S. D. Carter

INTEGRINS AND STRETCH ACTIVATED ION CHANNELS; PUTATIVE COMPONENTS OF FUNCTIONAL CELL SURFACE MECHANORECEPTORS IN ARTICULAR CHONDROCYTES

Perception of mechanical signals and the biological responses to such stimuli are fundamental properties of load bearing articular cartilage in diarthrodial joints. Chondrocytes utilize mechanical signals to synthesize an extracellular matrix capable of withstanding high loads and shear stresses. Recent studies have shown that chondrocytes undergo changes in shape and volume in a coordinated manner with load induced deformation of the matrix. These matrix changes, together with alterations in hydrostatic pressure, ionic and osmotic composition, interstitial fluid and streaming potentials are, in turn, perceived by chondrocytes. Chondrocyte responses to these stimuli are specific and well coordinated to bring about changes in gene expression, protein synthesis, matrix composition and ultimately biomechanical competence. In this hypothesis paper we propose a chondrocyte mechanoreceptor model incorporating key extracellular matrix macromolecules, integrins, mechanosensitive ion channels, the cytoskeleton and subcellular signal transduction pathways that maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte‐specific gene expression.

Apoptosis and the loss of chondrocyte survival signals contribute to articular cartilage degradation in osteoarthritis.

Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis (OA). Apoptotic pathways in chondrocytes are multi-faceted, although some cascades appear to play a greater in vivo role than others. Various catabolic processes are linked to apoptosis in OA cartilage, contributing to the reduction in cartilage integrity. Recent studies suggest that beta1-integrin mediated cell-matrix interactions provide survival signals for chondrocytes. The loss of such interactions and the inability to respond to IGF-1 stimulation may be partly responsible for the hypocellularity and matrix degradation that characterises OA. Here we have reviewed the literature in this area of cartilage cell biology in an effort to consolidate the existing information into a plausible hypothesis regarding the involvement of apoptosis in the pathogenesis of OA. Understanding of the interactions that promote chondrocyte apoptosis and cartilage hypocellularity is essential for developing appropriately targeted therapies for inhibition of chondrocyte apoptosis and the treatment of OA.

Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle

Bovine digital dermatitis (BDD) is a severe infectious cause of lameness which has spread through dairy cattle populations worldwide, causing serious welfare and agricultural problems. Spirochetes are the main organisms implicated and have previously proven difficult to isolate. This study aimed to isolate and characterise the range of spirochetes associated with BDD in the UK. Twenty-three spirochete isolates were obtained from 30 BDD lesions, which by 16S rRNA gene and flaB2 gene analysis clustered within the genus Treponema as three phylogroups; groups 1 (Treponema medium/Treponema vincentii-like), 2 (Treponema phagedenis-like) and 3 (Treponema denticola/Treponema putidum-like). The treponemes displayed large genotypic and phenotypic diversity between phylogroups and differed from named treponeme species. A previously isolated contagious ovine digital dermatitis spirochete was located within one of the three phylogroups, group 3, and could also be identified within this group on the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis may share the same aetiological agent. A strain isolated from a bovine interdigital dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine interdigital dermatitis and BDD may have the same causative agent. Two common enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD associated treponemes suggesting common metabolic pathways for sharing this novel niche or even common virulence traits. Further studies are required to determine whether the three groups of novel treponemes are representative of new treponeme taxa and to delineate how they interact with bovine tissues to cause disease.

Susceptibility to visceral leishmaniasis in the domestic dog is associated with MHC class II polymorphism

Zoonotic visceral leishmaniasis (VL) is a disease of dogs, humans and other animals caused by the intracellular macrophage parasite Leishmania infantum. We examined the relationship between DLA class II alleles (DRB1, DQA1, DQB1) and the course of infection in a cohort of Brazilian mongrel dogs exposed to natural L. infantum infection. DLA alleles were typed by sequence-based typing. DLA-DRB1 genotype was significantly associated with levels of anti-Leishmania IgG and parasite status assessed by PCR. Dogs with DLA-DRB1*01502 had higher levels of specific IgG and an increased risk of being parasite positive compared with dogs without this allele, controlling for other alleles and significant variables. No significant associations were seen for DLA-DQA1 or DLA-DQB1 alleles. These results suggest that the DLA-DRB1 locus plays a role in determining susceptibility to canine VL. As the domestic dog is the main reservoir for human infection, the identification of genetic factors influencing canine resistance or susceptibility to VL may provide insights into the immunology and potential control through vaccination of VL.

Dog MHC alleles containing the human RA shared epitope confer susceptibility to canine rheumatoid arthritis.

Abstract. To determine whether canine rheumatoid arthritis (CRA) is associated with dog MHC (DLA-DRB1) alleles which contain the QRRAA/RKRAA conserved third hypervariable region (3HVR) sequence, DNA samples were extracted from 61 dogs with clinically diagnosed small-joint polyarthritis and from 425 controls. Breed-matched controls were available for 41 cases. DLA-DRB1 genotypes were identified using molecular typing methods. Phenotype frequencies were compared between cases and controls and odds ratios with 95% confidence intervals calculated. Several DLA-DRB1 alleles were associated with increased risk for CRA: DLA-DRB1*002, DRB1*009, and DRB1*018. This was also observed for the presence of any shared epitope (SE)-bearing allele. The associations with DLA-DRB1*002 and the SE were maintained when only breed-matched cases and controls were compared. This study suggests that a conserved amino acid motif in the 3HVR present in some DRB1 alleles of both dogs and humans is associated with rheumatoid arthritis in both species.

Association of Unique, Isolated Treponemes with Bovine Digital Dermatitis Lesions

This study used a PCR-based approach targeting 16S rRNA gene fragments to determine the occurrence and association of the three bovine digital dermatitis (BDD) treponeme phylogroups within lesions found in cattle from the United Kingdom. Examination of 51 BDD lesions collected from infected cattle across the United Kingdom revealed that BDD treponeme group 1 (Treponema medium/Treponema vincentii-like), group 2 (Treponema phagedenis-like), and group 3 (Treponema putidum/Treponema denticola-like) were present in 96.1%, 98%, and 76.5% of BDD lesions, respectively. The three phylogroups were present together in 74.5% of lesions. The PCR assays enabled the isolation of further treponeme strains from previously mixed primary BDD lesion cultures. Here a representative from each of the three distinct treponeme phylogroups was isolated from a single BDD lesion for the first time. These data highlight the extent to which this disease is polytreponemal. Immunohistochemistry and electron microscopy were used to investigate lesional hoof tissues, resulting in treponemes being identified copiously in hair follicles and sebaceous glands, suggesting a potential route of exit and/or entry for these pathogens. This study gives further evidence for the importance of the three treponeme groups in BDD pathogenesis and reiterates the value of molecular genetic approaches for isolating and identifying fastidious anaerobes.

Heterogeneity of tensile strength and matrix metalloproteinase activity in the wall of abdominal aortic aneurysms.

Purpose: To measure the tensile strength of the aneurysm wall and the matrix metalloproteinase (MMP) activity in similar samples of aortic tissue. Methods: Detailed mechanical testing was conducted on 124 standardized specimens of aneurysm wall harvested from 24 patients undergoing elective aneurysm repair. The intrasac pressure required to cause aneurysm rupture was calculated based upon the Law of Laplace. In addition, MMP-2 and 9 were assayed from these specimens. Sixty specimens of nonaneurysmal aorta from 6 cadaveric organ donors served as controls. Intrasubject and intersubject variations were analyzed. Results: In the aneurysm specimens, the Youngs modulus was 1.80times106 N/m2, the load at break was 6.36 N, the strain at break was 0.30, the ultimate strength was 0.53times106 N/m2, and the MMP activity was 312 for MMP-2 and 460 for MMP-9. In the controls, the circumferential measurements were a Youngs modulus of 1.82times106 N/m2, a load at break of 5.43 N, strain at break of 0.29, ultimate strength of 0.61times106 N/m2, and MMP activity of 395 for MMP-2 and 2019 for MMP-9. Longitudinal measurements in controls were a Youngs modulus of 1.38times106 N/m2, a load at break of 11.39 N, a strain at break of 0.33, and ultimate strength of 1.30times106 N/m2. Intra and intersubject variation of all parameters was very high. Based upon the lowest measured tensile strength for each aneurysm, the intrasac pressure required to cause rupture varied from 142 to 982 mmHg. Conclusions: Localized “hot spots” of MMP hyperactivity could lead to focal weakening of the aneurysm wall and rupture at relatively low levels of intraluminal pressure. These data suggest that tensile strength of the sac is just as important as intrasac tension in determining the risk of rupture. Moreover, these observations may explain why some small aneurysms rupture and larger aneurysms do not. Assessment of rupture risk based on computation or measurement of wall stress may be subject to error and inaccuracy due to variations in wall tensile strength.

Staphylococcal colonization of mucosal and lesional skin sites in atopic and healthy dogs.

Staphylococcal colonization was compared in healthy dogs and in dogs with atopic dermatitis. Bacterial swabs were collected from the nasal mucosa, ear and perineum of 43 healthy and 24 atopic dogs and also from potentially infected skin lesions of the atopic dogs. Coagulase positive staphylococcal isolates were identified to the species level. At the time of this study Staphylococcus intermedius was considered a single species but has since been recognized as comprising at least three species with canine isolates believed to belong to Staphylococcus pseudintermedius. Of atopic dogs, 87.5% were colonized with S. intermedius compared to only 37.2% of healthy dogs. The ear was the only carriage site that showed any significant difference in S. intermedius isolation between healthy and atopic dogs. The perineum represented the most frequently colonized mucosal site for both groups. Sampling the nasal mucosa alone identified 71.4% of atopic and 37.5% of healthy S. intermedius carriers. Inclusion of a perineal swab identified 100% of atopic and 93.8% of healthy carriers. S. intermedius was isolated from all the lesional sites sampled from atopic dogs. Significantly fewer dogs were colonized by Staphylococcus aureus than S. intermedius, and there was no significant difference between S. aureus colonization of atopic and healthy dogs. S. aureus was not recovered from any lesions in atopic dogs. The results show that S. intermedius carriage is more prevalent in atopic dogs compared to healthy dogs and that to identify staphylococcal carriers both the nasal mucosa and the perineum should be sampled.

Gastrin-stimulated gastric epithelial cell invasion: the role and mechanism of increased matrix metalloproteinase 9 expression

The gastric hormone gastrin regulates the organization of the gastric epithelium, but the cellular control mechanisms are yet unknown. Epithelial remodelling typically involves extracellular proteolysis mediated by the matrix metalloproteinases (MMPs). Since a gene-array analysis of the gastric cancer cell line AGS-G(R) suggested that gastrin increased MMP-9 expression, we examined the control of MMP-9 expression by gastrin. Gelatin zymography confirmed gastrin induction of MMP-9 in AGS-G(R) cells, but showed a small inhibition of MMP-2. Immunocytochemical studies showed that MMP-9 was localized to vesicles that appeared to traffic along the processes that were extended in response to gastrin. Gastrin stimulated the invasion of AGS-G(R) cells through artificial basement membrane, which was reduced by an inhibitor of MMP-2/-9. There was also an increase in MMP-9 in the stomach of patients with elevated plasma gastrin and multiple-endocrine neoplasia type 1 (MEN-1) syndrome, suggesting in vivo regulation of MMP-9 expression by gastrin. Finally, we showed that the expression of 1.9 kb of human MMP-9 gene promoter coupled with luciferase (MMP-9-luc) was increased 7.65+/-1.2-fold by gastrin, via a pathway which includes stimulation of protein kinase C, and activation of Raf and the mitogen-activated protein (MAP) kinase pathway. The tumour suppressor menin (which is mutated in MEN-1 syndrome) inhibited the expression of MMP-9-luc by gastrin. These results suggest that gastrin increases MMP-9 expression, which is associated with increased invasion, and this is a putative mechanism regulating remodelling of the gastric epithelium.

Relative IgA deficiency and small intestinal bacterial overgrowth in German shepherd dogs

Serum immunoglobulin concentrations and densities of IgA-producing immunocytes in intestinal mucosa were compared in a group of clinically healthy dogs of various breeds, a group of clinically healthy German shepherd dogs, and a group of German shepherds with bacterial overgrowth in the proximal small intestine. Serum concentrations of IgA, but not IgM or IgG, were significantly lower in the clinically healthy German shepherd dogs than in other purebreed and mixbreed dogs, indicating that production of IgA by gut-associated lymphoid tissue might be relatively low in this breed. However, densities of IgA-producing cells were not significantly different comparing these two groups, suggesting that any impairment of mucosal IgA production is more likely to be related to defective synthesis or secretion of IgA than to reduced numbers of IgA-producing immunocytes. Comparable findings in German shepherd dogs with small intestinal bacterial overgrowth provided further indirect evidence that local immunity might be defective in this breed, since these luminal bacteria would be expected to stimulate mucosal IgA production. However, it is not clear whether such a defect is directly responsible for the overgrowth, or whether there is an indirect relationship between defective local immunity and bacterial overgrowth in German shepherd dogs.