Incidence and prevalence of hepatitis B in patients with diabetes mellitus in the UK: A population-based cohort study using the UK Clinical Practice Research Datalink.

We assessed the incidence and prevalence of hepatitis B (Hep B) in patients with or without diabetes mellitus (DM) using the UK Clinical Practice Research Datalink (CPRD). This was a retrospective, observational study of diabetic and nondiabetic cohorts aged 0-80 years using CPRD (NCT02324218). Incidence rates (IR) for each cohort were calculated. Crude and adjusted (Poisson regression) IR ratios (IRR) were estimated with 95% confidence intervals (CI) to compare the cohorts. Hep B prevalence stratified by age, and hospitalization related to Hep B was also calculated. Of 7 712 043 subjects identified, 4 839 770 were included (DM: 160 760; non-DM: 4 679 010). Mean ages were 54.4 and 32.0 years, and 57.20% and 50.14% were male in the diabetic and nondiabetic cohorts, respectively. Hep B was identified in 29 diabetic and 845 nondiabetic subjects; IR was 4.03 per 100 000 person-years and 2.88 per 100 000 person-years, respectively. The adjusted IRR was 1.00 (95% CI: 0.70-1.50) between diabetic and nondiabetic cohorts. Hep B prevalence was higher in the diabetic cohort (0.01%-0.26%) than in the nondiabetic cohort (0.00%-0.03%) across the different age groups. Hep B-associated hospitalization IR was higher in the diabetic cohort (4.98-10.91) than the nondiabetic cohort (0.26-2.44). The Hep B IR, hospitalization and prevalence were higher in males in both cohorts. In conclusion, the risk of incident Hep B diagnosis in the diabetic cohort was not different from that in the nondiabetic cohort. However, prevalence of Hep B and Hep B-associated hospitalization rate was higher in the diabetic than in the nondiabetic cohort.

A splicing switch and gain-of-function mutation in FgfR2-IIIc hemizygotes causes Apert/Pfeiffer-syndrome-like phenotypes.

Intercellular signaling by fibroblast growth factors plays vital roles during embryogenesis. Mice deficient for fibroblast growth factor receptors (FgfRs) show abnormalities in early gastrulation and implantation, disruptions in epithelial-mesenchymal interactions, as well as profound defects in membranous and endochondrial bone formation. Activating FGFR mutations are the underlying cause of several craniosynostoses and dwarfism syndromes in humans. Here we show that a heterozygotic abrogation of FgfR2-exon 9 (IIIc) in mice causes a splicing switch, resulting in a gain-of-function mutation. The consequences are neonatal growth retardation and death, coronal synostosis, ocular proptosis, precocious sternal fusion, and abnormalities in secondary branching in several organs that undergo branching morphogenesis. This phenotype has strong parallels to some Apert's and Pfeiffer's syndrome patients.

Fibroblast growth factor receptor 2-IIIb acts upstream of Shh and Fgf4 and is required for limb bud maintenance but not for the induction of Fgf8, Fgf10, Msx1, or Bmp4.

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, was not affected in mice deficient for the IIIb isoform. FgfR2-IIIb(-/-) (lac)(Z) mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence did not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but did prevent induction of Shh and Fgf4, indicating that they are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. We propose that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth.

Cloning and chromosomal mapping of the mouse and human genes encoding the orphan glucocorticoid-induced receptor (GPR83).

The mouse glucocorticoid-induced receptor (GIR) is an orphan G protein-coupled receptor highly expressed in brain and thymus (Harrigan et al., 1989; 1991). We have cloned the mouse GIR gene (Gpr83), determined its genomic organization and compared it with the human gene. The genomic organization of the gene is similar in both species although differences leading to specific splicing variants in the mouse have been found. Three introns interrupting the coding sequence are common to both mouse and human. A short sequence in the second intron of the mouse gene can be alternatively spliced in, leading to an insertion in the second intracellular loop of the receptor. This insertion constitutes an additional exon which is not present in the human genome. The human GIR polypeptide shares 89.5% and 91.5% identity with its mouse and dog orthologs respectively. Splice variants lacking the first extracellular loop and the third transmembrane domain have been found in human and mouse species. The receptor variants resulting from these minor transcripts are likely to be non functional. Comparative genetic mapping of the Gpr83 gene showed that it maps to regions of conserved synteny on mouse chromosome 9 (A2-3 region) and human chromosome 11 (q21 region).

An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis.

The fibroblast growth factor receptor 2 gene is differentially spliced to encode two transmembrane tyrosine kinase receptor proteins that have different ligand-binding specificities and exclusive tissue distributions. We have used Cre-mediated excision to generate mice lacking the IIIb form of fibroblast growth factor receptor 2 whilst retaining expression of the IIIc form. Fibroblast growth factor receptor 2(IIIb) null mice are viable until birth, but have severe defects of the limbs, lung and anterior pituitary gland. The development of these structures appears to initiate, but then fails with the tissues undergoing extensive apoptosis. There are also developmental abnormalities of the salivary glands, inner ear, teeth and skin, as well as minor defects in skull formation. Our findings point to a key role for fibroblast growth factor receptor 2(IIIb) in mesenchymal-epithelial signalling during early organogenesis.

Skeletal disorders associated with fibroblast growth factor receptor mutations.

Mutations in three fibroblast growth factor receptor loci underlie several autosomal dominant skeletal disorders; these include dwarfism and various craniosynostosis syndromes affecting limb and craniofacial bone patterning. A functional analysis of several of these mutations has demonstrated that a constitutive activation of the receptor kinase is a common theme.

Expression of the bovine viral diarrhoea virus Osloss p80 protein: its use as ELISA antigen for cattle serum antibody detection.

The putative gene encoding the cytopathic bovine viral diarrhoea virus (BVDV) Osloss strain p80 protein was amplified by PCR and inserted into a T7 promoter-based vector for expression in Escherichia coli. Bacterial expression led to cytoplasmic insoluble inclusion bodies which were denatured by urea treatment and renatured by dialysis. Rabbit antisera were raised against this p80 recombinant antigen and assayed for the immunoprecipitation of either p120 or p80 protein from cytopathic or non-cytopathic BVDV biotype-infected bovine cells. The p80 gene sequence was also integrated into a baculovirus genome for its expression in Spodoptera frugiperda insect cells. The recombinant proteins isolated from bacteria or insect cells showed distinct antigenic properties when analysed by ELISA. Their ability to detect anti-BVDV specific antibodies was examined in a monoclonal antibody-based competitive ELISA performed on a series of field cattle sera. This comparative assay revealed the superiority of the insect cell-mediated expression to mimic the natural BVDV antigen produced by cell culture. The baculovirus/insect cell recombinant antigen gave the highest correlation between the ELISA-detected antibodies and the corresponding virus neutralization data.

Nucleotide sequence of the bovine viral diarrhoea virus Osloss strain: comparison with related viruses and identification of specific DNA probes in the 5' untranslated region.

The nucleotide sequence of the cytopathic Osloss isolate of bovine viral diarrhoea virus (BVDV) was deduced from overlapping cDNA clones and from PCR products. The Osloss genome is an RNA molecule of positive polarity containing 12,480 nucleotides and having the capacity to code for a polyprotein of 3975 amino acids. The presence of the previously described internal stop codon in this viral sequence was disproved after direct sequencing of the appropriate PCR-amplified fragment. Except for the previously reported insertion of a sequence coding for a ubiquitin-like protein, the viral genome shares great similarity with those of three other strains of the pestivirus genus. Computer-assisted sequence analyses and comparisons of known pestiviral genomic sequences led us to identify selected PCR primers in the 5' untranslated region. These primers were used successfully to amplify 18 distinct pestivirus isolates and potential DNA probes were noted from the deduced sequences. The possible use of a well conserved 26 base fragment as a diagnostic probe was confirmed in hybridization experiments. The 5' untranslated region was further studied and compared with those of other members of the Flaviviridae family, which includes the flaviviruses and the hepatitis C virus group. These sequence analyses support the possibility of discrimination amongst the closely related ruminant pestiviruses, border disease virus and BVDV.

Stabilization of T7-promoter-based pARHS expression vectors using the parB locus.

We describe a modification of the pAR3040 vector which results in its efficient stabilization during cell division. The parB locus of the plasmid R1 was introduced into the plasmid, pAR3040, to construct the pARHS vectors. These vectors are stable for at least 60 cell generations, even in the absence of selection by an antibiotic present in the culture media, both with or without IPTG induction.

Identification and production of pestivirus proteins for diagnostic and vaccination purposes.

Using a panel of monoclonal antibodies (MAbs) previously characterized by seroneutralization, immunofluorescence and radioimmunoprecipitation, we have identified Pestivirus proteins useful for diagnostic purposes from the cytopathic Osloss isolate of bovine viral diarrhea virus (BVDV). Proteins that should be useful for vaccination have also been analysed. Cell-free translation of RNA from glycoprotein-coding cDNA fragments produced, when synthesized in the presence of canine pancreatic microsomes, two glycosylated proteins that were independently recognized and immunoprecipitated by two distinct classes of neutralizing MAbs. A similar in vitro procedure was carried out on nonstructural protein-coding sequences and allowed to identify a viral translation product that specifically reacted with MAbs directed against the 80 kDA protein of a number of Pestivirus strains. Its positioning within the polyprotein encoded by the viral genome was refined by epitope scanning using synthetic hexameric peptides. This viral antigen was further expressed in E. coli, produced as inclusion bodies and used successfully as an ELISA antigen in both competitive and indirect assays for the detection of BVD antibodies in cattle sera.

The coding region for the 54-kDa protein of several pestiviruses lacks host insertions but reveals a "zinc finger-like" domain.

We sequenced cDNAs, amplified by the polymerase chain reaction (PCR), which correspond to the carboxy-terminal portion of the 54-kDa protein of various cytopathic (cp) or noncytopathic (ncp) pestiviral strains. Except for the previously described insertions in two cp strains of the bovine viral diarrhea virus (BVDV), we did not find comparable insertions in this gene in eight pestiviral strains. The predicted amino acid sequences of this 54-kDa protein portion contain a conserved cysteine-rich stretch remarkably similar to a "zinc finger-type" binding domain found in many gene-regulatory proteins. Thus, this protein may be involved in the binding to viral RNA.

ELISA detection of bovine viral diarrhoea virus specific antibodies using recombinant antigen and monoclonal antibodies.

A panel of monoclonal antibodies was prepared by immunization of BALB/c mice with Moredun (BD) virus strains. These antibodies were characterized by immunofluorescence and seroneutralization against BD, BVD and hog cholera (HC) virus strains, and radioimmunoprecipitation of BVD-infected cells extracts. The MAbs reacting with the majority of the Pestivirus strains recognize the 80 kDa antigen of the BVD cytophathic strains. The 80 kDa antigen of the BVD/Osloss virus strain has been cloned and expressed in E. coli as a fusion protein with beta-galactosidase. The fusion protein has been purified from inclusion bodies and used successfully as an antigen for ELISA detection of BVDV specific antibodies in bovine sera. A competitive ELISA using MAbs is more specific than a direct assay. These results compare well with the ones obtained with antigen extracted from BVDV-infected cells.