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Biomedical subjects

R M Leach

Publications and source records attributed to R M Leach.

At least 19 recordsLinked to original sources
PubMed2001

Parathyroid hormone-related peptide expression in the epiphyseal growth plate of the juvenile chicken: evidence for the origin of the parathyroid hormone-related peptide found in the epiphyseal growth plate.

Parathyroid hormone-related peptide (PTHrP) has been shown to be essential for normal endochondral bone formation. Along with Indian hedgehog (Ihh), it forms a paracrine regulatory loop that governs the pace of chondrocyte differentiation. However, the source of PTHrP for this regulatory loop is not clear. While one hypothesis has suggested the periarticular perichondrium as the source of PTHrP for growth plate regulation, other data utilizing immunohistochemistry and in situ hybridization would indicate that growth plate chondrocytes themselves are the source of this peptide. The data described in this report supports the view that postnatal growth plate chondrocytes have the ability to synthesize this important regulatory peptide. Immunohistochemistry of tissue sections showed that PTHrP protein was evident throughout the chick epiphysis. PTHrP was seen in chondrocytes in the periarticular perichondrium, the perichondrium adjacent to the growth plate, the prehypertrophic zone of the growth plate, and the hypertrophic zone of the growth plate. However, cells in the proliferative zone, as well as some chondrocytes in the deeper layers of articular cartilage were predominantly negative for PTHrP. PTHrP was detected by Western blotting as a band of 16,400 Da in extracts from hypertrophic chondrocytes, but not from proliferative cells. RT-PCR detected PTHrP mRNA in both proliferative and hypertrophic growth plate chondrocytes, as well as in articular chondrocytes. PTH/PTHrP receptor mRNA was detected by Northern blotting in growth plate, but not articular chondrocytes. Thus, we conclude that most of the PTHrP present in the epiphyseal growth plate of the juvenile chick originates in the growth plate itself. Furthermore, the presence of large amounts of PTHrP protein in the hypertrophic zone supports the concept that PTHrP has other functions in addition to regulating chondrocyte differentiation.

Animals↗
PubMed2001

Continuous venovenous haemofiltration for the treatment of theophylline toxicity.

In patients with severe theophylline toxicity charcoal haemoperfusion is the recommended method for rapid reduction of serum theophylline levels. However, access to this technique is limited in most hospitals. This case report shows that continuous venovenous haemofiltration, a technique available in most hospitals, is an effective alternative to charcoal haemoperfusion.

Bronchodilator Agents↗
PubMed2000

Effect of epidural blockade on indicators of splanchnic perfusion and gut function in critically ill patients with peritonitis: a randomised comparison of epidural bupivacaine with systemic morphine.

OBJECTIVES: (a) To measure gastric tonometry values in critically ill patients with peritonitis and to assess the impact of epidural analgesia on these values. (b) To assess the impact of epidural analgesia on gastro-intestinal motility by abdominal ultrasound and paracetamol absorption. (c) To observe any change in clinical outcome that may result from the use of epidural analgesia in such patients. DESIGN: A double-blinded, prospective, randomised and controlled study of general intensive therapy unit (ITU) patients. PATIENTS: Twenty-one patients admitted with peritonitis and adynamic small bowel following abdominal surgery were randomly allocated to receive either intravenous morphine or epidural bupivacaine for analgesia. MEASUREMENTS AND RESULTS: Gastric intramucosal pH (pHig) and the mucosal:arterial PCO2 gradient (Pg-PaCO2) were measured at admission and after 24 h of analgesia. Analysis of mean changes in tonometry values showed a rise in Pg-PaCO2 and a fall in pHig in the morphine group and a significant difference between groups in the Pg-PaCO2 trends (p = 0.024). Significant improvements in the ultrasound appearance of the small bowel were observed in the epidural group (p = 0.0037, Mann-Whitney U test of median changes in a locally developed scoring system). There were no significant differences between the groups in any of the variables derived from the paracetamol absorption test (n = 10); both groups showed persistently delayed gastric outflow throughout the study period. CONCLUSIONS: Epidural analgesia resulted in improvements in gastric mucosal perfusion and the ultrasound appearance of the small bowel, indicating potential clinical benefit in a group of patients in whom epidural catheterisation is traditionally contraindicated.

Analgesia, Epidural↗
PubMed2000

Gene expression and tibial dyschondroplasia.

Tibial dyschondroplasia (TD) is a skeletal deformity associated with rapid growth in a number of avian species. The disease is the result of a disruption in the cascade of events that occur in the epiphyseal growth plate. Whereas the incidence of TD is susceptible to genetic selection, no specific genetic defect has been identified. Although there are extensive data describing the morphological and biochemical characteristics of the lesion, the mechanism of lesion formation is unknown. However, naturally occurring or induced genetic mutations in other species can provide important clues to possible mechanisms responsible for lesion development. Disruption of normal chondrocyte differentiation by constitutive activation of the parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor, inactivation of the fibroblast growth factor receptor-3 (FGFR-3) receptor, and blocking vascular endothelial growth factor (VEGF) signaling all result in lesions that resemble TD. Impairment of vascular penetration due to the ablation of matrix metalloproteinase-9 (MMP-9) or tartrate-resistant acid phosphatase (TRAP) activity also results in similar cartilage abnormalities. We have integrated these observations with our current knowledge of TD to describe a hypothesis for the sequence of events responsible for the development of tibial dyschondroplastic lesions.

Animals↗
PubMed2000

Energy state, pH, and vasomotor tone during hypoxia in precontracted pulmonary and femoral arteries.

To assess effects of smooth muscle energy state and intracellular pH (pH(i)) on pulmonary arterial tone during hypoxia, we measured ATP, phosphocreatine, P(i), and pH(i) by (31)P-NMR spectroscopy and isometric tension in phenylephrine-contracted rings of porcine proximal intrapulmonary arteries. Hypoxia caused early transient contraction followed by relaxation and late sustained contraction. Energy state and pH(i) decreased during relaxation and recovered toward control values during late contraction. Femoral arterial rings had higher energy state and lower pH(i) under baseline conditions and did not exhibit late contraction or recovery of energy state and pH(i) during hypoxia. In pulmonary arteries, glucose-free conditions abolished late hypoxic contraction and recovery of energy state and pH(i), but endothelial denudation abolished only late hypoxic contraction. NaCN had little effect at 0. 1 and 1.0 mM but caused marked vasorelaxation and decreases in energy state and pH(i) at 10 mM. These results suggest that 1) regulation of tone, energy state, and pH(i) differed markedly in pulmonary and femoral arterial smooth muscle, 2) hypoxic relaxation was mediated by decreased energy state or pH(i) due to hypoxic inhibition of oxidative phosphorylation, 3) recovery of energy state and pH(i) in hypoxic pulmonary arteries was due to accelerated glycolysis mediated by mechanisms intrinsic to smooth muscle, and 4) late hypoxic contraction in pulmonary arteries was mediated by endothelial factors that required hypoxic recovery of energy state and pH(i) for transduction in smooth muscle or extracellular glucose for production and release by endothelium.

Adenosine Triphosphate↗
PubMed1999

Parathyroid receptor gene expression by epiphyseal growth plates in rickets and tibial dyschondroplasia.

PTH/PTHrP receptor gene expression was evaluated in situ in avian epiphyseal growth plates taken from normal, rachitic and tibial dyschondroplasia (TD) afflicted chicks induced by thiram or by genetic selection. In the normal growth plates, PTH/PTHrP receptor gene expression was localized to the maturation zone as demonstrated by the expression of collagen type II (col II), osteopontin (OPN) genes and alkaline phosphatase activity (AP). In TD, either induced by thiram or by genetic selection, normal levels of PTH/PTHrP receptor gene expression were observed up to 21 days post-hatch. In rickets, on the other hand, no PTH/PTHrP receptor gene expression was observed in the growth plate from day 8 of a vitamin D-deficient diet. In cultured chondrocytes, PTH caused time-dependent down-regulation of its own receptor. These results suggest that alterations in the PTH/PTHrP receptor gene expression are associated with rickets but not with TD. The reduction in the PTH/PTHrP receptor gene expression in rickets may be due to the high plasma levels of PTH.

Animals↗
PubMed1998

Detection of endogenous biotin-containing proteins in bone and cartilage cells with streptavidin systems.

When utilizing streptavidin systems with Western blots of chondrocyte, osteoblast and osteoclast lysates, proteins of the molecular weights 116 kDa, 75 kDa and 67 kDa were observed to be bound by streptavidin alone. Streptavidin binding could not be blocked by pre-incubation with an RGD containing peptide. The same proteins were bound by ExtrAvidin which lacks the RGD sequence present in streptavidin. Pre-incubation with free biotin completely abolished the binding of both streptavidin and ExtrAvidin. The three proteins observed are believed to be the biotin containing carboxylases: pyruvate carboxylase, 3-methylcrotonyl carboxylase, and propionyl carboxylase. The findings of this study underscore the need to apply vigorous controls to distinguish between endogenous biotinylated proteins and biotin used as a means to amplify avidin detection systems since a wide variety of proteins with relevance to bone and cartilage biology have molecular weights similar to the biotin carboxylases.

Animals↗
PubMed1998

Relationship between TISS and ICU cost.

OBJECTIVE: To determine whether the therapeutic intervention scoring system (TISS) reliably reflects the cost of the overall intensive care unit (ICU) population, subgroups of that population and individual ICU patients. DESIGN: Prospective analysis of individual patient costs and comparison with TISS. SETTING: Adult, 12 bedded general medical and surgical ICU in a university teaching hospital. SUBJECTS: Two hundred fifty-seven consecutive patients including 52 coronary care (CCU), 99 cardiac surgery (CS) and 106 general ICU (GIC) cases admitted to the ICU during a 12-week period in 1994. A total of 916 TISS-scored patient days were analysed MAIN OUTCOME MEASURES: A variable cost (VC) that included consumables and service usage (nursing, physiotherapy, radiology and pathology staff costs) for individual patients was measured daily. Nursing costs were calculated in proportion to a daily nursing dependency score. A fixed cost (FC) was calculated for each patient to include medical, technical and clerical salary costs, capital equipment depreciation, equipment and central hospital costs. The correlation between cost and TISS was analysed using regression analysis. RESULTS: For the whole group (n = 257) the average daily FC was pound sterling 255 and daily VC was pound sterling 541 (SEM 10); range pound sterling 23-pound sterling 2,806. In the patient subgroups average daily cost (FC + VC) for CCU was pound sterling 476 (SEM 17.5), for CS pound sterling 766 (SEM 13.8) and for GIC pound sterling 873 (SEM 13.6). In the group as a whole, a strong correlation was demonstrated between VC and the TISS for each patient day (r = 0.87, p < 0.001) and this improved further when the total TISS score was compared with the total VC of the entire patient episode (r = 0.93, p < 0.001). This correlation was maintained in CCU, CS and GIC patient cohorts with only a small median difference between actual and predicted cost (2.2 % for GIC patients). However, in the individual patient, the range of error was up to +/- 65 % of the true variable cost. For the whole group the variable cost per TISS point was pound sterling 25. CONCLUSION: These results demonstrate that TISS reliably measures overall ICU population costs as well as those of the subgroups CCU, CS and GIC. However, the relationship between TISS and cost is less reliable for the individual patient.

Adolescent↗
PubMed1998

Development of avian tibial dyschondroplasia: gene expression and protein synthesis.

Age-dependent gene expression and protein synthesis associated with chondrocyte differentiation were evaluated in the epiphyseal growth plates of normal and tibial dyschondroplasia (TD)-afflicted chickens. In the normal growth plate, collagen type II gene is expressed mainly by chondrocytes at the upper zone of the growth plate and by the chondrocytes in the articular cartilage. Collagen type X and osteopontin (OPN) genes are expressed in the lower zone of the growth plate and in the zone of cartilage-to-bone transition. No age-dependent changes in the pattern of OPN and collagen type II or X gene expression were observed up to 20 days of age. In the TD-afflicted growth plates, the lesion is enlarged with age, and chondrocytes expressing the collagen type II gene were observed in the hypertrophic zone as early as 8 days posthatching. Abnormal expression of OPN and collagen type X genes was also observed starting at 13 days of age. At day 20, the entire TD lesion-which was significantly enlarged-was surrounded by collagen type II, collagen type X, and OPN expressing cells. The level of OPN in TD was reduced with increasing age, and at 20 days almost no OPN could be detected in either the upper or the lower hypertrophic zones. The level of bone sialoprotein (BSP) also diminished with increasing age in the TD growth plates. In contrast to OPN, the age-dependent reduction in BSP levels was mainly in the lower hypertrophic zone (LHZ), and at 20 days of age, BSP was barely detected in the LHZ, whereas in the upper hypertrophic zone, the levels of BSP were similar to those in normal growth plate. In summary, our results suggest that the primary event of the TD lesion occurs in cells of proliferative phenotype within the hypertrophic zone. These cells divide and form the TD lesion, which consists of cells that do not express the genes associated with hypertrophy.

Animals↗
PubMed1998

Type X collagen biosynthesis and expression in avian tibial dyschondroplasia.

OBJECTIVE: Tibial dyschondroplasia (TD) is an abnormality of growth plate cartilage characterized by the presence of non-vascularized, non-mineralized tissue. The objective of this study was to examine structural and functional alterations of the growth plate-specific type X collagen in TD cartilage. DESIGN: Collagen biosynthesis was examined in organ cultures and in cultured chondrocytes from normal growth plate and TD cartilage. Thermal stability of type X collagen extracted from normal and TD cartilage organ cultures to protease digestions by trypsin plus chymotrypsin or bacterial collagenase was determined. The expression of collagen genes was examined in cultured normal and TD chondrocytes. RESULTS: Synthesis of total collagen and of type X collagen was greater than threefold higher in organ cultures from the TD lesion compared with normal growth plate. The increase in type X collagen synthesis in the lesion was compensated by a reduction in the relative proportions of types II and XI collagens. The thermal denaturation and collagenase cleavage properties of purified types II and X collagens from TD cartilage were normal. The expression of type X collagen gene was threefold higher in cultured TD chondrocytes compared to chondrocytes from normal growth plate. Normal growth plate chondrocytes in primary cultures synthesized predominantly type X collagen (80% of total collagen). In contrast, TD chondrocytes synthesized mainly types I and II collagens and type X collagen represented only 22% of total collagen. TD cells initiated the synthesis of type I collagen within 5 days of primary culture, whereas normal chondrocytes did not synthesize this collagen during the same culture period. Although type X collagen synthesis was reduced in TD chondrocytes, the mRNA levels for type X collagen were substantially higher than in normal chondrocytes. CONCLUSION: Accumulation of type X collagen in TD cartilage results from its increased biosynthesis which is due largely to increased expression of the gene for this collagen, although, the chondrocyte culture studies suggest the possibility of postranscriptional defect in type X collagen synthesis or processing in TD lesion. Moreover, the TD chondrocytes in contrast with normal chondrocytes display evidence of prompt loss of their specific phenotype during short-term primary cultures.

Animals↗
PubMed1998

Effects of hypoxia on energy state and pH in resting pulmonary and femoral arterial smooth muscles.

To determine the effects of hypoxia on energy state and intracellular pH (pHi) in resting pulmonary and systemic arterial smooth muscles, we used 31P nuclear magnetic resonance spectroscopy and colorimetric and enzymatic assays to measure pHi; intracellular concentrations of ATP, phosphocreatine, creatine, and Pi; and phosphorylation potential in superfused tissue segments from porcine proximal intrapulmonary and superficial femoral arteries. Under baseline conditions (PO2 467 +/- 12.1 mmHg), energy state and total creatine (phosphocreatine + creatine) concentration were lower and pHi was higher in pulmonary arteries. During hypoxia (PO2 23 +/- 2.4 mmHg), energy state deteriorated more in femoral arteries than in pulmonary arteries. pHi fell in both tissues but was always more alkaline in pulmonary arteries. Reoxygenation reversed the changes induced by hypoxia. These results suggest that production and/or elimination of ATP and H+ was different in resting pulmonary and systemic arterial smooth muscles under baseline and hypoxic conditions. Because energy state and pHi affect a wide variety of cellular processes, including signal transduction, contractile protein interaction, and activities of ion pumps and channels, further investigation is indicated to determine whether these differences have functional significance.

Adenosine Triphosphate↗
PubMed1998

Membrane potential-dependent and -independent vasodilation in small pulmonary arteries from chronically hypoxic rats.

Chronic hypoxia is associated with altered pulmonary vasoreactivity, and it has been suggested that an increased response to voltage-dependent vasodilators may relate to enhanced Ca++ entry via voltage-dependent channels, secondary to depolarization. Few studies have been performed on small pulmonary arteries, and it is unknown whether they are depolarized after chronic hypoxia. We examined the resting membrane potential, and the actions of voltage-dependent (verapamil, levcromakalim) and -independent (isoproterenol, forskolin, papaverine) vasodilators in small ( approximately 300 microm internal diameter) pulmonary arteries from chronically hypoxic rats. The resting membrane potential was more positive in arteries after chronic hypoxia (control: -60 +/- 0.5 mV; hypoxic: -54.4 +/- 1.1 mV; P < .01), and this was reflected by a shift to the left of the response curves for K+ and 4-aminopyridine. In arteries constricted with prostaglandin F2alpha the response to verapamil and levcromakalim was increased after chronic hypoxia, although maximum prostaglandin F2alpha-induced tension was unchanged, which implies a reduction in voltage-independent constrictor mechanisms. Although vasorelaxation to isoproterenol was depressed in arteries from hypoxic rats, forskolin-induced relaxation was enhanced substantially, and because the response to the phosphodiesterase inhibitor papaverine was unchanged, we suggest that this reflects an up-regulation of adenylate cyclase. In conclusion, chronic hypoxia resulted in a significant depolarization in small pulmonary arteries, but this may explain only partly the increased efficacy of voltage-dependent vasodilators. Whether the reduction in voltage-independent constrictor mechanisms is related to the apparent up-regulation of adenylate cyclase remains to be elucidated.

Animals↗
PubMed1997

Immunolocalization of basic fibroblast growth factor in porcine epiphyseal growth plate.

Recent evidence clearly indicates that basic fibroblast growth factor (bFGF) signal transduction is essential for normal skeletal development. Here, we report that bFGF and its receptor are specifically localized in the terminal hypertrophic chondrocytes of the porcine epiphyseal growth plate, the tissue responsible for longitudinal bone growth. Similar observations were obtained with the chondrocytes immediately adjacent to resorbing blood vessels in the secondary center of ossification of the epiphysis. These results are consistent with a recent proposal that bFGF functions in coupling osteogenesis with chondrogenesis by attracting the vascular invasion of cartilage from adjacent trabecular bone.

Animals↗
PubMed1997

Evidence of increased cholecalciferol requirement in chicks with tibial dyschondroplasia.

A series of experiments was conducted to test the hypothesis that vitamin D utilization may not be as efficient in chicks with tibial dyschondroplasia (TD). The basal diet contained 1.0% Ca and 0.45% available P with no supplemental cholecalciferol (D3). Chicks from low TD (LTD) and high TD (HTD) selected lines were fed diets supplemented with various levels of vitamin D compounds and examined for rickets and TD. When chicks were fed a D3-deficient diet containing only 1.25 micrograms/kg added D3, HTD chicks had a greater incidence of severe rickets than LTD chicks (P < 0.05). The LTD chicks did not exhibit TD when fed a diet containing adequate (20 micrograms/kg) D3. The LTD chicks fed a diet supplemented with 5 micrograms/kg D3, however, had 22% incidence of TD. When HTD chicks were fed diets supplemented with 5 micrograms/kg D3 [control diet that meets NRC (1994) requirement for D3], 20 micrograms/kg D3, 5 micrograms/kg 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] or the combination of both D3 (20 micrograms/kg) and 1,25-(OH)2D3 (5 micrograms/ kg), TD incidence was highest in HTD chicks fed the control diet. When HTD chicks were fed diets with an increased dietary level of 1,25-(OH)2D3 (10 micrograms/kg) further reduction of TD incidence (P < 0.05) occurred. A potentially toxic level (Soares et al., 1983) of 1,25-(OH)2D3 (15 micrograms/kg) fed to HTD chicks resulted in still greater suppression of incidence of TD even though growth and feed intake in HTD chicks was greater than those of LTD chicks. It is concluded that the development of TD in HTD chicks is associated with subnormal ability to metabolize vitamin D.

Animals↗