The effect of bisphosphonate treatment on the biochemical and cellular events during bone remodelling in response to microinjury stimulation.

Osteoporosis is one of the most prevalent bone diseases worldwide and is characterised by high levels of bone turnover, a marked loss in bone mass and accumulation of microdamage, which leads to an increased fracture incidence that places a huge burden on global health care systems. Bisphosphonates have been used to treat osteoporosis and have shown great success in conserving bone mass and reducing fracture incidence. In spite of the existing knowledge of the in vivo responses of bone to bisphosphonates, the cellular responses to these drugs have yet to be fully elucidated. In vitro model systems that allow the decoupling of complex highly integrated events, such as bone remodelling, provide a tool whereby these biological processes may be studied in a more simplified context. This study firstly utilised an in vitro model system of bone remodelling and comprising all three major cell types of the bone (osteocytes, osteoclasts and osteoblasts), which was representative of the bone's capacity to sense microdamage and subsequently initiate a basic multicellular unit response. Secondly, this system was used to study the effect of two commonly utilised aminobisphosphonate treatments for osteoporosis, alendronate and zoledronate. We demonstrated that microinjury to osteocyte networks being treated with bisphosphonates modulates receptor activator of nuclear factor kappa-B ligand and osteoprotegerin activity, and subsequently osteoclastogenesis. Furthermore, bisphosphonates increased the osteogenic potential following microinjury. Thus, we have shown for the first time that bisphosphonates act at all three stages of bone remodelling, from microinjury to osteoclastogenesis and ultimately osteogenesis.

Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor.

Mechanically stimulating cell-seeded scaffolds by flow-perfusion is one approach utilized for developing clinically applicable bone graft substitutes. A key challenge is determining the magnitude of stimuli to apply that enhances cell differentiation but minimizes cell detachment from the scaffold. In this study, we employed a combined computational modeling and experimental approach to examine how the scaffold mean pore size influences cell attachment morphology and subsequently impacts upon cell deformation and detachment when subjected to fluid-flow. Cell detachment from osteoblast-seeded collagen-GAG scaffolds was evaluated experimentally across a range of scaffold pore sizes subjected to different flow rates and exposure times in a perfusion bioreactor. Cell detachment was found to be proportional to flow rate and inversely proportional to pore size. Using this data, a theoretical model was derived that accurately predicted cell detachment as a function of mean shear stress, mean pore size, and time. Computational modeling of cell deformation in response to fluid flow showed the percentage of cells exceeding a critical threshold of deformation correlated with cell detachment experimentally and the majority of these cells were of a bridging morphology (cells stretched across pores). These findings will help researchers optimize the mean pore size of scaffolds and perfusion bioreactor operating conditions to manage cell detachment when mechanically simulating cells via flow perfusion.

Prevalence of exon 11 internal tandem duplications in the C-KIT proto-oncogene in Australian canine mast cell tumours.

OBJECTIVE: To measure the prevalence of internal tandem duplications (ITDs) in exon 11 of the proto-oncogene C-KIT in a sample of Australian cutaneous canine mast cell tumours (MCTs) drawn from general practice and to evaluate relationships between tumour mutation status and prognostic factors including signalment, tumour histological grade, tumour anatomical location and tumour size. METHODS: C-KIT exon 11 ITDs were detected by PCR in DNA extracted from formalin-fixed, paraffin-embedded canine MCTs sourced from three veterinary diagnostic laboratories in Adelaide and Melbourne. Tumours were graded according to two different systems (Patnaik and Kiupel systems) by board-certified anatomical pathologists blinded to the PCR results. Relationships between tumour mutation status and prognostic factors were evaluated using a generalised binary logistic regression analysis. RESULTS: ITDs were identified in 13 of 74 cutaneous canine MCT samples, giving an overall prevalence of 17.6% (95% confidence interval: 8.9-26.2%). ITDs were detected in 10 of 18 Patnaik grade III MCTs (55.6%) and 11 of 22 Kiupel high-grade MCTs (50%). Wald chi-square analysis revealed that detection of tumour ITDs was significantly associated with both Patnaik's and Kiupel's histologic grading systems (each: P < 0.001). The presence of the ITDs in MCTs was not associated with signalment, tumour anatomical location or tumour size. CONCLUSION: The prevalence of C-KIT exon 11 ITDs in Australian canine MCTs is similar to the prevalence in overseas canine populations (overall prevalence in Australia approximately 18%). ITDs were more frequently identified in higher grade MCTs.

Protection against Helicobacter pylori infection by intestinal immunisation with a 50/52-kDa subunit protein.

A mouse model of Helicobacter pylori infection was used to evaluate the vaccine antigen potential of the citrate synthase homologue protein purified from the H. pylori NCTC 11637 strain. Mice were immunised with the protein by intra-Peyer's patch immunisation. This route gives maximal intestinal immunisation and was used to screen oral vaccine candidate antigens without the added complication of simultaneously testing oral delivery systems. Two weeks post-immunisation mice were infected with Sydney strain H. pylori and 4 weeks after infection the mice were killed and the level of H. pylori infection in the stomach determined. Pre-immunisation with the 50/52-kDa protein led to a 84-91% reduction in H. pylori infection compared to unimmunised controls.

Nucleotide and amino acid sequence analysis of the porcine adenovirus 23K protein.

The genomic location of the viral encoded protease (23K) of porcine adenovirus serotype 3 (PAV3) was determined and the appropriate fragment cloned and sequenced. An open reading frame (ORF) coding for a polypeptide of 203 amino acids and a calculated molecular weight of 23.3 kDa was found. The ORF was situated in a position similar to that of the human adenovirus 23K, that is, between a putative stop codon for the hexon gene and the polyadenylation signal, AATAAA, for the late region 3. Amino acid sequence alignment of the predicted polypeptide with the sequences of the 23K proteins from other mammalian adenoviruses revealed homology of between 50% and 60% for all except the bovine adenovirus type 7, which displayed appreciable variance from the PAV3 putative 23K with an overall sequence homology of approximately 35%. Conserved cysteine, histidine and proline residues believed to be important in the activity of the 23K protein of human adenoviruses were also present in the PAV3 protein. The genomic location and amino acid sequence of the characterised reading frame suggests that this gene is that of the 23K protein of PAV3.

Nucleotide and amino acid sequence analysis of the 100K protein of a serotype 3 porcine adenovirus.

The genomic region between map units 69 and 78 of a type 3 porcine adenovirus (PAV3) was sequenced and analysed. An open reading frame (ORF) of 2514 nucleotides encoding a polypeptide of 838 amino acids and approximately 94.1 kDa was found. The size and location of the ORF suggested it was the PAV3 homologue of the 100K gene and this was confirmed by nucleotide sequence comparison with the 100K of human adenovirus type 2. Amino acid sequence alignment of the predicted polypeptide with the sequences of the 100K proteins of four human adenoviruses and type 10 fowl adenovirus revealed sequence identities of between 31% and 52%. Although amino acid conservation was present throughout the entire sequences compared, lower identity was noted in both the amino- and carboxy-termini.

Evaluation of two commercial enzyme-linked immunosorbent assays for detection of bovine viral diarrhoea virus in serum and skin biopsies of cattle.

AIM: To assess the ability of two commercial bovine viral diarrhoea (BVD) virus (BVDV) antigen-capture enzyme-linked immunosorbent assays (ELISAs) to detect virus in serum and skin biopsies. METHODS: Thirty cattle persistently infected (PI) with BVDV were identified using routine diagnostic laboratory testing. Additional ear-notch skin biopsies and blood samples were collected from these animals to confirm the diagnosis, and from 246 cohorts, to determine their BVDV status. Skin biopsies were soaked overnight in buffer and the eluate collected. All sera and eluate were tested using two commercially available ELISAs for detecting BVDV antigen, and a subsample of positive and negative sera was tested using a polymerase chain reaction (PCR) test. A study was also performed to ascertain the risk of cross contamination occurring during the collection and processing of skin biopsies. RESULTS: Both serum and skin samples tested using either ELISA resulted in the detection of all cattle identified as PI and no non-infected cattle were incorrectly classified as infected using either method. Agreement between all assays (ELISAs, whether performed on serum or skin, and PCR) was 100%. No cross-contamination of skin samples between animals was evident using routine biopsy methods. CONCLUSIONS: Viraemic cattle infected with BVDV were accurately identified using either of the two commercial ELISAs evaluated on either serum or skin samples. CLINICAL RELEVANCE: Either skin biopsies or serum samples can be collected from cattle to determine their BVDV status. This should overcome problems in accurately identifying the infection status of young calves in which colostral antibodies might interfere with the antigen-capture ELISA.

DNA sequence analysis of an avian adenovirus terminal protein precursor.

Nucleotide sequence of the genomic region between map units 25 and 31 of the fowl adenovirus serotype 10 (FAV 10) was determined and analyzed. An open reading frame (ORF) running from right to left (that is on /-strand) of 1806 nucleotides in length was found. This ORF encoded a polypeptide of 602 amino acids with a molecular weight (M[r]) of approximately 70.4 kilo-Daltons. The genomic location of the ORF was determined to be between map units 25.5 and 29.5, similar to the genomic position of the human adenovirus (HAV) terminal protein precursor (pTP). From its size, approximate genomic location and direction of transcription, this ORF was suspected to be the FAV10 homologue of the pTP. Amino acid sequence comparison with the HAV2 pTP revealed an amino acid sequence similarity of 32.4% but was 51 amino acids shorter in length. A potential proteolytic cleavage site was identified which would create a post-cleavage terminal protein of 316 amino acids, again comparable to the 322 amino acids of the post-cleavage TP of HAV.

Genomic mapping and sequence analysis of the fowl adenovirus serotype 10 hexon gene.

The gene for the major capsid protein (hexon) of fowl adenovirus serotype 10 (FAV-10) has been identified by the use of the expression vector pGEX and rabbit polyclonal antisera raised against FAV-10. The nucleotide sequence of the entire hexon gene has been determined. Sequence analysis revealed an open reading frame of 2808 bp coding for a putative polypeptide 936 amino acids long with a molecular mass of 105.5 kDa. The translation initiation codon has a local sequence which conforms with the optimal translation start sequence of CC(A/G)CCATGG. The location of the hexon gene in the FAV genome was from 46.85 to 52.81 map units, which is to the left of the hexon gene in the genomes of both bovine and human adenovirus (52.4 to 60.5 map units.). A splice acceptor site was identified 12 bp upstream of the initiation codon by using mRNA and PCR. It had the sequence TAGG which conforms to the consensus sequence of (C/T)AGG. Comparison of the amino acid sequence of the FAV-10 hexon with those of the bovine, human and murine hexon gene products revealed highest levels of identity occurring in the regions corresponding to the pedestals which form the base region of the hexon, and the lowest levels of identity in the regions corresponding to the loops which are exposed to the external environment.

The major late promoter and bipartite leader sequence of fowl adenovirus.

The region of the fowl adenovirus serotype 10 (FAV-10) genome containing the major late promoter (MLP) and leader sequences was determined and appropriate genomic fragments were cloned and sequenced. A TATA box was identified and the location of the putative transcription start site was determined. By using synthetic primers from the transcription start site in conjunction with oligonucleotides from the coding regions of the penton base and hexon genes, cDNA was produced from late mRNA isolated from cell cultures infected with FAV-10 at 24 h post-infection. The resulting cDNA was cloned and sequenced and the leader sequences thus identified. It was found that the FAV-10 MLP utilized only two leader sequences (a bipartite leader). By comparison with human adenoviruses (HAVs) it appeared that the second leader in HAVs was absent from the FAV-10. The second leader sequences of FAV-10 was larger than either the second or third leaders of HAVs, but was 29 basepairs shorter than the combined size of the leader sequences 2 and 3 from HAV-2. To confirm the transcription start site and leader sequences, single stranded cDNA was produced from mRNA using the primers from within the coding sequence for the penton base or hexon. A tail of dGTP's was added and cDNA synthesis was completed using an oligonucleotide from within the hexon or penton base coding sequence and a second poly-dCTP oligonucleotide. Sequencing of the resultant G-tailed DNA confirmed the location of the transcription start site as an adenosine residue 24 basepairs upstream from the 3-prime (3') end of the TATA box. Sequencing 5' of the TATA box failed to reveal any sequence similarity with the human adenovirus upstream stimulatory factor (USF). Various plasmids were constructed which placed the determined sequences of the MLP, leader, and the region upstream of the TATA box linked to the co-acetyl acid transferase (CAT) gene. These expression plasmids in transient expression assays of CAT activity in primary chicken kidney cell culture with or without FAV-10 co-infection were determined. These experiments showed that the cassette containing sequences 5' of the TATA box expressed CAT to a much greater level than cassettes not containing this upstream region and that the presence of virus significantly increased the activity of the promoter following the onset of viral DNA replication. Without the 5' region, cassettes failed to express above background levels. These results suggest that the basic structure of the fowl adenovirus MLP is similar to that of the human adenovirus although it utilizes a bipartite rather than a tripartite leader sequence.

Genomic location and nucleotide sequence of a porcine adenovirus penton base gene.

The putative penton base gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and sequenced. The genomic location of the PAV3 penton base was deduced by probing a Southern blot with a polymerase chain reaction generated product containing the human adenovirus type 2 (HAV2) penton base gene. Sequencing revealed an open reading frame (ORF) of 1527 nucleotides coding for a polypeptide of 509 amino acids. However, cDNA analysis indicated an acceptor splice site one nucleotide upstream of the second ATG in the ORF. This produced an ORF of 1452 nucleotides coding for a polypeptide of 484 amino acids with a calculated molecular weight of 54.5 kDa. Comparison with the HAV2 penton base amino acid sequence revealed the putative PAV3 penton base homologue to be 87 amino acids shorter with an overall amino acid homology of approximately 65%. Comparison with the penton base proteins of other HAV types revealed a region between amino acid positions 283 and 379 with no similarity.

Genomic location and nucleotide sequence of a serotype 3 porcine adenovirus hexon gene.

The putative hexon gene of a porcine adenovirus serotype 3 (PAV3) has been identified, cloned and the nucleotide sequence determined. The genomic location of the PAV3 hexon gene was determined and an open reading frame (ORF) encoding a polypeptide of 939 amino acids identified. Comparison of the nucleotide sequence of the putative PAV3 hexon gene with the sequence of the HAV2 hexon gene returned an overall identity of approximately 63%. A stop codon 144 nucleotides upstream and a start codon 18 nucleotides downstream of the ORF were identified and comparison with the HAV genome demonstrated that their positions corresponded to the stop site of the pVI gene and start site of the 23K gene, respectively. To confirm the correct start codon of the putative PAV3 hexon gene, the acceptor splice site for the putative PAV3 hexon gene was determined from cDNA and found to be between the two guanines immediately upstream of the first ATG in the ORF. Comparison with the HAV2 hexon protein showed overall identity of approximately 65%, with higher identity in the carboxy-terminus of approaching 76% over 380 amino acids. Multiple alignment of the PAV3 hexon amino acid sequence with other known HAV and animal adenovirus hexon sequences indicated that conservation is generally maintained but that identity is much lower within the loop structures of the protein.

Vaccination with a single dose of a recombinant porcine adenovirus expressing the classical swine fever virus gp55 (E2) gene protects pigs against classical swine fever.

A recombinant porcine adenovirus (rPAV) with the gp55 (E2) gene from the classical swine fever virus (CSFV) 'Weybridge' strain inserted into the right hand end of the PAV serotype 3 (PAV3) genome was constructed. Expression of gp55 was directed by the major late promoter and tri-partite leader sequences located and cloned from PAV3. No compensatory deletions of PAV DNA sequences were made. Vaccination of outbred pigs with a single dose of the recombinant virus (rPAV-gp55) resulted in complete protection from lethal challenge with CSFV. No adverse clinical signs were observed in vaccinated animals following administration of rPAV-gp55 and following challenge, no clinical signs of CSF were observed prior to, or at, post mortem. The insert made into the rPAV increased the genome length to 106.8% of wild type and therefore exceeded the expected maximum insert size for a stable recombinant by almost 2%. Thus rPAV-gp55 contains the largest stable insertion made into a non-deleted Mastadeno virus recombinant so far reported.