplegia at 20 min deeper for Phenyltrope. Recovery from induced cycloplegia was greater for tropicamide 60 min after drug administration. For both Phenyltrope and tropicamide, no significant differences in any of the parameters investigated were observed as a function of iris color. We conclude that even though Phenyltrope induced a measurably deeper cycloplegia than did tropicamide, the amount of residual accommodation present at 20 min (about 38%) is insufficient for most refractive purposes.

Topical anaesthesia of gingival mucosa by 5% eutectic mixture of lignocaine and prilocaine or by 10% lignocaine spray.

The analgesic effect of topical application of local anaesthetics on the gingival mucosa and the absorption of the local anaesthetics into the blood were investigated in healthy volunteers by using a 5% eutectic mixture of the local anaesthetics lignocaine (100 mg) and prilocaine (100 mg) plus emulsifier (EMLA) or 10% lignocaine (200 mg) spray (Xylocain). The pain threshold on labial gingiva was measured by using the stimulator of an EMG-apparatus and a pair of stimulating electrodes, specially constructed for this purpose. There were no differences between the two methods in producing analgesia which was at its maximum in 13 to 14 minutes, on average. Sensation of the gingiva (pain thresholds), had usually returned to normal within 30 minutes. In the dosages used, the absorption of the local anaesthetics was more rapid after the mixture application than after spray application. No toxic reactions occurred.

[Effects of cotrimoxazole on some macrophage functions: microbicide, tumoricide, production of free oxygen radicals, prostaglandins and leukotrienes]. / Effets du cotrimoxazole sur certaines fonctions de macrophages: microbicidie, tumoricidie, production de radicaux libres oxygénés, de prostaglandines et de leucotriènes.

In order to demonstrate an immunomodulating effect of cotrimoxazole, we investigated its influence on some macrophage (M phi) functions in culture: P815 tumor cells killing, Toxoplasma gondii killing, production of free oxygen radicals by luminol-dependent chemiluminescence, prostaglandins and leukotrienes secretion evaluated after incorporation of tritiated arachidonic acid. In vitro, cotrimoxazole inhibited in a dose-dependent fashion the chemiluminescence of murine resident peritoneal or guinea pig alveolar M phi. Production of prostaglandin (PG) 6-keto-F1 alpha, PGF2 alpha, and 5-hydroxyeicosatetraenoic acid by resident peritoneal M phi was also inhibited. However, PGD2 synthesis by alveolar M phi was enhanced. A second study was performed on peritoneal M phi, resident or elicited in vivo by one intra-peritoneal injection of an extract from Mycobacterium Tuberculosis membranes and obtained from mice pretreated or not by cotrimoxazole per os. Resident M phi from cotrimoxale-treated animals showed increased production of leucotriene B4 compared to M phi from controls. 6-keto-PGF1 alpha and free oxygen radicals production by elicited M phi was greatly enhanced by cotrimoxazole whereas thromboxane B2 was reduced. Finally cotrimoxazole enhanced intracellular killing of Toxoplasma gondii and cytotoxicity for tumor cells P815 by resident but not by elicited M phi. It is concluded that cotrimoxazole can modulate MO activation and some M phi functions involved in immune homeostasis. This data could help to understand why an antibiotic such as cotrimoxazole, which is known to be frequently used in immunocompromised hosts, is also efficient in Wegener's granulomatosis.

BIPP--how does it work?

The antibacterial activity of BIPP and its constituents against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa was measured by growth inhibition tests. BIPP was found to have negligible antibacterial activity. In addition, no release of iodine from BIPP was detected over a 4-week period. It is proposed that much of the evident antibacterial activity of BIPP may be a reflection of the meticulous surgical debridement that accompanies its use. In addition, BIPP makes the impregnated gauze impervious to blood and body fluids ensuring little nutrition for bacteria to thrive in its interstices.