Raloxifene and estrogen inhibit neointimal thickening after balloon injury in the carotid artery of male and ovariectomized female rats.

The effects of raloxifene and 17alpha-ethinyl estradiol (EE2) on intimal thickening in response to balloon injury were tested in male and ovariectomized female rats. In male rats, oral raloxifene and EE2, administered either by gavage or in the diet, inhibited arterial intimal thickening in response to balloon injury to a maximum of approximately 60 and 50%, respectively. The effect of oral raloxifene to decrease cholesterol was observed at doses (> or = 3 mg/kg/day) higher than those required to inhibit intimal thickening (> or = 0.03 mg/kg/day). Coadministration of the estrogen receptor antagonist, ICI 182,780 (5 mg/kg/day, s.c.), blocked the inhibition of balloon injury by raloxifene and EE2. Direct adventitial delivery of raloxifene (0.03 mg/kg/day) and EE2 (0.001 mg/kg/day) to the vascular wall inhibited intimal thickening by 63 and 53%, respectively. In ovariectomized female rats, oral raloxifene (0.01-3.0 mg/kg/day) and EE2 (0.08 mg/kg/day) inhibited intimal thickening to a maximum of 32 and 60%, respectively. Together, these data suggest that raloxifene and EE2, inhibit balloon arterial injury in the rat through direct effects on the vascular wall that involve the estrogen receptor and are at least partially independent of serum cholesterol.

LY320135, a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1 receptor to stimulation of cAMP accumulation.

LY320135 is a selective antagonist for the brain CB1 receptor, having greater than 70-fold higher affinity for the CB1 than the peripheral CB2 receptor. The Ki values for LY320135 at the CB1 and CB2 receptors, transfected and stably expressed in cell lines, were 224 nM and > 10 microM, respectively. Similar Ki values were measured in binding studies performed on cerebellum and spleen membrane preparations endogenously expressing the CB1 (203 nM) and CB2 (> 10 microM) receptors, respectively. LY320135 functionally reversed anandamide-mediated adenylate cyclase inhibition in Chinese hamster ovary (CHO) cells stably expressing the CB1 receptor. Pertussis toxin treatment of CHO cells expressing the CB1 receptor attenuated the anandamide-mediated inhibition of adenylate cyclase and unmasked a stimulatory effect of anandamide on adenylate cyclase. The stimulatory component was blocked with LY320135. This compound also blocked WIN 55212-2-mediated inhibition of N-type calcium channels and activation of inwardly rectifying potassium channels in N18 and AtT-20-CB2 cells, respectively. LY320135 is a promising lead compound for the further development of novel, potent and selective cannabinoid antagonists of novel structure.

Immunopathogenesis and physical maps of fowl adenovirus serotype 9.

Restriction endonuclease maps of the genome of fowl adenovirus (FAV) serotype 9 have been constructed for the restriction endonucleases NdeI, NotI and XbaI. The total size of the FAV-9 genome was estimated to be 44.5 kb pairs, consistent with previous reports that FAV genomes are approximately 10 kb larger than human adenovirus (HAV). The pathogenicity of this virus in day-old chickens was intermediate between the pathogenicity of the non-pathogenic and the highly pathogenic FAVs.

Cloning and sequencing of the chicken anaemia virus (CAV) ORF-3 gene, and the development of an ELISA for the detection of serum antibody to CAV.

Chicken anaemia virus (CAV) is a small, unclassified virus involved in anaemia and suspected of causing immunosuppression in young chickens. We have developed an ELISA for the detection of serum antibody to CAV based on cloned antigen. The gene for ORF-3 (the putative capsid protein) was cloned, sequenced and expressed in a bacterial expression system, pGEX. An ORF-3 fusion protein was used to produce an indirect ELISA.

Molecular characterisation of peanut agglutinin-binding glycoproteins from Eimeria tenella.

A group of glycoproteins, which strongly bind peanut agglutinin (PNA) was found in Eimeria tenella. Two major antigenic glycoproteins, Et110gp and Et35gp, were identified in sporulated oocysts and sporozoites. Molecular characterisation of carbohydrate moieties (lectin binding, enzymic hydrolysis and monosaccharide composition) revealed that both glycoproteins are rich in galactose and N-acetylgalactosamine, and appear to be sialylated. Both glycoproteins were susceptible to treatment with neuraminidase followed by O-glycosidase, suggesting that the oligosaccharide chains are attached to the protein by an O-glycosidic linkage to serine and/or threonine. Purified Et35gp contained a large number of serine (14) and threonine (33) residues, and was rich in glycine. This protein aggregated after repetitive lyophilisation and migrated on SDS-PAGE gels as an 85,000 protein. Sera against purified Et35gp raised in chickens and rabbits, and anti-E. tenella immune chicken serum recognised both antigens on blots and on the surface of sporozoites. Chickens immunised with purified Et35gp were not protected against coccidial infection.

Serological relationships within the group E fowl adenoviruses.

Antisera were raised in chickens to six group E fowl adenoviruses (FAV) which have been divided into a highly virulent (hypervirulent) and a mildly virulent subgroup using restriction endonuclease analysis. Virus neutralisations showed that these two distinct restriction endonuclease groups were distinguishable serologically, and indicated a possible vaccine candidate for use against the hypervirulent FAV. The suitability of this candidate was established in challenge experiments where vaccination with this virus protected against challenge from another hypervirulent virus as well as one of the mildly virulent FAV.

Enhanced H2O2 release from immune chicken leucocytes following infection with Eimeria tenella.

Cell-mediated immunity is thought to be important in the resistance of chickens to infection by coccidia, and it has been demonstrated that sporozoites of Eimeria tenella are very sensitive to superoxide ions. Therefore an investigation into the cellular responses in naive specific pathogen-free and hyperimmune birds was carried out with particular attention to their ability to produce reactive derivatives of oxygen. Leucocytes were isolated from the blood, spleen and caecal mucosa of chickens infected with E. tenella and assessed for their ability to release H2O2. Leucocytes obtained from the blood and spleen of hyperimmune birds 1 day after challenge showed an elevated ability to produce reactive oxygen intermediates. In contrast, the ability of leucocytes from naive chickens to produce these molecules was transiently depressed after challenge. Prior to challenge, mucosal leucocytes from immune chickens were also able to release heightened levels of H2O2 when compared with cells from naive chickens.

Infectious laryngotracheitis virus growth, DNA replication, and protein synthesis.

The polypeptides associated with infection of primary chicken kidney (CK) cells with infectious laryngotracheitis virus (ILTV) were examined by metabolic labelling with [35S]methionine and SDS-PAGE. Polypeptide synthesis was followed over the first 24 h post-infection (p.i.) as this was shown to be the period of viable virus production. A total of 16 ILTV encoded or induced polypeptides were identified using metabolic labelling. The use of inhibitors of protein and DNA synthesis in conjunction with metabolic labelling and viral DNA replication studies enabled a cascade pattern of gene expression, similar to that of herpes simplex virus type 1 (HSV-1), to be established for ILTV. Representatives of alpha, beta, gamma 1 and gamma 2 classes of genes were identified. In contrast to infection with HSV types 1 and 2, which leads to a rapid inhibition of total host cell polypeptide synthesis, ILTV infection of CK cells did not result in a complete inhibition of cellular protein synthesis, with only a small number of host cell polypeptides absent from infected cells.

Molecular characterization of highly virulent fowl adenoviruses associated with outbreaks of inclusion body hepatitis.

Restriction enzyme analysis of DNA was used to characterize fowl adenoviruses (FAVs) consistently associated with outbreaks of acute inclusion body hepatitis. When low doses of these FAVs were administered via a natural route to chickens they caused IBH. A strong genomic relationship was demonstrated between these virulent FAVs. In contrast, the genomes of serologically related, but non-virulent or mildly virulent FAVs were found to differ substantially from those of the virulent FAVs.

Vaccination with affinity-purified glycoproteins protects chickens against infectious laryngotracheitis herpesvirus.

Glycoproteins of infectious laryngotracheitis virus (ILTV) were purified from detergent extracts of virus-infected cells by lectin affinity chromatography. In these preparations, glycoproteins of 205, 160, 115, 90, 85, 74, 60 and 50 kDa were recognized by immune chicken serum in Western blotting. Vaccination of chickens with these ILTV glycoproteins protected up to 83% of chickens against replication of the challenge virus as demonstrated by the absence of viral antigen in the trachea. Both neutralizing antibody and delayed-type hypersensitivity responses were induced by vaccination with glycoprotein preparations, but neither correlated with protection.

Physicochemical and immunological characterization of recombinant host-protective antigen (VP2) of infectious bursal disease virus.

Small fusions to the N-terminal end of the host-protective antigen (VP2) of infectious bursal disease virus lead to stable expression of VP2 in Escherichia coli and yeast, and reduce the levels of inclusion body formation in E. coli in comparison to VP2 constructs with larger N-terminal fusions. VP2 produced with small N-terminal fusions, like native viral VP2, can be fractionated into a high molecular weight 'multimeric' form and a monomeric form. A virus-neutralizing monoclonal antibody that only recognizes undenatured VP2 preferentially reacts with multimeric forms of recombinant VP2. Both native and recombinant monomeric forms of VP2 are non-immunogenic. The multimeric forms of viral and yeast-derived VP2 are highly immunogenic, while those produced in E. coli are not.

A recombinant subunit vaccine that protects progeny chickens from infectious bursal disease.

The major protective immunogen of infectious bursal disease virus (IBDV), VP2, was produced in a highly immunogenic form by expression in the yeast Saccharomyces cerevisiae. The recombinant protein, formulated as an oil-emulsion vaccine, induced both virus neutralising and ELISA antibodies in specific pathogen free hens. These antibodies were passed, via the egg yolk, to progeny chickens and protected them against a challenge infection with virulent IBDV. The protective efficacy of maternal antibodies to the recombinant VP2, as assessed by ELISA, was comparable to that of antibodies to the whole virus. The recombinant subunit vaccine induced anamnestic serum antibody responses in hens primed previously with live virus, and hence can replace the conventional inactivated vaccines administered to breeding hens.

Nucleotide sequence of the gene encoding infectious laryngotracheitis virus glycoprotein B.

The nucleotide sequence of the infectious laryngotracheitis virus (ILTV) gene encoding the 205K complex glycoprotein (gp205) was determined. The gene is contained within a 3-kb EcoRI restriction fragment mapping at approximately map coordinates 0.23 to 0.25 in the UL region of the ILTV genome and is transcribed from right to left. Nucleotide sequence analysis of the DNA fragment identified a single, long open reading frame capable of encoding 873 amino acids. The predicted precursor polypeptide derived from this open reading frame would have a calculated Mr of 98,895 Da and contains nine potential glycosylation sites. Hydropathic analysis indicates the presence of an amino terminal hydrophobic sequence and hydrophobic carboxyl terminal domain which may function as a signal peptide and a membrane anchor sequence, respectively. Comparison of the predicted ILTV gp205 protein sequence with those of other herpesviruses revealed a significant sequence similarity with gB-like glycoproteins. Extensive homology was observed throughout the molecule except for the amino and carboxyl termini. The high homology in predicted primary and secondary structures is consistent with the essential role of the gB family of proteins for viral infectivity and pathogenesis.

Humoral responses and salmonellosis protection in chickens given a vitamin-dependent Salmonella typhimurium mutant.

Humoral responses in chickens inoculated with an aromatic vitamin dependent (Aro-) Salmonella typhimurium mutant (STM) were studied to ascertain the efficacy of the organism as a vaccine for salmonellosis and possibly as a delivery system for antigens from enteric pathogens of chickens. Serum antibody responses in chickens that were given oral or subcutaneous inoculations of the bacterium followed the classic order of antibody production, with IgM being detected first, followed by IgG and IgA. Antibody responses in the gut of orally inoculated chickens were restricted to IgG and IgA. Weight gain measured in chickens given high doses of STM (up to 5 x 10(9)) orally, revealed that the bacterium did not adversely affect the chickens; in fact, inoculated chickens had significantly higher body weights than controls at the same age. Salmonellosis protection of chickens by oral vaccination with STM was examined in a vaccination/challenge experiment. The experiment revealed that oral vaccination reduced excretion of a virulent S. typhimurium used as the challenge organism.

Genetic susceptibility of chicken macrophages to in vitro infection with infectious laryngotracheitis virus.

Chicken macrophages are susceptible to infection with infectious laryngotracheitis virus (ILTV), the level of infection being dependent, in part, on the genotype of the host cell. Following infection in vitro a greater proportion of macrophages from the ILTV-resistant J1(B113/113) and N1(B114/114) inbred lines of chickens were found to be positive for ILTV antigens, than macrophages from the ILTV-susceptible M1(B15/15) chickens. The proportion of ILTV-positive macrophages was found to be genetically regulated, in part by the chicken major histocompatibility complex (MHC), although alloantisera to class I and class II MHC antigens did not reduce the number of macrophages infected. Similarly, the phagocytic activity of the cultured macrophages from chickens of different MHC genotype did not correlate with their susceptibility to infection with ILTV.

Genetic resistance to infectious laryngotracheitis in inbred lines of White Leghorn chickens.

The susceptibility of inbred lines of chickens to graded levels of infection with a virulent isolate (wild-strain) of infectious laryngotracheitis virus (ILTV) was investigated. An association was found between the level of resistance to ILTV and the line of inbred chickens, with chickens from the J1 inbred line being more resistant to clinical ILTV than M1 and N1 inbred chickens. The N1 chickens, however, became susceptible with higher doses of ILTV, while J1 chickens remained relatively resistant to clinical disease. Studies using other inbred lines of chicken which had the same major histocompatibility complex (MHC) antigens showed similar levels of resistance or susceptibility, suggesting a possible association of the chicken MHC with resistance or susceptibility to ILTV.

Virus-neutralizing and passively protective monoclonal antibodies to infectious bursal disease virus of chickens.

Murine monoclonal antibodies (MAbs) were produced to assist in the identification and characterization of the virus-neutralizing epitopes of infectious bursal disease virus (IBDV). Only MAbs that reacted in Western blotting with viral protein 2 (VP2) or immunoprecipitated VP2 neutralized the infectivity of the virus in cell culture and passively protected young chickens from infection. Three of the neutralizing MAbs did not react with denatured viral proteins. Additivity enzyme-linked immunosorbent assays indicated that the six virus-neutralizing MAbs recognized two spatially independent epitopes. The ability of two of the virus-neutralizing MAbs to neutralize a variant of IBDV that had escaped neutralization by all the other MAbs confirmed the existence of two distinct neutralizing epitopes. The results support the hypothesis that there are at least two non-overlapping epitopes recognized by the virus-neutralizing MAbs reported in this study, although these may still be within one conformational site on VP2 of IBDV.

Detection of infectious laryngotracheitis virus in chickens using a non-radioactive DNA probe.

A DNA hybridization assay using a non-radioactive probe has been developed for the detection of infectious laryngotracheitis virus (ILTV) DNA. A 1.4-kilobase pair BamHI fragment of ILTV genomic DNA was cloned and then labeled by one of two methods; nick translation using 32P-dATP or non-radioactive labeling using a commercially available DNA labeling and detection kit. The non-radioactive DNA labeling method proved to be as sensitive as the radioactive method. Using the non-radioactive probe, ILTV DNA was readily detected in tracheal samples from acutely infected chickens and also from convalescent chickens at a time when viral antigen could no longer be detected by the enzyme-linked immunosorbent assay or the virus could no longer be reisolated. This technique provides a safe and effective means of identifying field outbreaks of ILTV and also may detect latent ILTV infections relatively quickly and inexpensively.

Gallid herpesvirus 1 (infectious laryngotracheitis virus): cloning and physical maps of the SA-2 strain.

Clones representing 90% of the genome of Gallid herpesvirus 1 (infectious laryngotracheitis virus; ILTV) were obtained and used in hybridization experiments to construct EcoRI, KpnI amd SmaI physical maps. The genome was 155 kilobase pairs (kbp) and comprised of a long unique sequence (120 kbp) and a short unique sequence (17 kbp) bounded by repeat sequences each of 9 kbp. An unrelated second pair of repeat sequences was located at 0.67 and 0.88 map untis. A terminal repeat of the unique long region (UL) was also detected, but no isomerization of UL was detected.

The effect of infectious bursal disease virus on B lymphocytes and bursal stromal components in specific pathogen-free (SPF) White Leghorn chickens.

The effect of infectious bursal disease virus (IBDV) was studied on adult specific pathogen-free (SPF) white Leghorn chickens through analysis of peripheral blood cell suspensions and histological staining patterns on various tissue types, with specific mAbs. A rapid, progressive loss of B lymphocytes was observed in the bursal cortex and medulla, peripheral blood and thymic medulla. There was, however, a resistant population of MUI-36+ cells at the bursal cortico-medullary junction and scattered around splenic periellipsoidal sheaths. These resistant cells were suggested to be a subpopulation of macrophages which expressed the MUI-36 marker; alternatively these may have phagocytosed virally infected B cells or their remnants. Throughout the period of infection, T lymphocytes appeared nonsusceptible. Further, while the distribution of stromal cell antigens within the bursal cortex remained unaltered, particular epitopes on the surface epithelium and in the medulla were lost as a consequence of viral infection. The data presented therefore suggests that immunodepression of chickens post-IBDV infection, may arise as a direct consequence of infection of B lymphocytes; additionally, it is possible that the elimination of certain crucial elements within the bursal microenvironment may contribute to this state.