Repeated measurements of blood lactate concentration as a prognostic marker in horses with acute colitis evaluated with classification and regression trees (CART) and random forest analysis.

The objective of this study was to investigate the prognostic value of single and repeated measurements of blood l-lactate (Lac) and ionised calcium (iCa) concentrations, packed cell volume (PCV) and plasma total protein (TP) concentration in horses with acute colitis. A total of 66 adult horses admitted with acute colitis (<24 h) to a referral hospital in the 2002-2011 period were included. The prognostic value of Lac, iCa, PCV and TP recorded at admission and 6 h post admission was analysed with univariate analysis, logistic regression, classification and regression trees, as well as random forest analysis. Ponies and Icelandic horses made up 59% of the population, whilst the remaining 41% were horses. Blood lactate concentration at admission was the only individual parameter significantly associated with probability of survival to discharge (P < 0.001). In a training sample, a Lac cut-off value of 7 mmol/L had a sensitivity of 0.66 and a specificity of 0.92 in predicting survival. In independent test data, the sensitivity was 0.69 and the specificity was 0.76. At the observed survival rate (38%), the optimal decision tree identified horses as non-survivors when the Lac at admission was ≥4.3 mmol/L and the Lac 6 h post admission stayed at >2 mmol/L (sensitivity, 0.72; specificity, 0.8). In conclusion, blood lactate concentration measured at admission and repeated 6 h later aided the prognostic evaluation of horses with acute colitis in this population with a very high mortality rate. This should allow clinicians to give a more reliable prognosis for the horse.

Flow cytometry detection of vitamin D receptor changes during vitamin D treatment in Crohn's disease.

Crohn's disease (CD) is a chronic inflammatory disease associated with a dysregulated T cell response towards intestinal microflora. Vitamin D has immune modulatory effects on T cells through the nuclear vitamin D receptor (VDR) in vitro. It is unclear how oral vitamin D treatment affects VDR expression. The aim of this study was to establish a flow cytometry protocol, including nuclear and cytoplasmic VDR expression, and to investigate the effects of vitamin D treatment on T cell VDR expression in CD patients. The flow cytometry protocol for VDR staining was developed using the human acute monocytic leukaemia cell line (THP-1). The protocol was evaluated in anti-CD3/CD28-stimulated peripheral blood mononuclear cells (PBMCs) from vitamin D3- (n = 9) and placebo-treated (n = 9) CD patients. Anti-VDR-stained PBMCs were examined by flow cytometry, and their cytokine production was determined by cytokine bead array. VDR, CYP27B1 and RXRα mRNA expression levels in CD4(+) T cells were measured by quantitative reverse transcriptase polymerase chain reaction. The flow cytometry protocol enabled detection of cytoplasmic and nuclear VDR expression. The results were confirmed by confocal microscopy and supported by correlation with VDR mRNA expression. VDR expression in CD4(+) T cells increased following stimulation. This VDR up-regulation was inhibited with 30% by vitamin D treatment compared to placebo in CD patients (P = 0027). VDR expression was correlated with in-vitro interferon-γ production in stimulated PBMCs (P = 0.01). Flow cytometry is a useful method with which to measure intracellular VDR expression. Vitamin D treatment in CD patients reduces T cell receptor-mediated VDR up-regulation.

Polymorphisms of muscle genes are associated with bone mass and incident osteoporotic fractures in Caucasians.

The interaction between muscle and bone is complex. The aim of this study was to investigate if variations in the muscle genes myostatin (MSTN), its receptor (ACVR2B), myogenin (MYOG), and myoD1 (MYOD1) were associated with fracture risk, bone mineral density (BMD), bone mineral content (BMC), and lean body mass. We analyzed two independent cohorts: the Danish Osteoporosis Prevention Study (DOPS), comprising 2,016 perimenopausal women treated with hormone therapy or not and followed for 10 years, and the Odense Androgen Study (OAS), a cross-sectional, population-based study on 783 men aged 20-29 years. Nine tag SNPs in the four genes were investigated. In the DOPS, individuals homozygous for the variant allele of the MSTN SNP rs7570532 had an increased risk of any osteoporotic fracture, with an HR of 1.82 (95 % CI 1.15-2.90, p = 0.01), and of nonvertebral osteoporotic fracture, with an HR of 2.02 (95 % CI 1.20-3.41, p = 0.01). The same allele was associated with increased bone loss (BMC) at the total hip of 4.1 versus 0.5 % in individuals either heterozygous or homozygous for the common allele (p = 0.006), a reduced 10-year growth in bone area at the total hip of 0.4 versus 2.2 and 2.3 % in individuals heterozygous or homozygous for the common allele, respectively (p = 0.01), and a nonsignificantly increased 10-year loss of total-hip BMD of 4.4 versus 2.7 and 2.9 % in individuals heterozygous or homozygous for the common allele, respectively (p = 0.08). This study is the first to demonstrate an association between a variant in MSTN and fracture risk and bone loss. Further studies are needed to confirm the findings.

Functional polymorphisms in the P2X7 receptor gene are associated with osteoporosis.

UNLABELLED: The P2X(7) receptor is an ATP-gated cation channel. We investigated the effect of both loss-of-function and gain-of-function polymorphisms in the P2X(7) receptor gene on BMD and risk of vertebral fractures and found that five polymorphisms and haplotypes containing three of these polymorphisms were associated with BMD and fracture risk. INTRODUCTION: The P2X(7) receptor is an ATP-gated cation channel. P2X(7) receptor knockout mice have reduced total bone mineral content, and because several functional polymorphisms have been identified in the human P2X(7) receptor gene, we wanted to investigate the effect of these polymorphisms on BMD and risk of vertebral fractures in a case-control study including 798 individuals. METHODS: Genotyping was carried out using TaqMan assays. BMD was measured using dual energy X-ray absorptiometry, and vertebral fractures were assessed by lateral spinal X-rays. RESULTS: The rare allele of a splice site polymorphism, 151 + 1: G-T, was associated with increased fracture risk and reduced BMD in women. Two other loss-of-function polymorphisms, Glu496Ala and Gly150Arg, were also associated with BMD. The Glu496Ala variant allele was associated with decreased lumbar spine BMD in women and decreased total hip BMD in men. The 150Arg allele was associated with decreased total hip BMD in women and men combined. The minor allele of the gain-of-function polymorphism, Ala348Thr, was associated with reduced fracture risk and increased BMD at all sites in men. The Gln460Arg variant allele, which has been associated with increased receptor function in monocytes, was associated with increased total hip BMD in women. With the exception of His155Tyr for which we found conflicting results in men and women, our results are consistent with the phenotype of the knockout mouse. Analysis of a haplotype containing Ala348Thr, Gln460Arg, and Glu496Ala showed that the effects of the haplotypes on BMD and fracture were driven by Ala348Thr in men and by Gln460Arg and Glu496Ala in women. CONCLUSION: In conclusion, we found that functional polymorphisms in the P2X(7) receptor gene and haplotypes containing three of these polymorphisms are associated with osteoporosis.

The expression and regulation of bone-acting cytokines in human peripheral adipose tissue in organ culture.

The humoral cross-talk between bone and fat is an area of increasing interest. We investigated the expression and regulation of the bone-acting cytokines; bone morphogenetic protein 2 (BMP2), connective tissue growth factor (CTGF), osteoprotegerin (OPG), and transforming growth factor beta (TGFB1). Subcutaneous adipose tissue was aspirated from lean, healthy women. Tissue samples were incubated with interleukin 1-ß (IL1-ß), tumor necrosis factor-α (TNF-α), cortisol, troglitazone, IL1-ß + troglitazone, or vehicle. Gene expression in the adipose tissue was analyzed using qPCR and protein levels in the incubation media were analyzed using ELISA. OPG expression and secretion was diminished by 40.8% and 43.1% respectively, by cortisol, and OPG expression was diminished by 67.5% by troglitazone (p<0.05). The proinflammatory cytokines IL1-ß and TNF-α significantly increased the expression of CTGF (p<0.05) by 65.1% and 101.3%, respectively, and the expression and secretion of OGP by 62.3-165.8% (p<0.05). This interleukin 1-ß mediated increase in CTGF- and OPG expression and secretion was ameliorated by troglitazone. Troglitazone and related drugs are known to have adverse effects on bone. We suggest that this could be mediated via altered cytokine production in adipose tissue. Moreover, obese individuals have a low-grade inflammation in their adipose tissue and have higher bone mineral density than lean individuals. We suggest that this inflammation may increase the expression and secretion of OPG and CTGF and thereby increase BMD. In conclusion, bone acting cytokines are produced in the adipose tissue and may affect bone through endocrine mechanisms.

Effects of COLIA1 polymorphisms and haplotypes on perimenopausal bone mass, postmenopausal bone loss and fracture risk.

UNLABELLED: One thousand seven hundred seventeen perimenopausal women from the Danish Osteoporosis Prevention Study were genotyped for the -1997G/T, -1663indelT and +1245G/T polymorphisms in the COLIA1 gen. We found that the -1997T allele and a haplotype containing it were associated with reduced bone mineral density (BMD) and increased bone turnover at menopause and after 10 years of follow-up. INTRODUCTION: We wanted to investigate whether the -1997G/T, -1663indelT and +1245G/T polymorphisms in the COLIA1 gene are associated with perimenopausal bone mass, early postmenopausal bone loss and interact with hormone treatment. METHODS: One thousand seven hundred seventeen perimenopausal women from the Danish Osteoporosis Prevention Study were genotyped, and haplotypes were determined. BMD was examined by dual X-ray absorptiometry. RESULTS: Women carrying the -1997T variant had lower BMD at all measured sites: lumbar spine BMD 1.030 ± 0.137 g/cm(2), 1.016 ± 0.147 g/cm(2) and 0.988 ± 0.124 g/cm(2) in women with the GG, GT and TT genotypes, respectively (p < 0.05) and total hip BMD 0.921 ± 0.116 g/cm(2), 0.904 ± 0.123 g/cm(2) and 0.887 ± 0.109 g/cm(2) in women with the GG, GT and TT genotypes, respectively (p = 0.01). The effect remained after 10 years although statistical significance was lost. Haplotype 3 (-1997T-1663ins + 1245G) was associated with lower bone mass and higher levels of bone turnover. Compared with haplotype 1, haplotype 3 carriers had lower BMD at the lumbar spine, femoral neck and total hip by 0.016 ± 0.007 g/cm(2), 0.015 ± 0.006 g/cm(2) and 0.017 ± 0.006 g/cm(2), respectively (p < 0.05-0.005). No association with postmenopausal changes in bone mass and fracture risk and no overall interaction with the effects of hormone therapy could be demonstrated for any of the polymorphisms in COLIA1. CONCLUSIONS: The -1997G/T polymorphism and haplotype 3 are significantly associated with perimenopausal bone mass, and these effects were sustained up to 10 years after menopause. No association between the -1663indelT or +1245G/T polymorphisms and peri- or postmenopausal bone mass could be demonstrated.

Polymorphisms of the peroxisome proliferator-activated receptor γ (PPARγ) gene are associated with osteoporosis.

UNLABELLED: Stimulation of PPARγ turns mesenchymal stem cells into adipocytes instead of osteoblasts. We investigated the effect of polymorphisms in the PPARγ gene on BMD and fracture risk in two Danish cohorts and found opposing effects of certain SNPs and haplotypes in the two cohorts probably owing to environmental factors. INTRODUCTION: Stimulation of PPARγ causes development of mesenchymal stem cells to adipocytes instead of osteoblasts leading to decreased osteoblast number and BMD. The aim of this study was to examine the effect of PPARG polymorphisms on BMD and fracture risk in two Danish cohorts: AROS, a case-control population comprising 809 individuals and DOPS, a population comprising 1,716 perimenopausal women allocated to hormone therapy or not at baseline and followed for 10 years. On the basis of linkage disequilibrium between SNPs throughout the gene and previous studies we chose 10 polymorphisms for investigation. METHODS: In AROS, individuals heterozygous for the polymorphisms rs12497191, rs4135263, and rs1151999 had an increased risk of vertebral fractures (OR = 1.48-1.76, p = 0.005-0.04) compared with individuals homozygous for the common allele. In DOPS, individuals heterozygous for rs1151999 had an increased BMD at the hip sites (p ≤ 0.02). An interaction between rs1151999 and diet was found on BMD in both cohorts. RESULTS: For the polymorphism rs1152003 there was an interaction with body weight on BMD at all sites in both cohorts (p ≤ 0.07). Stratified analyses revealed that in the high weight group in AROS individuals homozygous for the variant allele had a decreased BMD (p ≤ 0.02), whereas the same pattern was found in the low weight group in DOPS (p ≤ 0.03). A number of haplotype associations were found as well, the direction of which was opposite in the two cohorts. CONCLUSION: Our study suggests an association SNPs in PPARG and haplotypes thereof and BMD and fracture risk. The effect however appears to be modifiable by environmental factors.

Polymorphisms in the ALOX12 gene and osteoporosis.

UNLABELLED: ALOX12 produces ligands for PPARγ thereby turning mesenchymal stem cells into adipocytes instead of osteoblasts. We investigated the effect of polymorphisms in the ALOX12 gene on BMD and fracture risk in two Danish cohorts and found four polymorphisms and a haplotype thereof to be associated with BMD and fracture risk. INTRODUCTION: Stimulation of the PPARγ with ligands produced by the ALOX enzymes drives mesenchymal stem cells in an adipocyte direction at the expense of osteoblasts leading to decreased osteoblast number and BMD. Previously, polymorphisms in the ALOX12 gene have been associated with osteoporosis. METHODS: We examined the effect of ALOX12 polymorphisms on BMD and the risk of fractures in two Danish cohorts: AROS, a case-control population comprising 809 individuals and DOPS, a population comprising 1,716 perimenopausal women allocated to hormone therapy or not at baseline and followed for up to 10 years. On the basis of linkage disequilibrium (LD) between SNPs throughout the gene and previous genetic association studies we chose ten polymorphisms for investigation. Genotyping was carried out using the Sequenom MassARRAY genotyping system and TaqMan assays. RESULTS: In AROS, individuals heterozygous for the polymorphisms rs3840880, rs9897850, rs2292350 and rs1126667 had a 3.0-4.7% decreased lumbar spine BMD (p = 0.02-0.06) and an increased risk of vertebral fractures (p < 0.05) compared with individuals homozygous for either allele. In DOPS, none of the individual SNPs were associated with BMD or incident fractures. In both cohorts, the above-mentioned SNPs comprised an LD-block (pairwise D´ = 1.0, r (2) = 0.45-0.97). A haplotype comprising all the common alleles (frequency 9%) was associated with decreased bone loss at the hip (p < 0.05) and decreased incidence of osteoporotic fractures (p < 0.05) in DOPS and increased femoral neck BMD in AROS (p < 0.05). CONCLUSION: Our study suggests that genetic variants in ALOX12 may influence BMD and fracture risk.

Genotypes and haplotypes of the estrogen receptor genes, but not the retinoblastoma-interacting zinc finger protein 1 gene, are associated with osteoporosis.

Osteoporosis is a common age-related disease with a strong genetic influence. Polymorphisms of ESR1 have consistently been shown to be associated with bone mineral density (BMD) and fracture; however, in regulating bone metabolism, ESR1 interacts with both ESR2 and RIZ1. We therefore examined the effects of polymorphisms in the ESR1, ESR2, and RIZ1 genes and their haplotypes on vertebral fractures and BMD in a case-control study comprising 462 osteoporotic patients and 336 controls. In ESR1, we found the variant C allele of the XbaI polymorphism to be associated with decreased risk of vertebral fractures in women (P < 0.01), whereas in men, the T allele seemed protective (P = 0.05). The variant G allele of the PvuII polymorphism decreased the risk of vertebral fractures independently of lumbar spine BMD in women (P = 0.04) but had no effect in men. Haplotype X-P-H (XbaI:C, PvuII:G, and a high number of TA repeats) was associated with decreased risk of vertebral fractures in women (P = 0.04) but not men. In ESR2, the G allele of the AluI polymorphism was associated with increased fracture risk (P = 0.04), and the haplotype that comprises rs1256031:T and AluI:A increased lumbar spine BMD by 0.04 +/- 0.02 g/cm(2) (P < 0.05) and decreased the risk of vertebral fractures (P = 0.04). There was no effect of the RIZ1 polymorphism on BMD or fracture risk and no evidence of interaction between the polymorphisms and haplotypes thereof. We confirm that genetic variants in ESR1 and ESR2, but not RIZ1, are important in osteoporosis. We found no evidence of interaction between polymorphisms, but we found that the effects of genetic variants in ESR1 might be sex dependent.

Effect of a feed/fast protocol on pH in the proximal equine stomach.

REASONS FOR PERFORMING STUDY: Risk factors for the development of gastric squamous ulcers include various management procedures, such as intermittent feed deprivation that can occur during weight management regimens or stall and dry lot confinement. OBJECTIVES: To investigate the effect of intermittent feed deprivation relative to continuous feed intake on proximal intragastric pH, specifically in the region of the squamous mucosa of the lesser curvature. METHODS: In 6 horses, pH electrodes were placed just inside of the oesophageal sphincter in the stomach for each of two 72 h protocols (A and B) in a randomised, cross-over design. Protocol A consisted of 12 h fed, 12 h fasted, 24 h fed and 24 h fasted, in sequence. Protocol B consisted of 72 h fed. During the fed periods of each protocol, horses had ad libitum access to coastal Bermuda hay and were fed sweet feed (1 kg, b.i.d.). Horses had ad libitum access to water at all times. RESULTS: Proximal intragastric pH was significantly lower during protocol A, than during protocol B. However, hourly mean pH was significantly different only during the day and evening hours between protocols. During protocol B, mean proximal pH decreased significantly from 03.00 to 09.00 compared to 19.00 to 23.00 h. A moderate positive correlation of hay intake vs. proximal gastric pH could be established. CONCLUSIONS: Intermittent feed deprivation decreased proximal gastric pH in horses relative to those horses for which feed was not restricted. However, the effect was only significant when fasting occurred during the day and evening hours, as a nocturnal decrease in pH occurred simultaneously in the fed horses. POTENTIAL RELEVANCE: Episodes of daytime feed deprivation should be avoided if possible, as proximal gastric acid exposure rapidly increases during such events.

Haplotypes of promoter and intron 1 polymorphisms in the COLIA1 gene are associated with increased risk of osteoporosis.

Osteoporosis is a common age-related disease with a strong genetic influence. COLIA1 is one of the most extensively studied candidate genes and has consistently been associated with BMD and fracture. We examined the effects of the polymorphisms -1997G>T, -1663indelT, and +1245G>T and their haplotypes on vertebral fractures and bone mineral density (BMD) in a case-control study comprising 462 osteoporotic patients and 336 controls. The -1663indelT polymorphism was associated with a decreased lumbar spine (ls) BMD, 0.75 +/- 0.14 g/cm(2), in individuals with the del/del genotype versus 0.83 +/- 0.18 and 0.85 +/- 0.18 g/cm(2) in individuals with the ins/del and ins/ins genotypes, respectively (p = 0.02). The T-allele of the +1245G>T polymorphism, which was in strong linkage disequilibrium (LD) with -1663indelT, was also associated with a decreased lsBMD (p = 0.02). -1997G>T was not significantly associated with lsBMD. The three most common haplotypes accounted for 98.5% of the alleles. Individuals with one or two copies of haplotype 1 (-1997G/-1663ins/+1245G) had a significantly higher lsBMD, 0.84 +/- 0.18 and 0.85 +/- 0.15 g/cm(2), respectively, versus 0.78 +/- 0.15 g/cm(2) in noncarriers (p = 0.01). Individuals with two copies of haplotype 2 (-1997G/-1663del/+1245T) had a significantly lower lsBMD, 0.76 +/- 0.14 g/cm(2), versus 0.85 +/- 0.18 and 0.82 +/- 0.18 g/cm(2), respectively, in individuals with zero or one copy (p = 0.03). The odds ratio for vertebral fracture in individuals carrying the variant T-allele of the -1997G>T polymorphism was 1.49 (CI, 1.03-2.16; p = 0.03). Logistic regression revealed that this effect was partly independent of BMD. In conclusion, the -1663del and +1245T alleles influence BMD negatively, whereas the -1997T-allele has a minor effect on BMD but increases the risk of vertebral fractures. These findings are in agreement with functional studies showing that these polymorphisms influence gene expression.

Effect of paddock vs. stall housing on 24 hour gastric pH within the proximal and ventral equine stomach.

REASONS FOR PERFORMING STUDY: Stall housing has been suggested as a risk factor for ulcer development in the equine stomach; however, the exact pathogenesis for this has not been established. OBJECTIVES: To investigate the effect of 3 environmental situations (grass paddock, stall alone or stall with adjacent companion) on pH in the proximal and the ventral stomach. METHODS: Six horses with permanently implanted gastric cannulae were used in a randomised, cross-over, block design. Each horse rotated through each of three 24 h environmental situations. Horses remained on their normal diet (grass hay ad libitum and grain b.i.d.) throughout the study. Intragastric pH was measured continuously for 72 h just inside the lower oesophageal sphincter (proximal stomach) and via a pH probe in the gastric cannula (ventral stomach). RESULTS: Neither proximal nor ventral 24 h gastric pH changed significantly between the 3 environmental situations. Mean hourly proximal gastric pH decreased significantly in the interval from 01.00-09.00 h compared to the interval from 13.00-20.00 h, regardless of environmental situation. Median hourly proximal pH only differed in the interval from 06.00-07.00 h compared to the interval 14.00-19.00 h. Neither mean nor median hourly ventral gastric pH varied significantly with the time of day. CONCLUSIONS: The change in housing status used in the current study did not affect acid exposure within either region of the equine stomach. The pH in the ventral stomach was uniformly stable throughout the study, while the proximal pH demonstrated a 24 h circadian pattern.

Sedation with detomidine and acepromazine influences the endoscopic evaluation of laryngeal function in horses.

REASON FOR PERFORMING STUDY: Endoscopy of the upper airways of horses is used as a diagnostic tool and at purchase examinations. On some occasions it is necessary to use sedation during the procedure and it is often speculated that the result of the examination might be influenced due to the muscle-relaxing properties of the most commonly used sedatives. OBJECTIVES: To evaluate the effect of detomidine (0.01 mg/kg bwt) and acepromazine (0.05 mg/kg bwt) on the appearance of symmetry of rima glottidis, ability to abduct maximally the arytenoid cartilages and the effect on recurrent laryngeal neuropathy (RLN) grade. METHODS: Forty-two apparently normal horses underwent endoscopic examination of the upper airways on 3 different occasions, under the influence of 3 different treatments: no sedation (control), sedation with detomidine and sedation with acepromazine. All examinations were performed with a minimum of one week apart. The study was performed as an observer-blind cross-over study. RESULTS: Sedation with detomidine had a significant effect on the RLN grading (OR = 2.91) and ability maximally to abduct the left arytenoid cartilages (OR = 2.91). Sedation with acepromazine resulted in OR = 2.43 for the RLN grading and OR = 2.22 for the ability to abduct maximally. The ability to abduct maximally the right arytenoid cartilage was not altered. CONCLUSIONS: Sedating apparently healthy horses with detomidine or acepromazine significantly impairs these horses' ability to abduct fully the left but not the right arytenoid cartilage. This resulted in different diagnosis with respect to RLN when comparing sedation to no sedation. POTENTIAL RELEVANCE: Since the ability to abduct the right arytenoid cartilage fully is not altered by sedation, it is speculated that horses changing from normal to abnormal laryngeal function when sedated, might be horses in an early stage of the disease. To confirm or reject these speculations, further studies are needed. Until then sedation during endoscopy should be used with care.

The effect of interleukin-1alpha polymorphisms on bone mineral density and the risk of vertebral fractures.

Interleukin-1alpha (IL-1alpha) stimulates bone resorption via osteoclasts. Mononuclear cells from patients with osteoporosis show increased IL-1alpha production, and IL-1alpha mRNA is more often detected in bone biopsies from osteoporotic compared to normal postmenopausal women. Polymorphisms have been identified in the IL-1alpha gene; however, none of these has been examined for an effect on bone phenotypes in Caucasians. We investigated if the polymorphisms in the IL-1alpha gene affect the risk of osteoporotic fractures, bone mineral density (BMD), and bone turnover in 462 osteoporotic patients and 336 normal controls. Based on previous studies of polymorphisms in the gene and data from the International Hap-Map Project, four polymorphisms needed examination in order to investigate the effect of known polymorphisms in the IL-1alpha gene. We examined C(-1202)-T(rs1800794), C(-889)-T(rs1800587), T(155 + 209)-C(rs2071373), C(155 + 320)-T(rs2856838), and G(398)-T(rs 17561) by Taqman and restriction fragment-length polymorphism assays. BMD was examined by dual-energy X-ray absorptiometry. Bone turnover was evaluated by serum osteocalcin, serum carboxy-terminal propeptide of human type I procollagen, serum bone-specific alkaline phosphatase, serum carboxy-terminal telopeptide of type I collagen, and urinary hydroxyproline/creatinine. Genotype distributions were in Hardy-Weinberg equilibrium. All polymorphisms were in strong linkage disequilibrium. The C allele of the C(155 + 320)-T polymorphism tended to be more common among patients with vertebral fractures (P = 0.06) and patients with BMD T score <-2.5 (P = 0.05). Furthermore, haplotype 1 was associated with reduced risk of having BMD T score <-2.5 (P = 0.02). None of the other polymorphisms or haplotypes was associated with fracture risk or BMD T score <-2.5. BMD and bone turnover were not associated with any of the genetic variants. In conclusion, all the polymorphisms within the IL-1alpha gene are in strong linkage disequilibrium and not convincingly associated with fracture risk, BMD, or bone turnover.

Risk factors for faecal sand excretion in Icelandic horses.

REASONS FOR PERFORMING STUDY: Sandy soil is often mentioned as a risk factor in the development of sand-related gastrointestinal disease (SGID) in the horse. There are other variables, but few studies confirm any of these. OBJECTIVE: To investigate soil type, pasture quality, feeding practice in the paddock, age, sex and body condition score as risk factors for sand intake in the horse. METHODS: Faeces were collected from 211 Icelandic horses on 19 different studs in Denmark together with soil samples and other potential risk factors. Sand content in faeces determined by a sand sedimentation test was interpreted as evidence of sand intake. Soil types were identified by soil analysis and significance of the data was tested using logistic analysis. RESULTS: Of horses included in the study, 56.4% showed sand in the faeces and 5.7% had more than 5 mm sand as quantified by the rectal sleeve sedimentation test. Soil type had no significant effect when tested as main effect, but there was interaction between soil type and pasture quality. Significant interactions were also found between paddock feeding practice and pasture quality. CONCLUSION: To evaluate the risk of sand intake it is important to consider 3 variables: soil type, pasture quality and feeding practice. Pasture quality was identified as a risk factor of both short and long grass in combination with sandy soil, while clay soil had the lowest risk in these combinations. Feeding practice in the paddock revealed feeding directly on the ground to be a risk factor when there was short (1-5 cm) or no grass. Also, no feeding outdoors increased the risk on pastures with short grass, while this had no effect in paddocks with no grass. More than 50% of all horses investigated in this study had sand in the faeces. POTENTIAL RELEVANCE: The identification of risk factors is an important step towards prevention of SGID. Further research is necessary to determine why some horses exhibit more than 5 mm sand in the sedimentation test and whether this is correlated with geophagic behaviour.

Semiquantitative mRNA measurements of osteoinductive growth factors in human iliac-crest bone: expression of LMP splice variants in human bone.

Although osteotropic growth factors are known to play an important role in bone metabolism, knowledge about their expression in relation to age, sex and smoking remains limited. In this study we report mRNA levels of the recently discovered Lim mineralization protein splice variants (LMP-1, LMP-2, LMP-3) and the established osteotropic growth factors BMP-2, BMP-6, BMP-7, TGF-beta, IGF-I, IGF-II and b-FGF in human iliac crest bone. Standardized bone biopsy specimens were obtained from the iliac crest during graft harvesting in 62 patients (38 males, 24 females, mean age 44.7 years, range 13-78 years) undergoing spinal surgery. Samples were immediately stored in liquid nitrogen for PCR analysis. Semi-quantitative RT-PCR was performed for TGF-beta, IGF-I, IGF-II, BMP2, BMP-6, BMP7, bFGF, LMP-1, LMP-2 and LMP-3 using beta-actin as internal standard. Triplicate measurements were made of each growth factor and beta-actin. mRNA for all examined growth factors was detected in 69% of the specimens. The lowest degree of detection was present for b-FGF and BMP-2, both of which were found in 85% of the specimens. LMP-1 was detected in 98% of the specimens. LMP-2 in 94% and LMP-3 in 27%, respectively. LMP-1 was generally expressed in higher amounts than LMP-2 and LMP-3. Nondetectable levels of the growth factors were more frequent in the >60-year-old males compared with >60-year-old females ( P < 0.05) and <60-year-old males ( P < 0.01). LMP-1 expression was more variable among young individuals, but mean values were similar between age groups. TGF-beta, BMP-2 and BMP-7 values did not differ between age groups, but generally a higher variation was found among older patients. IGF-I values were significantly higher ( P < 0.05) in males over 60 years, whereas the highest level of bFGF mRNA was present in males younger than 20 years ( P < 0.05). In addition, regression analysis revealed correlation between BMP-2 and BMP-7 (R2 = 0.74, P < 0.0005), LMP-2 and BMP-2 (R2 = 0.27, P < 0.0005) and LMP-2 and bFGF (R2 = 0.40, P < 0.0005). In conclusion, we have demonstrated expression of LMP-1 and LMP-2 in human bone. LMP-1 was expressed in higher amounts and showed a higher degree of variation among young individuals. LMP-2 was correlated to a number of other growth factors, suggesting that LMPs may also play a role in human bone metabolism. Higher variation in the expression of TGF-beta, BMP-2 and BMP-7 was found in the older age groups, but whether or not this can be correlated to age-related changes in bone turnover requires further studies.

Crystal structure of the receptor-binding domain of alpha 2-macroglobulin.

BACKGROUND: The large plasma proteinase inhibitors of the alpha 2-macroglobulin superfamily inhibit proteinases by capturing them within a central cavity of the inhibitor molecule. After reaction with the proteinase, the alpha-macroglobulin-proteinase complex binds to the alpha-macroglobulin receptor, present in the liver and other tissues, and becomes endocytosed and rapidly removed from the circulation. The complex binds to the receptor via recognition sites located on a separate domain of approximately 138 residues positioned at the C terminus of the alpha-macroglobulin subunit. RESULTS: The crystal structure of the receptor-binding domain of bovine alpha 2-macroglobulin (bRBD) has been determined at a resolution of 1.9 A. The domain primarily comprises a nine-strand beta structure with a jelly-roll topology, but also contains two small alpha helices. CONCLUSIONS: The surface patch responsible for receptor recognition is thought to involve residues located on one of the two alpha helices of the bRBD as well as residues in two of the beta strands. Located on this alpha helix are two lysine residues that are important for receptor binding. The structure of bRBD is very similar to the approximately 100-residue C-terminal domain of factor XIII, a transglutaminase from the blood coagulation system.

Localisation of the major reactive lysine residue involved in the self-crosslinking of proteinase-activated Limulus alpha 2-macroglobulin.

When alpha 2-macroglobulin (alpha 2M) from the American horseshoe crab, Limulus polyphemus, reacts with proteinases, its thiol esters, like those of other alpha-macroglobulins, become activated, leading to the formation of covalently crosslinked species that can be detected as high molecular weight bands in reducing SDS-PAGE. While other alpha-macroglobulins extensively form crosslinks to the reacting proteinase, Limulus alpha 2M does not. It rather becomes internally crosslinked. It was found from N-terminal sequence analysis of purified [14C]carboxymethylated peptides from Limulus alpha 2M-trypsin complexes that an isopeptide bond formed in approx. 60% yield from the thiol esterified Gln-1002 specifically to Lys-254 in the opposing monomer of the disulphide bridged dimer is the main cause of the internal crosslinking.

Typing the HLA-B locus by a nested primer approach and oligonucleotide hybridization.

A system for intermediate level identification of the HLA-B locus alleles was devised. This system can be extended to identify individual alleles in any sample. The first step used primers which amplify all HLA-B alleles. This amplicon was subjected to SSOP hybridization to allow intermediate level typing of samples. In the second step, group-specific primers were utilized to obtain specific amplification of groups consisting of a few alleles. The oligotypes within each group were identified by the use of SSOP. The separation of groups of alleles by amplification allowed the use of a limited number of probes to identify oligotypes present in a sample. Additional probes can be added as new alleles are identified, increasing the flexibility of the system. HLA typing software was developed to determine the resolution of the system and to identify HLA oligotypes. PCR-SSOP methods are in wide use and have been extensively validated. The procedures reported here will be relatively easy to implement for large-scale DNA-based typing of the HLA-B locus.

Pattern of repeating aromatic residues in synexin. Similarity to the cytoplasmic domain of synaptophysin.

Synexin was isolated from bovine liver by high resolution cation exchange chromatography and fragmented with cyanogen bromide or trypsin. Peptides were isolated and their amino acid sequences partially determined. Twenty percent of the synexin sequence was determined in one contiguous sequence of 61 residues and a nonoverlapping sequence of 20 residues. The sequence is characterized by a hexapeptide repeat of the form YPXXXX occurring eight times in series, with phenylalanine substituting for tyrosine in two positions. The intervening amino acids (X) are predominantly proline, glycine and alanine. This pattern of periodic aromatic residues suggests the presence of a novel secondary structure and is similar to repeats present in synaptophysin, gliadin and type II keratin.