Preparation and characterization of human thrombin for use in a fibrin glue.

Cryoprecipitate is frequently combined with thrombin to produce a fibrin sealant to enhance haemostasis during surgical procedures. We evaluated the thrombin produced from plasma using the Thrombin Processing Device (TPD)trade mark (Thermogenesis, Rancho Cordova, CA, USA). Plasma (250 mL) was processed in the CryoSeal FS System using the CP-3 disposable to produce cryoprecipitate by automated freezing and thawing. Simultaneously, thrombin was generated using the attached TPD. The cryoprecipitate and thrombin were harvested after approximately 50 min and then frozen and stored at -80 degrees C until analysis of total protein, fibrinogen, factor VIII (FVIII) activity, von Willebrand factor (vWF) and thrombin activity. Sodium dodecyl sulphate (SDS) gel electrophoresis was used to compare thrombin. After combining the thrombin with cryoprecipitate, the rate of clot initiation and strength was measured using a Thromboelastograph (TEG) (Haemoscope Corp, Skokie, IL, USA). Cryoprecipitate was produced, with a fibrinogen concentration of 22 +/- 7.7 g L(-1) (20 +/- 2% recovery), FVIII activity of 14.2 +/- 4.0 IU mL(-1) and vWF of 19.9 +/- 5.2 IU mL(-1). The separate thrombin product had a concentration of 64.3 +/- 16.7 IU mL(-1) of thrombin and a total protein of 0.39 +/- 0.1 g, with SDS gel electrophoresis showing a major band at 37 kD, as did the commercial human thrombin. The TEG curves of cryoprecipitate and TPD-produced or commercial thrombin were compared. The R values (time to clot initiation) were somewhat slower with the TPD-produced thrombin, but the maximum strength (MA) of the clots was similar. In conclusion, human thrombin can be produced during automated cryoprecipitate production. This thrombin is in sufficient concentration to initiate clotting and cross-linking of fibrin from cryoprecipitate to produce an entirely autologous fibrin glue.

Different culture media requirements of IVF and nuclear transfer bovine embryos.

Important differences exist between in vitro fertilized (IVF) and nuclear transfer (NT) bovine embryos. Studies have shown that although in vitro development is comparable, post-implantation survival is greatly reduced in NT embryos. In this study, we compare serum and bovine serum albumin (BSA) supplementation during oocyte maturation and embryo culture of IVF and NT embryos. In experiment 1, oocytes and embryos were randomly distributed into different treatment groups consisting of synthetic oviductal fluid (SOF) medium supplemented with either serum, fatty acid-free BSA (FAF) or fraction V BSA during maturation and/or culture to assess IVF embryo development. In experiment 2, oocytes were matured in SOF + serum or SOF + FAF and reconstructed embryos were cultured in SOF + FAF to assess NT embryo development. Among the IVF treatment groups, a greater number of blastocysts were observed in the steer serum (SER) group (IVM and IVC in SOF + serum) on day 6; however, no significant differences were seen in blastocyst development from day 8 onwards. Hatching frequencies on days 8 and 9 were significantly greater in groups with serum, with the exception of FAF (IVM and IVC in SOF + FAF) on day 9. For the NT treatment groups, the presence of serum during IVM resulted in a higher proportion of MII oocytes and increased blastocyst development and hatching rates were compared with supplementation of FAF. These results indicate that both serum and FAF provide comparable embryo development for IVF but not for NT bovine embryos.

Effect of cooling and warming rates during cryopreservation on survival of in vitro-produced bovine embryos.

The effect of cooling and warming rates during cryopreservation on subsequent embryo survival was studied in 607 bovine morulae and 595 blastocysts produced by in vitro maturation, fertilization and culture (IVM/IVF/IVC). Morulae and blastocysts were prepared by co-culturing presumptive zygotes with bovine oviductal epithelial cells (BOEC) in serum-free TCM199 medium for 6 and 7 d, respectively. The embryos in 1.5 M ethylene glycol in plastic straws were seeded at -7 degrees C, cooled to -35 degrees C at each of 5 rates (0.3 degrees, 0.6 degrees , 0.9 degrees, 1.2 degrees, or 1.5 degrees C/min) and then immediately plunged into liquid nitrogen. The frozen embryos were warmed either rapidly in a 35 degrees C water bath (warming rate > 1,000 degrees C/min) or slowly in 25 degrees to 28 degrees C air (< 250 degrees C/mm). With rapid warming, 42.1% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts. The proportions of rapidly wanned morulae that hatched decreased with increasing cooling rates (30.4, 19.0, 15.8 and 8.9% at 0.6 degrees , 0.9 degrees, 1.2 degrees and 1.5 degrees C/min, respectively). With slow warming 25.9% of the morulae that had been cooled at 0.3 degrees C/min developed into hatching blastocysts, while <10% of the morulae that had been cooled faster developed. The hatching rate of blastocysts cooled at 0.3 degrees C/min and warmed rapidly (96.3%) was higher than those cooled at 06 degrees and 0.9 degrees C/min (82.7 and 84.6%, respectively), and was also significantly higher than those warmed slowly after cooling at 0.3 degrees, 0.6 degrees or 0.9 degrees C/min (69.1, 56.6 and 51.8%, respectively). Cooling blastocysts at 1.2 degrees or 1.5 degrees C/min resulted in lowered hatching rates either with rapid (71.2 or 66 0%) or slow warming (38.2 or 38.9%). These results indicate that the survival of in vitro-produced bovine morulae and blastocysts is improved by very slow cooling during 2-step freezing, nevertheless, slow warming appears to cause injuries to morulae and blastocysts even after very slow cooling.

Ventilatory sensitivity to carbon dioxide: the influence of exercise and athleticism.

Endurance training reduces the ventilatory response to a given level of work, and there is evidence that endurance athletes possess attenuated chemosensitivity at rest; but it is unclear whether attenuation persists during exercise. We compared the carbon dioxide sensitivity (S) of endurance-trained (ETG), sprint-trained (STG), and control subjects (CG), at rest and during cycle ergometry. Steady-state carbon dioxide (CO2) inhalation was employed; ventilatory parameters were measured using an ultrasonic flowmeter linked to a computer. CO2 concentrations were measured at the mouth using an infrared CO2 analyzer or mass spectrometer. Mean resting CO2 sensitivity of the ETG was significantly lower than that of the STG (P < 0.05), but not the CG (P < 0.058). S increased from rest to exercise in all endurance-trained subjects, but the responses of the STG and CG were varied. Compared to rest, mean S was significantly higher during exercise for the ETG, but not for the STG or CG. S was the same in all groups during exercise. During air breathing exercise all subjects were mildly hypercapnic. The ETG showed the greatest rise in mean alveolar PCO2, but this could not be attributed to attenuated chemosensitivity since responsiveness during exercise was identical in all three groups.

Proto-oncogene expression in cAMP and TPA-mediated neuronal differentiation in a human retinal cell line KGLDMSM.

PURPOSE: A human retinal cell line, KGLDMSM, developed by SV-40T antigen gene transfection, is stable in culture for a long period, unlike the primary cells. The cell line shows some degree of morphological differentiation with limited extension of stublike neurites upon transfer to defined medium. In our effort to explore genes implicated in neuritic extension and neuronal differentiation seen in response to cAMP and TPA, we have analyzed time dependent induction of a variety of proto-oncogenes: c-myc, H-ras, c-ras, and c-fos. METHODS: Cells were adapted to grow in defined media and exposed to differentiation inducing agents cAMP, TPA, Retinoic Acid, and sodium butyrata. Cells were assessed for phenotypic changes and altered expression of proto-oncogenes as evaluated by Northern Blot analysis and immunocytochemistry. RESULTS: Exposure of the cells to cAMP and TPA induced dramatic changes, with 100% of the cells extending neuritic processes. However, other differentiation inducing agents such as retinoic acid and sodium butyrata failed to elicit any response. We report that agents that promote neuritic extension also induce expression of c-fos. Transcriptional activation of c-fos in response to cAMP (30 min) and TPA (1hr) is also accompanied by expression of fos gene product as evaluated by using fos antibody. No fos expression was seen in uninduced cells. CONCLUSION: In retinal cell line KGLDMSM, agents that enhance neuronal differentiation (cAMP, TPA) also induce c-fos expression. Expression of c-fos may be a necessary prerequisite in neuronal differentiation and the established retinal cell line offers an excellent cell model for dissecting the molecular events underlying neuronal differentiation.

Three year results of in vitro production of bovine embryos in serum-poor bovine oviduct conditioned medium. An overview.

This paper presents a synthesis of 3 year results of in vitro production of bovine embryos in medium previously conditioned by bovine oviduct epithelial cells. In Louvain-la-Neuve, Belgium, a total of 18356 oocytes were matured and inseminated in vitro: 13967 (76%) had cleaved at 3 days post-insemination and 3593 (26%) became blastocysts using this culture system. Our data show that conditioned medium can be stored frozen for up to 3 years without significant loss of activity and is resistant to lyophilization. One single batch of conditioned medium was tested within the same period in four different laboratories and yielded variable results: 27 and 37% blastocysts/cleaved embryos in two of them and only 7 and 0% in the two others whereas in each case more than 30% blastocysts were obtained with the local reference co-culture system. In one laboratory, the batch of oil used to overlay the culture drops had a detrimental effect on the blastocyst rate in conditioned medium but not in co-culture.

Comparison of the effects of oviductal cell co-culture and oviductal cell-conditioned medium on the development and metabolic activity of cattle embryos.

The objective of this study was to compare the development and metabolic activity of cattle embryos co-cultured with bovine oviductal cells or cultured in serum-free medium previously conditioned by bovine oviductal cells. Zygotes were produced by in vitro fertilization of oocytes from bovine ovaries obtained from an abattoir. Development to the four-cell stage occurred by 48 h after fertilization in both culture systems, but co-cultured embryos reached the 16-cell stage by 96 h, whereas those cultured in conditioned medium did not do so until 24 h later. Similarly, the morula and blastocyst stages were reached 24 h earlier in co-culture than in conditioned medium. There were significantly more cells in the blastocysts from co-culture (96.8 +/- 6.1 versus 56.7 +/- 3.3; P < or = 0.0001). The metabolism of glutamine did not differ between embryos cultured in the two systems, but the metabolism of glucose was significantly greater in embryos cultured in conditioned medium. The first significant increase in glucose metabolism occurred between the four-cell and the 16-cell stages in embryos cultured in conditioned medium, but occurred between the 16-cell and morula stages in the co-cultured embryos, such that the glucose metabolism was significantly greater at the 16-cell stage in embryos cultured in conditioned medium compared with co-cultured embryos (6.5 +/- 1.0 versus 1.5 +/- 0.4 pmol per embryo per 3 h, P < or = 0.0001). The concentration of glucose was significantly less, and that of lactate significantly greater, in co-culture medium than in conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Practical audit workshops for nurses.

Six practical audit workshops were run for nurses working in the Greater Glasgow Health Board. Between 15 and 25 nurses came for a morning session and returned 6 weeks later for an afternoon session. The participants came from all grades, specialties and units across the health board. The morning session included a basic introduction to the principles and stages of the audit cycle and information on different audit methods. The majority of the time was devoted to small group discussions where individuals were able to develop their own ideas for audit projects relevant to their working situations. Together with one of the organizers, the members of the groups helped each other in the design of the different projects. Almost 65% of attenders (78 out of 122) returned 6 weeks later for the afternoon session and most had begun an audit project in the time interval. These projects were at various stages around the audit cycle. The afternoon sessions started with a talk by a nurse involved in an audit project, followed by the participants returning to their small groups to discuss the successes and problems involved in their own audits. The groups provided further support and advice based on a wide range of different experiences during the intervening time. The projects carried out were impressive both in the quality and the variety of topics covered. Ninety per cent of those attending felt that the knowledge gained from the exercise was of practical use in their own working environment. A measure of the success of the workshops was that several of the later afternoon talks were given by nurses who had attended the earlier workshops.

Establishment of a human retinal cell line by transfection of SV40 T antigen gene with potential to undergo neuronal differentiation.

Recently, a number of laboratories have been interested in developing cell lines of ocular tissues to understand the pathogenesis of ocular diseases. Toward this end, we report here the generation of cell lines of human retina by transfection of simian virus SV40 T antigen gene. Established retinal cells grow as a monolayer and exhibit limited serum dependence. Phase-contrast and electron microscopic studies revealed distinct morphological cell types. Immunofluorescence studies showed that the established retinal cells were positive for neuron-specific enolase, neurofilament protein, glycine receptor, synaptophysin, and secretogranin. Cells were negative for glial fibrillary acidic protein, glutamine synthetase, galactocerebroside, and carbonic anhydrase II. In addition to neuronal features, a small percentage of flat cells were, however, positive for cellular retinaldehyde binding protein, and cells with the phenotype of rod and cone photoreceptor coexpressed opsin and interphotoreceptor retinoid-binding protein. An important feature of this cell line is that addition of phorbol ester and cAMP induced dramatic changes, with 100% of the cells extending long, thin neuritic processes. Thus, the established retinal cells would be useful for studies dealing with differentiation and plasticity of the cells of the nervous system.

The changing role of an audit support group.

An audit support group was established within a University Department of Public Health in 1991 to provide a resource for clinical audit. A postal survey of 300 consultants and senior registrars (59% response rate) showed 72% had participated in audit, 47% currently involved, 40% implementing change. 40% had no experience in standard setting or implementing change. Comparison of the workload of the group for August 91-March 92 and April 92-March 93 showed 4 requests for support per month, 4/5 from doctors during first period compared with 6 requests per month and 1/3 from doctors during second period. Since the group was established demand from clinicians has decreased and from professions allied to medicine increased. This may reflect either clinicians' increased experience or decline in enthusiasm for audit. Education in the basic skills for audit for all healthcare professionals, improved communications and practical support for clinical audit is necessary.

Ventilatory responses to exercise and carbon dioxide in elderly and younger humans.

The present investigation examined the relationship between CO2 sensitivity [at rest (SR) and during exercise (SE)] and the ventilatory response to exercise in ten elderly (61-79 years) and ten younger (17-26 years) subjects. The gradient of the relationship between minute ventilation and CO2 production (delta VE/delta VCO2) of the elderly subjects was greater than that of the younger subjects [mean (SEM); 32.8 (1.6) vs 27.3 (0.4); P < 0.01]. At rest, SR was lower for the elderly than for the younger group [10.77 (1.72) vs 16.95 (2.13) l.min-1 x kPa-1; 1.44 (0.23) vs 2.26 (0.28) l.min-1 x mmHg-1; P < 0.05], but SE was not significantly different between the two groups [17.85 (2.49) vs 19.17 (1.62) l.min-1 x kPa-1; 2.38 (0.33) vs 2.56 (0.21) l.min-1 x mmHg-1]. There were significant correlations between both SR and SE, and delta VE/delta VCO2 (P < 0.05; P < 0.001) for the younger group, bot none for the elderly. The absence of a correlation for the elderly supports the suggestion that delta VE/delta VCO2 is not an appropriate index of the ventilatory response to exercise for elderly humans.

The cancer-initiating potential of the fumonisin B mycotoxins.

The cancer-initiating potential of the fumonisin B (FB) mycotoxins produced by Fusarium moniliforme was screened in rat liver for their ability to induce rare hepatocytes with an acquired resistance to the mitoinhibitory effect of 2-acetyl-aminofluorene (2-AAF). Two different initiating protocols were used: a feeding regimen during which FB1 was fed at a dietary level of 0.1% for 26 days, and another where single or multiple doses of FB1 and FB2 (varying from 200 to 50 mg/kg) were administered (by gavage) to hepatectomized rats. In both cases promotion was effected by a 2-acetylamino-fluorene/carbontetrachloride treatment. Cancer initiation was only obtained after the prolonged feeding regimen, indicating that the fumonisins are poor cancer initiators. FB1 and FB2 also lack genotoxic effects in the in vivo and in vitro DNA repair assays in primary hepatocytes. Although FB1 primarily affects the liver, it is not very cytotoxic to primary hepatocytes when compared to aflatoxin B1.

Stimulation of DNA synthesis by rat plasma following in vivo treatment with three liver mitogens.

The present study was undertaken to determine the effect of two different types of liver cell proliferative stimuli, namely compensatory regeneration and direct hyperplasia on DNA synthesis of normal and preneoplastic isolated hepatocytes. Platelet-poor plasma (PPP) isolated from male Wistar rats treated with three different hepato-mitogens, lead nitrate (LN), cyproterone acetate (CPA) and ethylene dibromide (EDB), or subjected to surgical partial hepatectomy (PH), was tested for its ability to stimulate DNA synthesis in normal and preneoplastic hepatocytes in primary cultures. Induction of DNA synthesis was detected as early as 30 min after CPA, EDB and PH administration and persisted up to 5 days after the LN administration. In addition, hepatocytes isolated from preneoplastic liver nodules were also able to respond in culture to the DNA synthesis stimulus induced by these factors.

Counteracting effects of dexamethasone and alpha 2-macroglobulin on inhibition of proliferation of normal and neoplastic rat hepatocytes by transforming growth factors-beta type 1 and type 2.

Primary cultures of hepatocytes isolated from normal F-344 rats or from F-344 rats with hepatocellular carcinomas generated by a 2-step model of chemical carcinogenesis were used to determine if dexamethasone (DEX) or alpha 2-macroglobulin (alpha 2M) modify the ability of transforming growth factors-beta type I (TGF-beta I) and type 2 (TGF-beta 2) to inhibit labelling index of hepatocytes cultured continuously with or without epidermal growth factor (EGF). Both TGF-beta 1 and beta 2 were equivalently potent inhibitors of S-phase DNA synthesis in normal and neoplastic hepatocytes as determined by 3H-thymidine autoradiography. Both DEX (1 to 100 microM) and alpha 2M (50-200 microM) partially counteracted the mito-inhibitory effect of both TGF-betas on the proliferation of normal and surrounding hepatocytes. In contrast, neoplastic hepatocytes cultured with DEX released much less immunoreactive alpha 2M and were less able to overcome the inhibitory effect of TGF-beta than normal or surrounding hepatocytes. Purified bovine alpha 2M partially counteracted the inhibition of TGF-beta 1 or beta 2 of both surrounding and neoplastic hepatocytes. Both DEX and alpha 2M were more effective against the mito-inhibitory activity of TGF-beta 2. Our data suggest that alpha 2M released by DEX-treated normal hepatocytes contributes to the counteraction of the TGF-beta effect by DEX. Our results support the hypothesis that glucocorticoids and growth-factor-binding proteins may have important roles in modulating the effects of TGF-beta on normal hepatocyte proliferation and suggest that under some conditions hepatocellular neoplasms can be more sensitive than normal hepatocytes to inhibition of proliferation by TGF-beta.

Replication of HIV in human fetal retinal cultures and established pigment epithelial cell lines.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in cells derived from ocular tissue was studied. Primary retinal cultures (containing both glial and neuronal cells) were found to support the replication of HIV upon transfection with molecularly cloned proviral DNA. In addition, established retinal pigment epithelial (RPE) cell lines also produced HIV particles upon transfection. HIV released by these cell lines was able to infect and induce characteristic cytopathic effects in T4+ cells. An indicator plasmid containing the HIV long terminal repeat sequences (LTR) linked to the chloramphenicol acetyltransferase gene showed barely detectable activity in RPE cells and was transactivated by the addition of the HIV "tat" gene. Based on these observations, direct infection of ocular tissue derived cells such as RPE, fetal retinal cells, retinoblastoma cells (Y 79, WER1), choroidal endothelial cells (Chor 55) (mix culture) and corneal fibroblasts (K61) by HIV was attempted. HIV replication in these cells was not detected by reverse transcriptase, antigen and transactivation function assays.

In vitro phenotypic and functional characterization of human pigment epithelial cell lines.

We have characterized human retinal pigment epithelium (HRPE) for the expression of cell surface antigens. Primary HRPE cultures, established cell lines, and freshly brushed pigment epithelial cells all express HLA-ABC but not HLA-DR antigens. However, both primary cultures and established cell lines can be induced by gamma interferon stimulation to express HLA-DR in a dose dependent manner. Only freshly brushed HRPE cells express Fc, and no cells demonstrated the presence of C3b. Our results show that HRPE cells change in culture, as reflected by the loss of Fc receptors, but retain the ability to synthesize HLA-ABC spontaneously and HLA-DR upon stimulation.

Inhibition of DNA synthesis in primary cultures of hepatocytes by orotic acid.

Orotic acid has been shown to promote carcinogenesis in the liver and the intestine of the rat. In an attempt to determine whether orotic acid promotes liver carcinogenesis by creating differential mitoinhibition, experiments were designed to study the effect of orotic acid on the labeling index of isolated hepatocytes in response to epidermal growth factor. The results indicated that orotic acid added in vitro inhibited epidermal-growth-factor-induced labeling index of isolated hepatocytes. In addition, isolated hepatocytes from rats exposed to orotic acid under promoting conditions also exhibited a decreased response to epidermal growth factor. These data suggest that orotic acid may exert its promoting effect by differentially inhibiting the response of normal hepatocytes to one or more endogenous growth stimuli while permitting the initiated hepatocytes to respond to such stimuli and grow to form hepatic nodules.

Inhibition of proliferation of normal, preneoplastic, and neoplastic rat hepatocytes by transforming growth factor-beta.

The influences of purified transforming growth factor beta (TGF-beta) on proliferation of normal, preneoplastic, and neoplastic rat hepatocytes were examined in primary monolayer culture with or without prior stimulation with epidermal growth factor (EGF). Hepatocytes from normal livers or discrete preneoplastic nodules or carcinomas generated in F344 rats by the Solt-Farber model were isolated and cultured in serum-free modified Williams' E. medium for up to 72 h. Proliferation was quantified by labeling index by [3H]thymidine autoradiography. The majority of normal hepatocytes became labeled in response to EGF (20 ng/ml) between 24 and 72 h. TGF-beta had a dose-dependent inhibitory effect which was virtually complete at concentrations above 0.5 ng/ml added at 0 h together with EGF. Hepatocytes from all nodule and carcinoma populations were less stimulated by EGF but also strongly inhibited by TGF-beta. Hepatocytes isolated from normal livers 24 h after partial hepatectomy were similarly inhibited by TGF-beta. The minimal initial exposure period for TGF-beta to maximally inhibit was 2 h. TGF-beta added at various times between 8 and 48 h after EGF partially inhibited the labeling index to levels that were constant but substantially greater than the labeling index at the time TGF-beta was added. A proportion of hepatocytes from normal and nodular livers became resistant to the inhibitory effects of TGF-beta between 48 and 72 h, suggesting that the inhibitory effect is transient. TGF-beta added at 0 h also virtually completely inhibited the labeling of normal and nodular hepatocytes that were not exposed to EGF. These studies demonstrate that TGF-beta is a potent negative regulator of proliferation of normal, regenerating, preneoplastic, and neoplastic hepatocytes. This suggests that persistent proliferation of neoplastic hepatocytes in vivo cannot be explained by a difference in response to TGF-beta.

Mitogenic activity in platelet-poor plasma from rats with persistent liver nodules or liver cancer.

Platelet-poor plasma (PPP) from F-344 rats with chemically-induced preneoplastic liver nodules or hepatocellular carcinoma stimulated S-phase DNA synthesis in monolayer cultures of normal rat hepatocytes. Similar mitogenic activity was detected in PPP 6 hrs to 1 week after partial hepatectomy (PH) or after necrotizing doses of CCl4 or diethylnitrosamine (DENA). Very little activity was found in PPP4 from control rats. The mitogenic activity in PPP from animals with nodules was non-dialyzable (greater than 14 kd) and bound to a heparin-sepharose affinity column. None of the mitogenic PPPs competed with [125I] epidermal growth factor (EGF) for binding sites on A431 cells or normal rat hepatocytes. These studies indicate that persistent proliferation of preneoplastic and neoplastic hepatocytes is associated with increased circulating levels of mitogenic hepatocyte growth factor.