Detection of influenza A virus nucleoprotein antibodies in oral fluid specimens from pigs infected under experimental conditions using a blocking ELISA.

In commercial swine populations, influenza is an important component of the porcine respiratory disease complex (PRDC) and a pathogen with major economic impact. Previously, a commercial blocking ELISA (FlockChek(™) Avian Influenza Virus MultiS-Screen(®) Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME, USA) designed to detect influenza A nucleoprotein (NP) antibodies in avian serum was shown to accurately detect NP antibodies in swine serum. The purpose of this study was to determine whether this assay could detect NP antibodies in swine oral fluid samples. Initially, the procedure for performing the NP-blocking ELISA on oral fluid was modified from the serum testing protocol by changing sample dilution, sample volume, incubation time and incubation temperature. The detection of NP antibody was then evaluated using pen-based oral fluid samples (n = 182) from pigs inoculated with either influenza A virus subtype H1N1 or H3N2 under experimental conditions and followed for 42 days post inoculation (DPI). NP antibodies in oral fluid were detected from DPI 7 to 42 in all inoculated groups, that is, the mean sample-to-negative (S/N) ratio of influenza-inoculated pigs was significantly different (P < 0.0001) from uninoculated controls (unvaccinated or vaccinated-uninoculated groups) through this period. Oral fluid versus serum S/N ratios from the same pen showed a correlation of 0.796 (Pearson's correlation coefficient, P < 0.0001). The results showed that oral fluid samples from influenza virus-infected pigs contained detectable levels of NP antibodies for ≥42 DPI. Future research will be required to determine whether this approach could be used to monitor the circulation of influenza virus in commercial pig populations.

Detection of bovine viral diarrhoea virus infected cattle--testing tissue samples derived from ear tagging using an Erns capture ELISA.

A new diagnostic approach testing tissue samples derived from cattle ear tagging for bovine viral diarrhoea virus (BVDV) antigen in a commercially available antigen capture enzyme-linked immunosorbent assay (ACE) was developed. To validate this method, 99 positive and 469 negative samples were tested. With those samples the assay yielded a sensitivity of 100% and specificity of >or=99.6%. Serum and ear tissue samples from 11 persistently infected (PI) BVDV calves were tested. While serum samples were negative after intake of colostrum, the ear tissue samples could be detected positive for BVDV all the time. Testing multiple samples derived from the same ear from PI cattle yielded positive results and low variation. Using cattle ear tags combining the ear tag application with sampling of a small ear tissue plug and testing those tissue samples with an ACE could be a reliable and economic way of BVDV testing.

Modelling the variability of lag times and the first generation times of single cells of E. coli.

A mathematical model combining deterministic and stochastic elements describes the growth and division of single cells. Its deterministic part is based on the model of Baranyi and Roberts [International Journal of Food Microbiology 23 (1994) 277] modelling the gradual adjustment of the cells to a new environment. The stochastic part assumes a random threshold size for the division of a single cell, which accounts for the variability of the individual generation times. Experimental results of the first division times of thousands of single cells using a microscopic flow system could be reproduced with this model, and it has the potential to be used to study the effects of different stress and environmental factors on the distribution of the lag and generation times of individual cells.

Observing growth and division of large numbers of individual bacteria by image analysis.

We describe a method that enabled us to observe large numbers of individual bacterial cells during a long period of cell growth and proliferation. We designed a flow chamber in which the cells attached to a transparent solid surface. The flow chamber was mounted on a microscope equipped with a digital camera. The shear force of the flow removed the daughter cells, making it possible to monitor the consecutive divisions of a single cell. In this way, kinetic parameters and their distributions, as well as some physiological characteristics of the bacteria, could be analyzed based on more than 1,000 single-cell observations. The method which we developed enabled us to study the history effect on the distribution of the lag times of single cells.

Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli.

We report a rapid, large-scale process for the purification of a recombinant Fab fragment specific for the tobacco mosaic virus coat protein (Fab57P). The fragment is expressed periplasmically in Escherichia coli. The expression level was optimized in 0.3-L fermentors. The highest levels were obtained using the following conditions: (1) low postinduction temperature (21 degrees C), (2) combined use of two beta-lactam antibiotics (carbenicillin and ampicillin), (3) IPTG concentration 0.1 mM, (4) regulated pH 7.2, (5) 17-h induction time, and (6) conditions that reduce mechanical stress. Optimized large-scale fermentations were done in 15- and 300-L capacity fermentors. The recombinant Fab fragment was purified by two chromatographic steps. After disruption of the bacteria using an APV Gaulin homogenizer, the crude E. coli homogenate was directly applied, without centrifugation, to an SP Sepharose Big Beads column. The recombinant Fab fragment was eluted as a single peak in a sodium chloride gradient. The fragment was further purified by affinity adsorption to a column packed with Epoxy-activated Sepharose 6B to which the antigen peptide NH(2)-CGS YNR GSF SQS SGLV-CONH(2) had been coupled through its N-terminal cysteine. The purified Fab57P fragment showed one band in SDS-PAGE. The overall purification yield was 35%.

Nuclear factor Y controls the basal transcription activity of the mouse platelet-derived-growth-factor beta-receptor gene.

To determine the regulatory mechanism of the expression of the mouse platelet-derived growth factor (PDGF) beta-receptor gene, a 1.9-kb 5' flanking genomic fragment was cloned and analyzed. Site-directed mutagenesis of a CCAAT motif, located 60 bp upstream of the transcriptional-start site, completely abolished the promoter activity [Ballagi, A. E., Ishisaki, A., Nelin, J.-O. & Funa, K. (1995) Biochem. Biophys. Res. Commun. 210, 165-1751. The sequence around the intact CCAAT motif was protected by in vitro DNase-I-footprinting analysis. Electrophoresis-mobility-shift assays with anti-[nuclear factor Y(NF-Y)]Ig revealed binding of the NF-Y complex to the CCAAT box. Furthermore, the double-stranded oligonucleotides corresponding to the sequence around the CCAAT motif were conjugated with DNA-affinity magnetic beads. The binding proteins were affinity purified and identified as the NF-Y transcription factor by western blotting. Our results indicate that NF-Y controls the basal transcription activity of the mouse PDGF beta-receptor gene.

Isolation and characterization of the mouse PDGF beta-receptor promoter.

The PDGF beta-receptor expression is tightly regulated during embryonic development and in several physiological and pathological situations. To determine the regulatory mechanism of the receptor, a 1.9 kb 5' flanking genomic fragment of the mouse PDGF beta-receptor gene was cloned and analyzed by functional promoter assays. The fragment was shown to exert promoter activity in the luciferase expression vector system in mouse NIH 3T3 fibroblast and NB41 neuroblastoma cell lines as well as rat ST15A cerebellar cell lines. Functional studies on deletion mutants revealed several putative regulatory sequences. The deletion mutants acted similarly in NB41 cells and in ST15A cells, both of neuronal origin, but differently in the NIH 3T3 fibroblasts. No TATA box was found in the analyzed promoter region, however, site directed mutagenesis of a CCAAT motif, located 60 basepair upstream of the transcriptional start site, almost completely abolished the promoter activity in all cell types.

Specific effects of platelet derived growth factor (PDGF) on fetal rat and human dopaminergic neurons in vitro.

The neurotrophic effects of the BB isoform of platelet-derived growth factor (PDGF) on rat and human fetal mesencephalic dopaminergic neurons have been characterized in vitro. A dose-response analysis demonstrated maximal responses at 30 ng/ml of PDGF-BB. This concentration resulted in a marked increase in the survival and neurite outgrowth from rat and human tyrosine hydroxylase-(TH) positive, presumed dopaminergic neurons after 7 days in vitro. The effects of PDGF-BB on survival of TH-positive neurons were comparable to those of brain-derived neurotrophic factor (BDNF), whereas neurite outgrowth was more pronounced after addition of BDNF. The combination of BDNF and PDGF-BB yielded no additive effects. Double immunohistochemical staining of rat cultures demonstrated PDGF beta-receptors on about 90% of the TH-positive neurons. PDGF-BB treatment of rat mesencephalic cultures induced an upregulation of c-fos and TH mRNA with maximal levels after 0.5-2 h as assessed by quantitative PCR analysis. An increased number of Fos protein-positive cells was detected immunohistochemically after 4 h of PDGF-BB treatment. The present results provide further evidence for specific and direct effects of PDGF-BB on gene expression, survival and neurite outgrowth of mesencephalic dopaminergic neurons of rat and human origin.

Platelet-derived growth factor receptor expression after neural grafting in a rat model of Parkinson's disease.

Platelet-derived growth factor (PDGF) has trophic effect on dopaminergic neurons in vitro. We have previously shown dynamic changes in the expression of PDGF in embryonic mesencephalic grafts and surrounding host striatal tissue following intracerebral transplantation in a rat model of Parkinson's disease. In this study the expression of the PDGF receptors was examined in the same model using immunohistochemistry. Most ventral mesencephalic (VM) cells from E13-E15 rat embryos possessed both PDGF alpha- and beta-receptors before implantation. Double immunofluorescence staining revealed that about 10% of the cells also expressed tyrosine hydroxylase (TH). The PDGF alpha-receptor was detectable in the graft up to 1 wk after transplantation but had disappeared at 3 wk. In the host tissue, scattered glial cells were positive for the alpha-receptor but the expression was unchanged following transplantation. The beta-receptor expression almost completely disappeared from the grafted tissue by 4 h following transplantation, and only a few cells of the host striatum showed immunoreactivity. However, after 3 wk beta-receptor positive cells were again detectable in the graft. These cells appeared to be endothelial cells as identified by an antibody against von Willebrand's factor. Our data suggest that PDGF might act locally on embryonic dopaminergic cells in an autocrine or juxtacrine manner before and shortly after transplantation, and on surrounding glial cells in a paracrine manner after transplantation. Furthermore, PDGF-BB might influence neovascularization in the graft.

Regulation of GAD expression in islets of Langerhans occurs both at the mRNA and protein level.

The expression of the autoantigen glutamate decarboxylase in islets of Langerhans was investigated under different culture conditions, which affect the functional activity of the beta-cell. Using immunoprecipitations and analyses of enzyme activity, an increase in glutamate decarboxylase was detected in rat islets cultured at a glucose concentration of 11 mmol/l compared with those cultured at 5.6 mmol/l glucose. To determine whether the change was induced at the level of mRNA expression, total RNA was extracted from rat islets cultured at 5.6 or 11 mmol/l glucose, reverse transcribed and amplified by the polymerase chain reaction. Comparative quantitation in a phosphor imager revealed a significantly higher (82%, P < 0.005) content of glutamate decarboxylase mRNA in islets cultured at 11 mmol/l glucose. In parallel, human recombinant interleukin-1 beta, and diazoxide were tested for their effects on the expression of glutamate decarboxylase. Islets cultured at 11 mmol/l glucose in the presence of 40 U/ml of interleukin-1 beta, showed a 63% decrease (P < 0.005) in enzyme activity compared with those cultured at 11 mmol/l glucose alone, and similar decreases were noted on analysis of glutamate decarboxylase biosynthesis and mRNA. Islets cultured at 11 mmol/l glucose in the presence of 22.5 mg/ml diazoxide exhibited a significant reduction in enzyme activity (59%; P < 0.001) compared with those cultured at 11 mmol/l glucose only. This reduction, however, was not accompanied by a decrease in the content of glutamate decarboxylase mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

PDGF-BB exerts trophic activity on cultured GABA interneurons from the newborn rat cerebellum.

Platelet-derived growth factor (PDGF) is a well known mitogen for mesenchyme-derived cells and glial cells. Its presence in neuronal cells of the central nervous system has only recently been described. We have shown earlier that neurons of newborn rat brains in culture express PDGF beta-receptors and that PDGF-BB, a homodimer of PDGF B-chain, increases survival and promotes neurite outgrowth of newborn cerebellar cells (Smits et al., Proc. Natl Acad. Sci. USA, 88, 8159-8163, 1991). In this study, the effects of PDGF on early postnatal rat cerebellar cells were further explored. By using chemically defined serum-free medium, we have established primary cell cultures of rat cerebella (postnatal day 4-5) containing 70-80% neuronal cells. During the first 10 days in vitro, no difference in total cell number was found between PDGF-BB-treated and untreated cultures. After this time period, however, increased survival of the PDGF-BB-treated cells was found. Within the first 10 days in vitro, the addition of PDGF-BB to the cultures resulted in a relative increase in survival of interneurons expressing glutamic acid decarboxylase (GAD), the GABA biosynthetic enzyme. Moreover, addition of PDGF-BB in the untreated cell culture resulted in a rapid increase of GAD mRNA. These results show that PDGF-BB acts as a trophic factor on GABAergic interneurons of the cerebellum by up-regulating GAD synthesis and prolonging the survival of these cells. Furthermore, in situ hybridization revealed that there are scattered cells present in the early postnatal cerebellum that express PDGF beta-receptor mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)