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Biomedical subjects

S D Carter

Publications and source records attributed to S D Carter.

At least 55 records · Page 3Linked to original sources
PubMed2000

Immunological and inflammatory characterisation of three canine cell lines: K1, K6 and DH82.

Three canine cell lines, K1, K6 and DH82, derived from canine malignant neoplasms, were characterised. They were examined for expression of surface antigens, cytokines, neuropeptide receptors, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The growth characteristics of the cell lines were established and bioassays used to detect production of TNF-alpha, IL-1 and IL-6. In the DH82 cell line, production of TNF-alpha and IL-6 was readily detected. Neither K1 or K6 cell lines produced any measurable amounts of TNF-alpha, IL-1 or IL-6. At a molecular level, using reverse transcription-polymerase chain reaction (RT-PCR) to detect specific mRNA, the DH82 cell line expressed TNF-alpha, IL-1 and IL-6, whereas the K1 and K6 cell lines expressed TNF-alpha. Canine IL-5, IL-8 and IL-10 mRNA were detected in the DH82 cell line but only IL-5 and IL-8 mRNA were detected in the K1 and K6 cell lines. Gelatin zymography was used for the detection of MMP-2 and MMP-9 and all three cell lines produced MMP-2 but only the DH82 cell line produced MMP-9. Reverse zymography was used to detect TIMP-1 and TIMP-2 and all three cell lines produced both proteins. The presence of these MMPs and TIMPs was confirmed at a molecular level using RT-PCR. Canine MMP-14 mRNA was detected in all three cell lines. For this investigation several genes for canine inflammatory molecules were cloned and sequenced for molecular detection; these included IL-1, IL-6, IL-8, TNF-alpha, MMP-9, MMP-14, TIMP-1, TIMP-2 and beta-actin. Of all the cell surface antigens tested, only CD14 was expressed on the DH82 cell line although CD5 and CD45 was partially expressed. The K1 and K6 cell lines were negative for all of the CD markers tested. K1 and K6 were negative for Neurokinin 1 receptor (NK1-R) but positive for Calcitonin gene related peptide receptor type 1 (CGRP-1R) and Calcitonin gene related peptide receptor component protein (CGRP-RCP). The DH82 cell line expressed neither NK1-R or CGRP-1R; however, it did express CGRP-RCP. Generally the DH82 cell line exhibited considerable similarity to canine monocytes, but all three cell lines will be useful as standards and for the purification of various immunological and inflammatory mediators in the dog.

Animals↗
PubMed2000

DLA-DQA1 polymorphisms in dogs defined by sequence-specific oligonucleotide probes (SSOP).

Several recent studies have identified DNA sequences for alleles of the DLA-DQA1 locus in the dog. To date, 10 DQA1 alleles have been reported. No data exists on the frequencies of these alleles within the general dog population, nor is there any indication of whether alleles are breed specific. We have addressed this issue by establishing a molecular-based sequence-specific oligonucleotide probing (SSOP) method to identify all published DQA1 alleles and have used these methods to type a large number of dogs. Oligonucleotide probes were designed to detect all the polymorphic sites in exon 2. This allowed assignment at the allele level. Three hundred and thirty dogs were typed for DQA1. All but two of the published DQA1 alleles were identified in these animals. One new allele was identified, and confirmed by DNA cloning and sequencing. This typing method provides a powerful tool for generating data that will be essential for studies investigating the genetic relationships between different breeds.

Animals↗
PubMed2000

Nomenclature for factors of the dog major histocompatibility system (DLA), 1998: first report of the ISAG DLA Nomenclature Committee.

A Nomenclature committee for Factors of the Dog Major Histocompatibility System or Dog Leukocyte Antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence based nomenclature for the genes of the DLA system. The remit of this committee includes: assignment of gene names rules for naming alleles assignment of names to published alleles assignment of names to new alleles rules for acceptance of new alleles DLA Nomenclature Committee, rules for acceptance, DLA genes and alleles, sequence based nomenclature.

Alleles↗
PubMed2000

Identification of further DLA-DRB1 and DQA1 alleles in the dog.

Three novel DLA-DRB1 alleles and one novel DQA1 allele have been identified in a panel of 367 dogs. These were suggested by unusual reaction patterns found in sequence specific oligonucleotide probing (SSOP) data. Four new alleles were confirmed using DNA cloning and sequencing.

Alleles↗
PubMed2000

Matrix metalloproteinases 2 and 9 activity in bovine synovial fluids.

Matrix metalloproteinases (MMPs) are important enzymes found in connective tissues and thought to be involved in cartilage degradation. They are detectable in bovine synovial fluid and may play a destructive role in bovine septic arthritis. The MMP gelatinase enzymes were detected by gelatin zymography using image analysis of the gels. The active gelatinase levels were determined by a gelatin degradation enzyme-linked immunosorbent assay (ELISA). Increased concentrations of MMP-9 activity were found in the synovial fluids of cows with septic arthritis (P < 0.001) in comparison with fluids from normal joints. Using the gelatin degradation ELISA the net active gelatinases were measured, and significant increases were found in gelatinase bioactivities in synovial fluids from septic joint disease cases (P < 0.001). Increased concentrations of MMP-2 activity were found in the synovial fluids of cows with aseptic arthritis, which appeared to be playing an important role in degradation of articular cartilage in joint disease. This finding required further investigation.

Animals↗
PubMed2000

Canine TIMP-2: purification, characterization and molecular detection.

Matrix metalloproteinases (MMPs), which degrade tissues in health and disease are under the control of the tissue inhibitors of MMPs, the TIMPs. TIMP-2 is particularly important for control of MMP-2 and both have been implicated in many pathological processes from arthritis to tumour invasion. This study characterized and detected TIMP-2 from canine cells; including synovial fibroblasts and three tumour-derived canine cell lines, K1, K6 and DH82. Gelatin zymography demonstrated that pro-MMP-2 is produced by synovial fibroblasts and the three cells lines. Reverse zymograms showed that all the cell sources tested secrete both TIMP-1 and TIMP-2. The 22 kDa band was purified and n-terminal amino acid sequencing showed it to be highly homologous to equine and human TIMP-2. Analysis of purified canine MMP-2 and MMP-9 showed that TIMP-2 is associated, and co-purifies with MMP-2. Polymerase chain reaction, using consensus primers, was used to detect TIMP-2 mRNA from the cell sources and proved positive in all cases. This work highlights the importance of TIMP-2 as the main inhibitor for MMP-2 and, therefore, opens the possibilities of targeting TIMP-2 for therapeutic intervention against connective amino acid tissue degradation in a range of diseases.

Animals↗
PubMed2000

Effects of dietary valine concentration on lactational performance of sows nursing large litters. NCR-42 Committe on Swine Nutrition.

A cooperative study, using 231 primiand multiparous crossbred sows from six experiment stations (IN, KS, MI, MN, ND, and OH), was conducted to determine the effects of elevating dietary valine concentration in corn-soybean meal diets on lactational performance of sows nursing large litters. Crossbred sows were fed diets containing a minimum of .60% lysine during gestation. Sows were allotted at farrowing to four dietary valine concentrations, .80, .95, 1.10, and 1.25%. Crystalline L-valine replaced cornstarch to maintain a constant ratio of corn:soybean meal across diets. Dietary lysine, provided by corn, soybean meal, and .15% crystalline L-lysine x HCl, was .90% in all diets. Sows were allowed ad libitum access to feed. Sows were weighed within 24 h after farrowing, and all litters were adjusted to > or = 10 pigs/litter by d 2 following farrowing. Average sow parity, number of pigs on d 2, and lactation length for the four treatments were, respectively, 2.3, 2.3, 2.3, 2.5; 10.9, 10.8, 10.8, 10.7; and 25.1, 24.5, 25.2, 25.0 d. The ADFI during lactation was 5.87, 5.77, 5.87, and 5.74 kg (P > .50); hence, valine intakes were 41, 48, 55, and 61 g/d (linear, P < .01). Lysine intake ranged from 51.5 to 52.7 g/d (P > .50). Sow weight after farrowing averaged 198 kg (P > .60). Overall pig survival to weaning was high (>92%), and the number of pigs weaned (10.1, 10.3, 10.3, 10.3) did not differ (P > .30) among treatments. Litter weaning weights (73.6, 73.6, 74.5, 72.6 kg), litter weight gains (55.1, 55.1, 56.0, 54.1 kg), sow weight change during lactation (-4.9, -5.4, -4.8, -6.3 kg), and return-to-estrus interval (7.5, 6.4, 6.9, 8.2 d) were not affected (P > .30) by dietary valine. There were no station x treatment interactions (P > .50). These results indicate no benefit of elevated dietary valine for lactating sows nursing > or = 10 pigs and consuming a corn-soybean meal diet containing .90% lysine and .80% valine.

Animal Feed↗
PubMed1999

Interbreed variation of DLA-DRB1, DQA1 alleles and haplotypes in the dog.

Although 36 DLA-DRB1 and 10 DLA-DQA1 allele sequences have been published to date, no data on individual allele frequencies exists, either for specific breeds or cross breeds, and the full extent of the polymorphism at each of these loci is still not known. We have used sequence-specific oligonucleotide probing (SSOP) to characterise a series of 367 dogs for their DRB1 and DQA1 alleles. These included individual animals from over 60 different breeds, with numbers per breed ranging from 1 to 39. DLA types were generated from 218 dogs for DRB1 and from 330 dogs for DQA1, while 181 dogs were characterised for both these loci. The frequency of individual DRB1 and DQA1 alleles showed considerable interbreed variation, e.g. 83% of West Highland White Terriers were DRB1*01 as opposed to 9% of Collies. No breed had >9 of the 22 DRB1 types defined in this study; several breeds had only two DRB1 types. DLA-DQA1 showed less variation in allele numbers per breed, but also showed considerable interbreed frequency variation. Haplotype analysis revealed over 44 different DRB1/DQA1 combinations. Of these, 25 were in a number of animals, and also in an animal that was homozygous for one or both of these loci. Some DRB1 alleles could be found in combination with several different DQA1 alleles, while others were only present in one haplotypic combination. DLA allele frequency data in normal dogs will be critical for disease association studies. It may also be possible to use haplotype data to establish the genetic relationships between different dog breeds.

Alleles↗
PubMed1999

Canine rheumatoid arthritis and inflammatory cytokines.

Bioassays were developed to detect canine pro-inflammatory cytokines. These enabled characterisation of these cytokines and their isoforms and provided means for their assay in the joints of dogs with different naturally occurring arthropathies. Canine IL-1 was detected by its induction of proliferation of D10(N4)M cell line, whilst IL-6 had a proliferative effect on B9 cell line. TNFalpha had a cytotoxic effect on WEHI 164 (13) cells. Partial purification of the cytokines was achieved by FPLC ion-exchange chromatography and two isoforms of IL-1 were shown, possibly corresponding to IL-1alpha and IL-1beta. TNFalpha only appeared as one isoform whereas IL-6 showed at least five isoforms, possibly corresponding to the other molecules in the IL-6 family, such as IL-11 and oncostatin M. Analysis of synovial fluids from dogs with osteoarthritis (OA) and rheumatoid arthritis (RA) showed that IL-1 and TNFalpha bioactivity was not readily detectable at increased levels in diseased joints but that IL-6 was significantly increased in both diseases. It is now important to determine the role of IL-6 in OA and RA in the dog, particularly in the induction of proteolytic enzymes which lead to cartilage loss.

Animals↗
PubMed1999

Immunocytochemical demonstration of lymphocyte subsets and MHC class II antigen expression in synovial membranes from dogs with rheumatoid arthritis and degenerative joint disease.

The study describes the distribution of canine leucocyte antigens in synovial membrane biopsies from six dogs with canine rheumatoid arthritis (CRA) and from eight dogs with osteoarthritis (OA) secondary to spontaneous rupture of the cranial cruciate ligament (CCL) (n = 5) or patellar luxation (n = 3). Synovial membranes from five dogs without evidence of joint lesions were used as control tissues. In the subsynovium of dogs with normal joints CD5+, CD4+, CD8+ and alpha beta TCR+ lymphocytes were present only in low numbers. With monoclonal antibody (mAb) to MHC class II antigen, either none or up to 20-30% of synovial lining cells were immunoreactive. Furthermore, scattered MHCII+ stromal cells were seen in the deeper subsynovial layer. In synovial membrane biopsies from dogs with CRA numerous diffusely and perivascularly distributed CD5+ lymphocytes were found in the subsynovium. CD4+ cells outnumbered CD8+ cells and were more numerous in the perivascular areas. In all the CRA cases examined, there were markedly higher numbers of alpha beta TCR+ cells compared with gamma delta TCR+ cells. With mAb to CD21, low numbers of immunoreactive lymphocytes were demonstrated. In all the CRA cases, a marked increase of MHC class II antigen expression was noted. In the majority of samples, 50% or more than 90% of the synovial lining cells were strongly MHC class II+. Throughout the subsynovial layer there were numerous MHC class II+ cells and included those with dendritic morphology and inflammatory mononuclear cells. Furthermore, marked perivascular immunoreactivity for MHC class II antigen was found. In biopsies from dogs with OA, there were markedly lower numbers of subsynovial CD5+, CD4+ and CD8+ lymphocytes. T-cells were mainly diffusely distributed. In three of the eight OA dogs examined, there was an increased percentage of synovial lining cells expressing MHC class II. The majority of OA cases had subsynovial major histocompatibility complex (MHC) class II+ cells with a dendritic morphology.

Animals↗
PubMed1999

DLA-DRB1 polymorphisms in dogs defined by sequence-specific oligonucleotide probes (SSOP).

To date, DNA sequences for 29 dog DLA-DRB1 alleles have been reported. However, no data exists on the frequencies of these alleles within the general dog population, nor is there any indication of whether there is interbreed variation of allele distribution. We have addressed this by establishing a molecular based sequence-specific oligonucleotide probing (SSOP) method to identify all of the known broad DRB1 types and we have used this to type a random panel of dogs. A series of oligonucleotide probes were designed to detect known polymorphisms in the three DRB1 hypervariable regions, together with two distinctive motifs in other regions of exon 2. This set of probes enabled us to assign broad DRB1 types. Two hundred and eighteen dogs were SSOP typed for DRB1. All but 4 of the published DLA-DRB1 alleles were identified in these animals. Interbreed variation in both allele distributions and allele frequencies were observed, which may be useful in the study of genetic variation between breeds. This variation also has implications for the selection of control groups for studies aimed at identifying MHC associations with disease susceptibility in the dog.

Animals↗
PubMed1999

Nomenclature for factors of the dog major histocompatibility system (DLA), 1998. First report of the ISAG DLA Nomenclature Committee. International Society for Animals Genetics.

A Nomenclature Committee for factors of the dog major histocompatibility system or dog leukocyte antigen (DLA) has been convened under the auspices of the International Society for Animal Genetics (ISAG) to define a sequence-based nomenclature for the genes of the DLA system. The remit of this committee includes: i) assignment of gene names; ii) rules for naming alleles; iii) assignment of names to published alleles; iv) assignment of names to new alleles; and v) rules for acceptance of new alleles.

Amino Acid Sequence↗
PubMed1999

Serological evidence of spirochaetal infections associated with digital dermatitis in dairy cattle.

A potentially infectious aetiology for digital dermatitis in dairy cattle was investigated and centred on the possible involvement of spirochaetes. An enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine anti-Borrelia burgdorferi (B31) and anti-Treponeme (USA bovine isolates) antibodies in the sera of cows; sera were further tested for antigen specificity by Western blotting. Compared to normal cows, those with digital dermatitis had a much higher seropositivity rate to B. burgdorferi and the treponemes. Significant correlations were shown between antibodies to B. burgdorferi and to Treponemes (P < 0.001), suggesting strong cross-reacting epitopes shared by these spirochaetes. In Western blotting of B. burgdorferi antigens, the main band detected by ELISA positive sera was the 41 kDa flagellar protein; lesser frequency of staining was seen with 34 (OspB), 39 and 55 kDa bands. For the USA treponeme antigens, ELISA positive sera gave reactions to the 34-kDa band and also bands at 41 and 55 kDa. Polyclonal antibodies to Treponema denticola and T. vincentii showed reactions with the bovine treponemes which were predominantly to the 34-kDa antigen. Monoclonal antibodies to B. burgdorferi flagella (41 kDa) antigen and OspA (31 kDa) did not detect any treponeme bands in Western blotting. The study has provided serological evidence that spirochaetes (which are related to human treponemes) may be involved in the pathogenesis of digital dermatitis.

Animals↗
PubMed1999

Matrix metalloproteinase-2 and -9 are activated in joint diseases.

A study was performed to identify the activation status of the gelatinase MMPs, MMP-2 and -9, in both normal and diseased equine articular tissues. In addition, the production and activation status of equine MMP-2 and -9 by equine articular cells and tissues in response to increasing IL-1beta concentrations was assessed. The study was performed to test the hypothesis that activation of MMPs is a fundamental step in the pathogenesis of joint diseases; and that this activation is mediated by the cytokine IL-1. Using purified equine MMP-2 and -9, the molecular weights of the zymogen and activated form of equine MMP-2 and -9 were identified by a combination of gelatin zymography and a gelatin degradation assay using aminophenylmercuric acetate as a chemical activator of the molecules. Normal equine articular tissues (cartilage and synovial membrane) maintained in short-term tissue culture produced MMP-2 zymogen alone, while similar tissues obtained from a variety of pathological conditions produce both zymogen and active MMP-2, as well as MMP-9 monomer and dimer. Activated MMP-9 was an inconsistent finding. Normal equine synovial fibroblasts in monolayer culture produced zymogen MMP-2 alone under basal conditions. A mild increase in active and zymogen MMP-2 levels occurred with IL-1beta treatment. Equine synovial membrane explants demonstrated a dose-dependent increase in active and zymogen MMP-2 and MMP-9 levels following IL-1beta treatment. Monolayer chondrocyte cell cultures demonstrated a dose-dependent mild increase in active and zymogen MMP-2 following IL-1beta treatment. Explant cartilage cultures demonstrated a dose-dependent mild increase in zymogen MMP-2 alone following IL-1beta treatment. This study supports the hypothesis that activation of MMPs is occurring in joint disease, and that in vitro stimulation of equine articular cells and tissues causes not only an increase in MMP production, but also an increase in amount of activated enzyme released. Further research is required to investigate the role of MMP activation in joint diseases, and to investigate the potential use of therapeutic agents, which inhibit MMP activation, in the treatment and prevention of joint diseases.

Animals↗
PubMed1999

Effects of porcine sometotropin on calcium and phosphorus balance and markers of bone metabolism in finishing pigs.

Six sets of four littermate barrows initially averaging 75.5 kg BW were equally fed (within blocks) fortified corn-soybean meal diets (1.30% lysine) containing two concentrations of Ca (.50 and 1.00%) and P (.45% and .90%) in a 34-d test. One-half of the pigs were injected with 4 mg of porcine ST (pST)/d. Following a 7-d adjustment period, total collection of feces and urine was performed during two periods (d 1 to 10 and d 20 to 30) for the determination of Ca and P apparent digestibility (absorption) and retention. Pigs were bled after each period (d 10, 20, and 30) for the determination of serum metabolites associated with Ca, P, and bone metabolism. Feed intake for the 30-d period averaged 2,020 g/d. There were no treatment x period interactions, so the absorption and retention data were pooled across periods. The absorption and retention of Ca and P were greater (P<.01) in pigs fed the higher Ca and P levels. Within each Ca and P level, pST reduced (P<.01) fecal Ca and P excretion. Administration of pST did not affect urinary P excretion, but it increased (P<.03) urinary Ca excretion in pigs fed the low-Ca diet. The absorption and retention of Ca and P were increased (P<.01) by pST; however, the increases in Ca retention and P absorption and retention on an absolute basis (g/d) were more pronounced in pST-treated pigs consuming the higher Ca and P diet (interaction, P<.10). Serum concentrations of 1,25-dihydroxyvitamin D3, osteocalcin, and IGF-I on d 10 and 30 were increased (P<.07) with pST administration. However, the increases in 1,25-dihydroxyvitamin D3 and osteocalcin in pST-treated pigs were more pronounced when the lower dietary Ca and P levels were fed (interaction, P<.08). Urinary excretion of hydroxyproline increased (P<.01) with pST administration, but this effect was more pronounced in pST-treated pigs fed the lower Ca and P diet (interaction, P<.09). These results suggest that pST increases the absorption and retention of Ca and P independent of dietary Ca and P level. However, serum measures associated with Ca, P, and bone metabolism in pST-treated pigs were dependent on the Ca and P content of the diet, suggesting an effect of pST on the homeostatic control of Ca, P, and bone metabolism.

Alkaline Phosphatase↗
PubMed1998

Matrix metalloproteinases 2 and 9 in canine rheumatoid arthritis.

Matrix metalloproteinases (MMPs) are considered important mediators of tissue damage in joint diseases. The levels of MMPs 2 and 9 were measured in samples of synovial fluid from 20 joints in seven dogs with rheumatoid arthritis by gelatin zymography. The results were compared with the actual gelatinolytic activity of the fluid measured in a gelatin-degradation ELISA. The gelatinolytic activity in synovial fluid from arthritic joints was markedly greater than that in fluid from disease-free joints. The zymographic activity attributable to MMP-9 (identified by Western blotting) was absent from synovial fluid from control joints but prominent in fluid from arthritic joints, and in these joints the presence of a 75 kDa form of MMP-9 was correlated with the gelatinolytic activity of the fluid measured by the ELISA (r = 0.81, P < 0.05). Synovial fluid from one dog with rheumatoid arthritis was examined before and after treatment with corticosteroids. After treatment its zymographic pattern had returned to normal.

Adrenal Cortex Hormones↗