A CRE/ATF-like site in the upstream regulatory sequence of the human interleukin 1 beta gene is necessary for induction in U937 and THP-1 monocytic cell lines.

Transfection of U937 and THP-1 cells with a recombinant plasmid, pIL1(4.0kb)-CAT, containing 4 kb of the interleukin 1 beta (IL-1 beta) gene upstream regulatory sequence resulted in inducer-dependent expression of chloramphenicol acetyltransferase activity. Treatment of the transfected cells with various combinations of the inducers lipopolysaccharide, phorbol myristate acetate, and dibutyryl cyclic AMP upregulated the IL-1 beta promoter. In U937 and THP-1 cells, maximum stimulation of both the endogenous IL-1 beta gene and pIL1(4.0kb)-CAT transfectants was observed following treatment with the combination of inducing agents lipopolysaccharide-phorbol myristate acetate-dibutyryl cyclic AMP. This combination of inducing agents was used to identify and study, at the molecular level, some of the regulatory elements necessary for induction of the IL-1 beta gene. A series of 5' deletion derivatives of the upstream regulatory sequence were used in transient transfection assays to identify an 80-bp fragment located between -2720 and -2800 bp upstream of the mRNA start site that was required for induction. Exonuclease III mapping, electrophoretic mobility shift assays (EMSA), and DNA sequence analysis of this region were used to identify a transcription factor binding sequence which contained a potential cyclic AMP response element (CRE/ATF)- and NF-kappa B-like binding site. Site-directed mutagenesis of the CRE/ATF-like site resulted in the loss of binding of a specific factor or factors as determined by EMSA. The loss of binding activity directly correlated with a loss of approximately 75% of promoter activity as determined in transient transfection assays. As determined by EMSA, the factor binding to the CRE/ATF-like site was present in nuclear extracts prepared from both uninduced and induced THP-1 and U937 cells. However, the intensity of the band appeared to be increased when nuclear extracts from induced cells were used. In contrast to the CRE/ATF mutation, which resulted in the loss of promoter activity, mutation of the NF-kappa B-like site resulted in a moderate increase in activity in U937 cells. A similar increase in promoter activity was not observed in THP-1 cells. From these studies, we conclude that a CRE/ATF-like site and a factor or factors interacting with this site are essential for the maximum induction of the IL-1 beta gene in stimulated U937 and THP-1 cells.

Characterization of human E4BP4, a phosphorylated bZIP factor.

In this report we described the isolation of transcription factor E4BP4 by lambda gt11 expression cloning using a probe containing the CRE/ATF-like sequence located between -2764 bp and -2753 bp in the upstream regulatory region for the human IL-1 beta gene. DNaseI protection, gel mobility shift analysis, and cotransfection studies were performed to investigate the binding and functional properties of E4BP4 using IL-1 beta promoter sequences. By DNaseI footprinting, a protection pattern was generated over the CRE/ATF-like site and the flanking sequences by bacterially produced E4BP4. Competition experiment by gel shift assay indicated that E4BP4 bound specifically to CRE/ATF-like site, not NF kappa B-like site. In cotransfection studies, E4BP4 repressed promoter activity and this repression was mediated through the CRE/ATF-like site. Mutational analysis of E4BP4 suggested that the DNA binding as well as repression activities required leucine heptad repeat domain. Analysis of E4BP4 produced in Escherichia coli and Sf9 cells infected with recombinant baculovirus indicated that baculovirus produced protein showed enhanced binding to the CRE/ATF-like site compared to the E. coli-produced protein. Analysis of posttranslational modifications indicated that E4BP4 produced in Sf9 cells was phosphorylated and this phosphorylation was important for the DNA binding activity of E4BP4.

Transmission of acute respiratory disease (rhinotracheitis) of turkeys.

Clinical signs of acute respiratory disease of turkeys were transmitted to susceptible day-old poults by direct contact, litter contact, and drinking water. Attempts failed to transmit the disease by air (cage-to-cage) or by oral and nasal inoculation with feces, nasal exudates, or nasal turbinate extracts. The disease was not transmitted by inoculation with white blood cells. Chicks were not affected by the disease, but young quail developed signs of respiratory disease when exposed to contaminated drinking water. Thus, acute respiratory disease in turkeys appears to be of an infectious nature, and the infectious agent(s) probably exist in the heavily contaminated environment used to house young commercial turkey poults.

A technique for isolating turkey respiratory adenoviruses.

Several turkey respiratory adenoviruses were isolated in turkey embryonic liver cells from nasal turbinate filtrate collected from clinically ill turkey poults during postmortem examination. Suspect adenovirus inoculum had to remain in cell culture for 5 to 7 days, and 9 blind passages were required before a sample was declared negative. Once isolated, the turkey adenoviruses adapted rapidly to turkey kidney cells.

In vitro cytotoxicity of an Alcaligenes faecalis and its relationship in in vivo tracheal pathologic changes in turkeys.

Two isolates of Alcaligenes faecalis from turkeys with respiratory disease were indistinguishable physically, biochemically, and for specific agglutinating antibodies. The isolates differed in in vitro cytotoxicity for turkey tracheal organ cultures and in ability to induce clinical rhinotracheitis in poults. The isolate designated NCDp induced in vitro cytotoxic changes in turkey tracheal organ cultures. Additionally, poults inoculated with NCDp developed severe clinical signs of rhinotracheitis, flaccid (collapsing) trachea, bacterial colonization of the cilia, and degeneration and loss of the columnar epithelium from the anterior one third to one half of the trachea. The isolate designated NCDm induced little or no cytotoxic changes in turkey tracheal organ culture. Isolate NCDm caused mild clinical signs of rhinotracheitis and colonized the trachea of inoculated poults, but it caused no other observable changes. A correlation seems to exist between in vitro cytotoxicity and in vivo pathogenicity for these isolates of A. faecalis.

Evidence for adherence-dependent cytotoxicity of Alcaligenes faecalis in turkey tracheal organ cultures.

A virulent isolate of Alcaligenes faecalis was examined in turkey tracheal organ cultures (TTOC) for adherence using immunofluorescent staining and for cytotoxicity using light microscopic observation. Treatment of the bacterial culture with trypsin, antiserum specific for A. faecalis, and N-acetylneuraminic acid inhibited the ability of the organism to adhere to TTOC. Treatment of the bacterium with D-galactose partly decreased adherence of the bacterium. Those treatments that inhibited the adherence of A. faecalis also inhibited the cytolytic activity in TTOC. Treatment of the bacterial culture with D-galactose only partly decreased the cytolytic activity. These data indicate that adherence of the organism to TTOC is necessary for the cytolytic activity characteristic of A. faecalis isolates capable of causing alcaligenes rhinotracheitis in turkeys.

Isolation of an etiologic agent of acute respiratory disease (rhinotracheitis) of turkey poults.

A small gram-negative motile bacillus was isolated from laboratory poults affected by acute respiratory disease (rhinotracheitis) of turkeys. The bacterium was inoculated intranasally into susceptible day-old poults; the poults developed typical clinical signs of acute respiratory disease, and the bacterium was reisolated. This same bacterium was isolated from commercial poults with typical signs of acute respiratory disease but not from poults of similar age which were clinically normal. The bacterium has not been identified taxonomically. We conclude that it is a primary etiologic agent for acute respiratory disease of turkey poults.

Adenovirus respiratory infection in turkey poults.

A virus from turkey poults with respiratory signs was isolated in specific-pathogen-free embryonated chicken eggs and subsequently adapted to chicken embryo fibroblast and turkey kidney cell cultures, where round cell formation was observed. The cloned virus was ether-resistant and incorporated tridiated thymidine. Intra-nuclear icosahedral virus particles of 80 nm were detected. These physicochemical characteristics place this isolant into the adenovirus group of viruses. The disease was experimentally reproduced by intratracheal inoculation of one-day-old turkey poults. Snicking occurred in 100% of the birds and mortality reached 50%. CELO (Phelps strain) antiserum neutralized uncloned and cloned CUA-2 in chicken embryos and uncloned virus in chicken embryo fibroblast cell cultures. Quail bronchitis virus antiserum neutralized cloned CUA-2 in TK cells. Agar-gel precipitin lines of identity were formed using CELO antiserum and postinoculation sera from experimentally infected turkeys. Serologically, this virus should be classified as a type-1 adenovirus.

Isolation and identification of a turkey respiratory adenovirus.

A virus with physical and biological characteristics of an adenovirus was isolated from turkey poults with respiratory disease. The virus was ether-resistant and incorporated [3H] thymidine. Electron microscopy revealed virions of icosahedral configuration, approximately 78 nm in diameter, within the nuclei of infected cells. The virus produced cytopathology in turkey kidney cells, but did not produce observable disease when inoculated into commercial turkey poults or specific-pathogen-free embryonated chicken eggs. Virus-neutralization tests indicated widespread exposure to the virus in North Carolina turkey populations.

An Alcaligenes faecalis isolate from turkeys: pathogenicity in selected avian and mammalian species.

An Alcaligenes faecalis isolate of known pathogenicity for turkeys was examined for adherence and cytotoxicity in tracheal organ cultures of turkeys, chickens, Japanese quail, guinea pigs, hamsters, and mice, and for colonization and pathogenicity in these 6 species. Adherence and colonization were detected by fluorescent antibody staining. Infected and noninfected tracheal rings were examined by phase-contrast microscopy for cytotoxicity (ciliostasis, blebing of the cell membrane, and sloughing of the ciliated epithelium). Alcaligenes faecalis adhered to the tracheal rings of all species examined. Cytotoxicity was apparent in the tracheal rings of turkeys, quail, and chickens. Cytotoxicity was not detected in tracheal rings from the mammalian species. Alcaligenes faecalis colonization of turbinates and tracheas of intact turkeys and quail was detected. Clinical signs of alcaligenes rhinotracheitis were observed and histopathologic characteristics of the disease were detected. Chickens, guinea pigs, hamsters, and mice were refractory to infection with this isolate of A faecalis.

Cytotoxic activity of pathogenic Alcaligenes faecalis in turkey tracheal organ cultures.

Turkey tracheal organ cultures were used to study the virulence of Alcaligenes faecalis isolants that have been shown to be pathogenic for turkey poults. Viable infected and noninfected tracheal rings were examined by phase-contrast microscopy, and fixed stained sections were examined by light microscopy. Alcaligenes faecalis at concentrations of 10(8) and 10(9) colony-forming units/ml caused ciliostasis, hydropic degeneration (characterized by blebbing of the plasma membrane, cellular swelling, and cytoplasmic vacuolation), and eventual sloughing of the ciliated epithelium. Only ciliated epithelial cells appeared affected. For comparison, other bacterial isolants not pathogenic for turkeys were tried in this system. These bacterial isolants included 3 isolants of A faecalis from human beings and isolants of Klebsiella pneumoniae, Escherichia coli, and A faecalis from turkeys. Inoculation of each of these bacterial cultures onto tracheal organ cultures failed to produce the lesions described.

Homidium (ethidium) bromide inhibition of avian reovirus replication.

Homidium bromide inhibited replication of avian reovirus in cell culture. Inhibition was dose dependent, and the critical event required that the dye be present during the replicative viral cycle and was not attributable to a cellular function.

Definitive emergency treatment of perforated duodenal ulcer.

A retrospective survey was made of all patients with a proved diagnosis of perforated duodenal ulcer admitted to a regional hospital during a recent six year period. The over-all mortality from this condition in the 192 patients was 11.4 per cent and mortality after operation, 6.3 per cent. One hundred and seventy-six patients were treated surgically. Seventy-seven patients underwent simple suture only with a mortality of 13 per cent and are compared with 99 patients treated by emergency vagotomy and pyloroplasty procedures with a 1 per cent mortality. Emergency definitive operations were performed without increased morbidity, mortality or hospital stay by junior surgeons with greatly improved long term results compared with simple suture. At the present time, emergency vagotomy and pyloroplasty procedures are the treatment of choice for a perforated duodenal ulcer.

The effect of topical povidone iodine on wound infection following abdominal surgery.

The effect of povidone iodine on wound sepsis following gastrointestinal surgery was studied in a trial involving 153 patients of whom 72 had their wounds sprayed with povidone iodine dry powder (Disadine DP) and 81 acted as a control group. The infection rate of 9.9 per cent in the patients treated with povidone iodine was significantly lower than that of 24.4 per cent in the control group (P less than 0.05). Bacterial contamination of the wound at the time of operation was shown to be of importance, being associated with a 52 per cent infection rate in the control group. However, spraying of contaminated wounds with povidone iodine reduced the infection rate to the significantly lower level of 11 per cent (P less than 0.05). We conclude that povidone iodine is a safe and effective means of reducing wound sepsis following gastrointestinal surgery.

Aspiration cytology and ultrasonography of cold thyroid nodules.

Over an 18 month period, 50 'high risk' patients with solitary or dominant cold thyroid nodules on 99m Tc-pertechnetate scanning, have undergone fine needle aspiration biopsy cytology (ABC) under general anaesthetic prior to thyroidectomy. Histological malignancy was confirmed in only four patients (8%). Cytological malignancy was suspected or confirmed in six patients, each having a false positive rate of 4%. There were no false negative reports. Ultrasonography, performed pre-operatively in 37 of the patients, did not significantly add to the overall patient management. ABC appears to be safe, simple and sufficiently accurate to incorporate its use routinely in the pre-operative assessment of thyroid nodules.