Cardiac troponin I is a sensitive, specific biomarker of cardiac injury in laboratory animals.

This study directly demonstrates that cardiac troponin I (cTnI) is a sensitive, specific, and persistent biomarker in laboratory animals. Histopathological and pathophysiological cardiac changes in dogs, rats and mice correlated with increased serum cTnI with various cardiac inotropic agents, and cardiotoxic drugs and with cardiac arrhythmias, tachycardia, cardiac effusion with dyspnoea, and ageing. A comparison of six immunoassays for cTnI and cardiac troponin T (cTnT) to detect and monitor cardiac injury in a rodent model indicated that enzyme-linked immunosorbent (Life Diagnostics Inc and TriChem Resources Inc, West Chester, Philadelphia, USA) and Immulite (Diagnostic Products Corporation, Llanberis, UK) assays had low sensitivity and less than 1% of the dynamic range of Centaur (Bayer Healthcare Diagnostics, Newbury, UK) cTnI and Elecsys (Roche Diagnostics, Basel, Switzerland) and M8 (Bioveris Europe, Whitney, UK) cTnT assays. In dogs, however, the Immulite assay was effective and correlated with the Centaur. Serum concentrations were highly correlated but 10-fold lower for cTnT compared with cTnI with cardiac injury. Centaur assay also detected cTnI in myocardium from marmosets, swine, cattle, and guinea pigs, indicating it to be candidate cardiac biomarker for these species as well. Purified rat cTnI was 50% more reactive than purified human cTnI in the Centaur assay. In the rat, an age- and gender-dependent variation in serum cTnI was found. Male rats aged six and eight months had a 10-fold greater serum cTnI than age-matched females and three-month-old rats. These increases correlated with minimal histopathological change. Isoproterenol-induced serum cTnI increased up to 760-fold the minimal detectable concentration of 0.07 microg/L, within 4-6 h and decreased with a half-life of 6 h, with an expected return to baseline of 60 h. Severity of histopathological change correlated with serum cTnI during the ongoing injury.

Corneal stromal sequestration and keratoconjunctivitis sicca in a horse.

A 19-year-old Shetland pony presented with unilateral ocular discomfort and abnormal ocular appearance. Keratoconjunctivitis sicca, ulcerative keratitis and brown discoloration of the corneal stroma were identified on ophthalmic examination. The etiology of keratoconjunctivitis sicca was not determined in this case. For practical and financial reasons, the owners requested enucleation of the affected eye. Histopathologic examination revealed extensive loss of corneal epithelium overlying a zone of hypereosinophilic, degenerate, and necrotic corneal stroma. This well-circumscribed region of corneal stromal sequestration was surrounded by stromal vascularization, and an intense inflammatory, predominantly polymorphonuclear, cellular infiltrate. The clinical and histopathologic features of this case were considered remarkably similar to those observed in feline corneal stromal sequestration.

A comparison of serum ionized calcium concentrations and serum total calcium concentrations in dogs with lymphoma.

Serum ionized calcium and total calcium concentrations were measured in 16 dogs with lymphoma and 49 healthy control dogs. Blood samples for all determinations of ionized calcium were collected into tubes containing silicone separator gel and processed under a closed anaerobic system. An ionized calcium analyser with ion selective electrodes was used to determine pH, ionized calcium and ionized calcium adjusted to pH 7.4. Reference ranges for ionized calcium (iCa pH 7.4) of 1.30-1.46 mmol/l, and for total calcium of 2.37-2.82 mmol/l were established. A stronger correlation (r = 0.85) was found between measured ionized calcium and total calcium levels in dogs with lymphoma and hypercalcaemia than in those with lymphoma and normocalcaemia (r = 0.64). Measurement of serum ionized calcium was diagnostically concordant with the measurement of serum total calcium in the determination of calcium status in all dogs with lymphoma. Serum ionized calcium did not provide a diagnostic advantage over total calcium in the detection of hypercalcaemia of malignancy in these dogs.

Evaluation of three pancreas specific protein assays, TLI(trypsin-like immunoreactivity), PASP (pancreas specific protein) and CA 19-9 (glycoprotein) for use in the diagnosis of canine pancreatitis.

Three radioimmunoassays (RIA) for the pancreas specific proteins TLI, PASP and CA 19-9 were evaluated in serum from normal control dogs (n = 40) and dogs with pancreatitis (n = 20). Statistically significant differences (P < 0.05) were found for serum TLI and PASP levels between the control and pancreatitis groups. However, only 3/20 dogs with pancreatitis had serum TLI concentrations greater than the highest concentration in control dogs. Concentrations of PASP in serum were higher in 15/20 dogs with pancreatitis than in the control dogs. The magnitude of the increase in concentrations of PASP in pancreatitis was small in the majority of cases. Thus these assays are of limited clinical value in the diagnosis of pancreatitis. There was no cross-reactivity with dog serum in the CA 19-9 assay.

Differences in electrophoresis patterns between plasma albumins of the cockatiel (Nymphicus hollandicus) and the chicken (Gallus gallus domesticus).

Unique migration patterns for chicken and cockatiel albumins were detected when electrophoresis was performed using cellulose acetate and agarose gel support media. Cockatiel albumin migrated to a position equivalent to chicken alpha globulins, while the migration of cockatiel prealbumin was similar to that of chicken albumin. A chicken prealbumin band was not detected. Cockatiel and chicken albumins purified by affinity chromatography had similar migration patterns when electrophoresis was performed under denaturing conditions [sodium dodecyl sulphate (SDS) ] in 7% polyacrylamide gel (PAGE). The molecular weights of both albumins were similar, and were estimated to be approximately 66,000 Da when compared to known molecular weight markers. The different migration patterns were attributed to variations in conformation and surface charge distribution of albumin molecules between the two species. The experimental and clinical consequences of these findings are briefly discussed.

Alkaline phosphatase bone isoenzyme and osteocalcin in the serum of hyperthyroid cats.

The effect of hyperthyroidism on serum markers for increased bone metabolism and turnover was evaluated in 36 cats with elevated serum levels of thyroxine and alkaline phosphatase. Serum was analyzed for total and ionized calcium and phosphorous. Alkaline phosphatase isoenzymes were separated by agarose gel electrophoresis and osteocalcin was measured by radioimmunoassay. Values for hyperthyroid cats were compared with those for healthy cats. Alkaline phosphatase bone isoenzyme was markedly increased in all 36 hyperthyroid cats. Osteocalcin was increased in 44% of the cats. There was no correlation among the magnitude of increase in alkaline phosphatase bone isoenzyme, osteocalcin, and serum thyroxine concentrations. Increased serum phosphorus was found in 35% of the cats. Total calcium was within the reference range in all cats, while 50% of the cats had reduced levels of serum ionized calcium. We conclude that hyperthyroid cats do have altered bone metabolism, although it is usually clinically insignificant.

Comparison of media and sera used for in vitro culture of equine superficial digital flexor tendon explants.

OBJECTIVE: To compare the effects of different commercial nutrient media and sera on protein synthesis and maintenance of cellular density in cultures of the equine superficial digital flexor tendon (SDFT). ANIMALS: 8 healthy 2- to 4-year-old horses. PROCEDURE: First Dulbecco's modified Eagle's medium, Ham's F12 nutrient mixture, RPMI 1640 medium, minimum essential medium with Earle's salts, minimum essential medium with Hanks' salts, and a Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture with 10% fetal bovine serum (FBS) were compared. Then FBS, fetal equine serum, and donor horse serum, each at 5, 10, and 15% in RPMI 1640 medium, were compared. Explants were cultured in roller bottles at 37 C and aerated (50% O2/45% N2/5% CO2) daily. Rates of [3H]-proline incorporation were used as a measure of the rates of total protein and collagen synthesis on days 13 and 28. Matrix cellular density of explants at days 14 and 28 was measured by computerized image analysis. RESULTS: Equine SDFT explants were cultured in all media for up to 4 weeks. Proline incorporation was greatest in Ham's F12 nutrient mixture and in RPMI 1640 medium, with the concentration of proline in medium correlating to the in vitro response. Total proline incorporation was greater in 15% FBS than in 5 or 10% FBS. Other differences among sera were not detected. Matrix cell density in 15% donor horse serum was equivalent to that in uncultured controls and higher than that in most other sera at week 2. CONCLUSION: The in vitro SDFT culture system described may be used in future studies to enhance knowledge of the biological and biochemical characteristics of intrinsic tendon healing.

Ascites and hepatic cirrhosis in a cockatiel (Nymphicus hollandicus).

A mature, female cockatiel (Nymphicus hollandicus) was examined because of respiratory difficulties. Clinical and laboratory findings included ascites and evidence of hepatic disease (i.e., increased plasma bile acid concentrations, aspartate aminotransferase, and alkaline phosphatase activities). Plasma protein electrophoresis results were consistent with chronic-active inflammation. The albumin-to-globulin (A:G) ratio, calculated from plasma electrophoresis, was 0.3. Postmortem examination revealed severe hepatic fibrosis and a diffuse, interstitial, granulomatous lipid pneumonia.

In vitro culture of preimplantation mouse embryos and day 12 limb-buds: effects of serum and albumin.

The effects of three different protein sources at different concentrations on the growth and development of preimplantation mouse embryos and day 12 mouse limb-buds in culture were studied. Mouse embryos and forelimb-buds were cultured with a range of concentrations (5.5 to 42%) of either donor bovine serum (DBS) or fetal bovine serum (FBS), or (0.2 to 0.8%) bovine serum albumin (BSA). After 48 h in culture, the rate of embryo development was significantly higher in 5.5% DBS than in all other groups (P < 0.05). The embryo hatching rate was higher in 21% FBS, 42% FBS, and all DBS groups than in serum-free medium, and all BSA groups (P < 0.05). Morphologic analysis of cultured limb-buds at 72 h revealed that total, paw, and cartilage area were greater (P < 0.05) in the serum-free medium than in all other groups. Shape factor analysis suggested that 5.5% DBS was most beneficial to mouse limb-bud development. No differences were seen in DNA or protein content of limb-buds among groups. Results suggest that mouse forelimb-buds can be successfully cultured in serum-free medium and that high concentrations of FBS and DBS may be detrimental for preimplantation embryo and/or limb-bud growth and development.

Aging of diabetic and nondiabetic skin fibroblasts in vitro: life span and sequential growth curves.

Skin fibroblasts from age-matched normal and juvenile diabetic (IDDM) subjects were studied throughout their life span in vitro. The number of mean population doublings (MPD) attained at senescence was determined. Sequential 5-day growth curves were constructed. Normal cells were compared to cells from diabetics with recent onset of disease, 1-5 years, 6-10 years, and greater than 10 years of insulin therapy. A trend of decreasing MPD in vitro with increasing duration of diabetes in vivo was found. The growth potential of all cells (sequential growth curves) decreased with increasing time in vitro. The confluent density (Day 5) of diabetic (10 years' duration) cells was significantly lower than that for normal cells at Stage 2 of growth (passage 6-10). The in vitro aging of cells from diabetics (recent onset) was essentially the same as that of normal cells.

Catheter infection. A comparison of two catheter maintenance techniques.

Incidence of catheter-related infections was studied using two techniques: changing catheters over a guide-wire or placing a new catheter at a new site every 3 days. Patients were randomized into two groups: Group 1 (new site) and Group 2 (guide-wire). Of the 105 catheterization sites (20 arterial and 85 central lines) in patients of Group 1, none were considered infected (i.e., having 15 or more colonies at the time of semi-quantitative microbiology analysis and clinical signs of infection at the catheter site). Of the 274 catheterization sites (56 arterial and 218 central) of patients of Group 2, eight (2.9%) were infected (chi 2 = 1.89, p greater than 0.05). Colonization (15 or more cultures without clinical signs of infection) occurred in three of 105 (2.9%) and in four of 274 (1.5%) of the catheterization sites of Groups 1 and 2, respectively (chi 2 = 0.23, p greater than 0.05). Study results indicate no significant difference in infection or colonization rates between the two methods of catheter replacement.

Monolayer culture of neonatal pig pancreatic islet cells.

Pancreatic islet cells from pigs (3-12 days old) were maintained in monolayer culture at 37 degrees C for greater than 30 days. Cultures were maintained in enriched Medium 199 with 5% fetal calf serum and with the glucose concentration being cycled between 11.10 and 27.75 mmol/l every 2 days. The presence of B cells in the cultures was demonstrated by insulin secretion into the culture medium, cell staining with aldehyde fuchsin and immunofluorescence. A cells in the cultures were monitored by glucagon secretion into the medium. The absence of exocrine cells was determined by the inability to detect alpha-amylase in the culture medium. Insulin secretion determined over a 34-day period, was found to diminish after 12 days in culture. The fall in levels of extractable B cell insulin with time correlated with the levels of insulin secreted into the medium. Response to short term glucose stimulation (2 h in serum-free medium) was determined on days 6, 10 and 12 in culture. Morphological changes appeared after 25 days in culture, although on day 33 insulin secretion was 62.5 microU X culture-1 X day-1. DNA synthesis (determined by 3H-thymidine incorporation and autoradiography) was demonstrated (day 12, 39.4 +/- 0.9 labelled cells/culture). These findings suggest that pig islet cell monolayer cultures are a useful tool for morphological and biochemical studies and that neonatal pig islets are a potential source of material for transplantation.

Cultured fibroblasts in Huntington's disease. I. Effects of L-glutamic acid.

Huntington's disease (HD) is associated with a defect in the CNS that may involve the "glutamine cycle." There is conflicting evidence that other cell types also manifest the abnormality. Thirty HD, 20 "at-risk," and 20 normal cell lines were used in studies of viability, plating efficiency, cell growth "glutamine rescue," and tritiated thymidine and tritiated leucine incorporation in the presence of O to 30mM L-glutamic acid. Cell viability, plating efficiency, and growth were decreased, with increasing glutamic acid concentrations. Tritiated thymidine and tritiated leucine incorporation was slightly affected by glutamic acid. Glutamine rescue was significantly more effective in normal cells than in HD cells. Fibroblasts in HD are a little more sensitive to L-glutamic acid than normal cells.

Cultured fibroblasts in Huntington's disease. II. Effects of glucosamine.

It has been proposed that there is a generalized membrane defect in Huntington's disease (HD) that is expressed in non-neuronal tissue. This membrane abnormality has been linked to a glucosamine dependence of HD cells that can be demonstrated in cultured skin fibroblasts. Twenty HD, "at-risk," and normal cell lines were used in studies of growth, viability, and adhesion in Eagle's Minimum Essential Medium with dialyzed serum or serum separated by a fractionating column (Sephadex G50). The effects of the supplementation of these media with serine, glutamine, glucosamine, and N-acetylglucosamine (0.1mM) on cell growth were determined. The growth of cells in the presence of glucosamine (0mM to 2mM) and N-acetylglucosamine (1mM) was monitored. The HD cells grew slightly better than normal or at-risk cells in the depleted media and attached to the culture surface better. However, the glucosamine dependence of HD cells was not demonstrated.