2-Deoxy-d-Glucose (2-DG)-Induced Cardiac Toxicity in Rat: NT-proBNP and BNP as Potential Early Cardiac Safety Biomarkers.

2-Deoxy-d-glucose (2-DG) is being developed as a potential anticonvulsant and disease-modifying agent for patients with epilepsy; however, during preclinical development, cardiac toxicity has been encountered in rats. This study was performed to determine whether cardiac troponin (cTnI and cTnT), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), N-terminal pro-brain natriuretic peptide (NT-proBNP), and/or creatine kinase (CK) could be useful as indicators of 2-DG cardiac toxicity. In addition, this study also investigated the association of cardiac histopathological changes with these biomarkers. F344 rats (4/sex/group/sacrifice point) were gavaged with either vehicle or 2-DG (50, 125, or 375 mg/kg twice daily; total daily dose of 100, 250, or 750 mg/kg/d) for 7, 14, 21, or 45 days followed by a 15-day recovery. Dose-dependent increases in NT-proBNP and BNP plasma concentrations were observed. Following recovery period, the NT-proBNP and BNP concentrations returned to baseline levels. There were no remarkable increases in CK, ANP, cTnI, or cTnT concentrations. There were no gross cardiac lesions observed at the necropsy. Microscopic findings of vacuolar degeneration and hypertrophy of the endothelial cells of the endocardium were present in the heart at doses of 250 and 750 mg/kg/d. Microscopic findings, in general, were associated with increases in NT-proBNP levels. Cardiac toxicity appeared to be reversible. In conclusion, NT-proBNP and BNP are potential early biomarkers for 2-DG-induced cardiac toxicity that can be useful to monitor 2-DG therapy in clinical trials.

Variable immune cell frequencies in peripheral blood of LEW.1AR1-iddm rats over time compared to other congenic LEW strains.

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes (T1D), which arose through a spontaneous mutation within the major histocompatibility complex (MHC)-congenic background strain LEW.1AR1. The LEW.1AR1-iddm rat is characterized by two phenotypes: diabetes development with a diabetes incidence of 60% and a variable T cell frequency in peripheral blood. In this study the immune cell repertoire of LEW.1AR1-iddm rats was analysed over time from days 30 to 90 of life and compared to the background strain LEW.1AR1 and the LEW rat strain as well as the LEW.1WR1 rat strain. The LEW.1AR1-iddm rats are characterized by a high variability of CD3(+), CD4(+) and CD8(+) T cell frequencies in peripheral blood over time, and the frequency is unique for each animal. The variability within the frequencies resulted in changes of the CD4(+) : CD8(+) T cell ratio. The other three rat strains studied were characterized by a stable but nevertheless strain-specific T cell frequency resulting in a specific CD4(+) : CD8(+) T cell ratio. The frequency of natural killer (NK) cells and B cells in LEW.1AR1-iddm rats was increased, with a higher variability compared to the other strains. Only monocytes showed no differences in frequency and variability between all strains studied. These variabilities of immune cell frequencies in the LEW.1AR1-iddm rats might lead to imbalances between autoreactive and regulatory T cells in peripheral blood as a prerequisite for diabetes development.

Prevention of spontaneous immune-mediated diabetes development in the LEW.1AR1-iddm rat by selective CD8+ T cell transfer is associated with a cytokine shift in the pancreas-draining lymph nodes.

AIMS/HYPOTHESIS: The LEW.1AR1-iddm rat is an animal model of spontaneous type 1 diabetes mellitus. This study analysed how adoptive transfer of selective T cell subpopulations affects the incidence of diabetes. METHODS: CD4(+) or CD8(+) T cells were isolated from diabetic LEW.1AR1-iddm rats or diabetes-resistant LEW.1AR1 rats. Cells were selectively transferred into athymic LEW.1AR1-Whn ( rnu ) or prediabetic LEW.1AR1-iddm rats. The animals were monitored for blood glucose, islet infiltration and immune cell composition of pancreas-draining lymph nodes. RESULTS: After adoptive transfer of CD4(+) T cells from diabetic LEW.1AR1-iddm rats into athymic LEW.1AR1-Whn ( rnu ) rats, 50% of the recipients developed diabetes. Transfer of CD8(+) T cells failed to induce diabetes. Only 10% of the athymic recipients became diabetic after co-transfer of CD4(+) and CD8(+) T cells. Adoptive transfer of CD8(+) T cells from LEW.1AR1 or diabetic LEW.1AR1-iddm rats into prediabetic LEW.1AR1-iddm rats significantly reduced the incidence of diabetes. In protected normoglycaemic animals regulatory CD8(+)/CD25(+) and CD4(+)/CD25(+) T cell subpopulations that were also FOXP3-positive accumulated in the pancreas-draining lymph nodes. In this lymphatic organ, gene expression of anti-inflammatory cytokines was significantly higher than in diabetic rats. CONCLUSIONS/INTERPRETATION: Our results show that adoptive transfer of CD4(+) but not CD8(+) T cells from diabetic LEW.1AR1-iddm rats induced diabetes development. Importantly, CD8(+) T cells from diabetic LEW.1AR1-iddm rats and diabetes-resistant LEW.1AR1 rats provided protection against beta cell destruction. The accumulation of regulatory T cells in the pancreas-draining lymph nodes from protected rats indicates that transferred CD8(+) T cells may have beneficial effects in the control of beta cell autoimmunity.

Viable podocytes detach in experimental diabetic nephropathy: potential mechanism underlying glomerulosclerosis.

BACKGROUND: A decrease in podocyte number contributes to the development of glomerulosclerosis in diabetic nephropathy. Although podocytes have been detected in the urine in certain glomerular diseases, their viability is poorly understood. METHODS: Diabetes was induced in rats with streptozotocin. Urine was collected from control rats (given citrate), and rats with diabetic nephropathy, and cells obtained by centrifugation were resuspended in tissue culture media, and seeded onto collagen-coated tissue culture plates. Cells were grown under standard cell culture conditions ex vivo. Cell number was measured, the cell type in the urine was identified by immunostaining with specific antibodies, and morphology was assessed by light and electron microscopy. RESULTS: Within 24 h, cells obtained from the urine of diabetic rats attached to tissue culture plates ex vivo. Cells were not detected in the urine from control rats. All cells from diabetic rats stained positive for the podocyte-specific proteins synaptopodin, nephrin, podocin and Glepp-1 and negative for mesangial (OX-7), tubular (Tamm-Horsfall protein) and endothelial (RECA) cell antigens. The cell number increased daily, which is consistent with cell growth ex vivo. CONCLUSIONS: Rats with diabetic nephropathy shed podocytes into the urine that attach and grow ex vivo. These results are consistent with the detachment of viable podocytes in diabetes and add new perspectives into our understanding of development of glomerulosclerosis in diabetes mellitus.

Further characterization of Pasteurella haemolytica-like bacteria isolated from swine enteritis.

DNA-DNA hybridization studies were conducted on six Pasteurella haemolytica-like (PHL) organisms recovered from cases of swine enteritis. Chromosomal-enriched fractions of PHL organisms served as the source of DNA for Southern blots or as whole-chromosomal DNA probes. Under stringent hybridization conditions, chromosomal DNA probes of a prototype PHL (strain 6213A) organism distinguished other PHL organisms from Pasteurella haemolytica types A1 and T3, Pasteurella multiocida types A:1 and A:3, Escherichia coli, Pseudomonas aeruginosa, Actinobacillus pleuropneumoniae type 1, and Salmonella cholerasuis. The guanine-cytosine content of the DNA of three PHL strains was 41.2 to 42.8 mol % as calculated from the thermal denaturation midpoint temperatures. The PHL strains are Gram-negative, nonmotile, beta-hemolytic, pleomorphic, oxidase-positive, urease- and indole-negative, fermentative rods with the key characteristics of the species Pasteurella haemolytica. None of the PHL strains reacted with the type-specific antisera of P. haemolytica types 1 through 12 as tested by an agglutination procedure. These swine strains differed in their biochemical differentiation from P. haemolytica types A1 and T3 in that all produced acid from M-inositol and failed to grow on MacConkey agar. Acid production from trehalose and L-arabinose was variable with PHL strains. Leukotoxicity of PHL strains was evaluated by a colorimetric micro-titration assay. Sterile culture supernatants of three of five PHL strains were toxic to bovine neutrophils. Results of these studies suggest that the PHL organisms may belong to a new group of organisms under the genus Pasteurella.

Recombinant DNA probe detecting Eperythrozoon suis in swine blood.

A genomic library to Eperythrozoon suis DNA was constructed in lambda gt11, and from this library, E suis clone KSU-2 was identified as a potential diagnostic probe. In hybridization experiments that used 100-microliters samples of blood collected in chaotropic salt solutions, the KSU-2 probe hybridized strongly with purified E suis organisms and blood samples from splenectomized swine that were parasitized with E suis. However, the probe under stringent conditions did not give radiographic indications of hybridizing with equine blood DNA, bovine blood DNA infected with Anaplasma marginale, canine blood DNA infected with Ehrlichia canis, feline blood DNA infected with Haemobartonella felis, or uninfected swine blood DNA.

Efficacy of ivermectin pour-on against Ostertagia ostertagi infection and residues in the American bison, Bison bison.

Sixteen American bison, Bison bison, were artificially infected with 10(5) infective stage larvae of Ostertagia ostertagi on 21 April 1993. At 42 days post-infection eight bison were treated with 0.5% ivermectin pour-on (500 micrograms/kg bodyweight) and eight treated with the carrier only. Bison were necropsied 17 and 18 days post-treatment (21 and 22 June 1993, respectively). Mean (+/- SE) of 5,413 (+/- 1,716) adults and 565 (+/- 305) immature O. ostertagi were recovered at necropsy from bison treated with the carrier. No O. ostertagi were detected in bison treated with ivermectin pour-on. Based on the levels of the ivermectin marker metabolite in liver and adipose tissue 18 days post-treatment, the established bovine withdrawal time of 48 days appears adequate to insure that violative residues do not occur.

Examination of brown adipose tissue thermogenesis of glutamate-induced obese rats by differential temperature measurements.

Differential temperature measurement between interscapular brown adipose tissue (BAT, Tbat), rectum (Trect) and a subcutaneous point in the back left of the vertebral column (Tsc) was useful for examination of BAT-thermogenesis in glutamate-induced obese Wistar-rats. Positive temperature gradients Tbat-Tsc pointed to a basal BAT-thermogenesis, whereas negative temperature gradients Tbat-Trect did not indicate that heat production in lean and obese rats. One may conclude from this, that inclusion of subcutaneous points outside the BAT improves sensitivity of differential temperature measurements for BAT-thermogenesis. Basal temperatures Tbat, Trect and Tsc were reduced in obese rats compared to lean rats, although thermoinsulation of obese rats is improved on account of their high fat content. This points to a diminished heat production in obese rats. Cold exposure at 4 degrees C elicited an increase of temperature gradients Tbat-Trect in lean as well as in obese rats, with positive values found only in lean rats. However, positive values Tbat-Tsc were calculated for both groups. Increases were noted only in lean rats. Injection of noradrenaline (0.5 mg/kg i.m.) was followed by positive temperature gradients Tbat-Trect and increased positive values for Tbat-Tsc, pointing to a remarkable activation of BAT-thermogenesis in lean and obese rats. These findings confirm, that glutamate-induced obese rats preserved the ability to activate BAT-thermogenesis. There were, however, hints of reduced heat production in BAT of obese rats, thus contributing to obesity despite normophagia.

Reduced, positive nitrogen balance and elevated plasma free fatty acid concentration in growing, glutamate-induced obese rats.

Glutamate-induced obesity of Wistar-rats is known to develop under normophagic and normoinsulinemic conditions, although hyperphagia and hyperinsulinemia are common to obese individuals. Rats of this obesity model show retarded growth, reduced mass of some organs, carcass and whole body as well as an extraordinary high fat content, whereas protein content is reduced. In this study, nitrogen (N) balance, urinary excretion of urea-N, ammonia-N, creatine-N and alpha-amino acid-N and plasma free fatty acid concentration of growing, glutamate-induced obese rats were determined. The main results were independent of frame of reference (mmol N/kg body mass; mmol N/kg0.75 metabolic body mass; N in % of nitrogen intake): Nitrogen intake, urinary excretion of alpha-amino acids and nitrogen excretion in faeces were equal between lean and obese rats. Nitrogen excretion in urine was elevated in obese rats, mainly resulting from increased amounts of urea and ammonia. Nitrogen balance was positive in both groups, but reduced in obese rats. These data point to normal digestion of food proteins, but an unusual high oxidative desamination rate of the absorbed amino acids in obese rats. Taking into account the various hormonal and nerval alterations in glutamate-induced obese rats, resulting e.g. in increased hepatic insulin concentration, the retained amino acid carbon should be channelled into hepatic fatty acid synthesis. Really, unfasted and overnight fasted obese rats showed elevated plasma free fatty acid concentrations. Channeling of amino acids into lipogenesis may explain the low muscle mass and striking fat accumulation--despite normophagia and peripheral normoinsulinemia--of growing, glutamate-induced obese Wistar-rats.

Urine-creatinine concentration as a marker of urine dilution: reflections using a cohort of 45,000 samples.

BACKGROUND: Statistical data from a wide cohort of subjects should provide arguements for a more valid interpretation of urine-creatinine concentrations as laboratory marker of urine dilution. METHODS: Unselected, consecutive urine-creatinine concentrations from 11,811 women and 13,009 men in a clinical chemistry laboratory (mainly from clinical trials and employment medicine departments) and from 7300 women and 12,456 men in a toxicological chemistry laboratory (mainly from drug screenings for re-issuing drivers licenses or from employment medicine departments) were evaluated by descriptive and comparative statistics. RESULTS: Women (clinical chemistry lab, toxicological chemistry lab): mean 723 mg/L, 921 mg/L; median 568 mg/L, 728 mg/L; 2.5-97.5% percentile range 189-2198 mg/L, 129-2690 mg/L. Men (clinical chemistry lab, toxicological chemistry lab): mean 975 mg/L, 1395 mg/L; median 802 mg/L, 1241 mg/L; 2.5-97.5% percentile range 256-2660 mg/L, 204-3520 mg/L. The rate of urine-creatinine concentrations of >3000 mg/L (up to 3-fold of the upper limit of the reference range) was higher for men in both laboratories and for both genders in the toxicological chemistry lab compared with the clinical chemistry lab (toxicological chemistry lab: 697 for men (5.6%) and 111 for women (1.5%), clinical chemistry lab: 200 for men (1.5%) and 93 for women (0.8%)). CONCLUSIONS: Utmost caution should be taken when interpreting urinary creatinine concentrations that fall below so-called cut-offs. Cut-offs greater than the gender-specific 2.5% percentiles bear a high risk of misinterpretation regarding urine adulteration. Such cut-offs are no longer acceptable. At present, the borderline range of >50mg/L to <200mg/L given by the Australian Standard AS/NZS4308:2008 and indicating dilute urines but are not implicated in adulteration seems to fit best with the clinical and forensic requirements. Nevertheless, using gender-independent urine-creatinine concentration cut-offs can discriminate women since women have in general lower muscle mass and thus lower urinary creatinine concentrations compared with men. Future concepts of drug screen in urine should use gender-specific and creatinine-adjusted decision limits.

Mechanical stretch induces podocyte hypertrophy in vitro.

BACKGROUND: Increased intraglomerular pressure is a final pathway toward glomerulosclerosis in systemic hypertension, diabetes, and focal segmental glomerulosclerosis (FSGS). Increased intraglomerular pressure causes stress-tension, or stretch, on resident glomerular cells. However, the effects of stretch on podocyte growth, and the mechanisms that underlie this, have not been elucidated. METHODS: To test the hypothesis that stretch alters podocyte growth, cultured mouse podocytes were exposed to cyclic mechanical stretch created by vacuum; control cells were grown under similar conditions, but not exposed to stretch. Proliferation (cell cycle phases) and hypertrophy (forward light scatter) were measured in stretched and control podocytes by flow cytometry. The role of the cyclin-dependent kinase (CDK) inhibitors, p21 and p27, was examined by stretching podocytes isolated from p21 and p27 knockout (-/-) mice, and the role of specific signaling pathways was assessed by Western blot analysis and blocking studies. RESULTS: Our results showed that stretch reduced cell cycle progression in wild-type and single p27-/- podocytes and induced hypertrophy in these cells in all phases of the cell cycle at 24, 48, and 72 hours. In contrast, stretch did not induce hypertrophy in single p21-/- and double p21/p27-/- podocytes. Stretch-induced hypertrophy required cell cycle entry, and was prevented by specifically blocking extracellular signal-regulated kinase 1/2 (Erk1/2) or Akt. Although stretch increased p38 activation, inhibition of this pathway had no effect on hypertrophy. CONCLUSION: Mechanical stretch induces hypertrophy in podocytes in vitro in all phases of the cell cycle. This effect is cell cycle dependent, and requires p21, Erk1/2, and Akt. Stretch may play a role in podocyte injury when intraglomerular pressure is increased.

Carbohydrate-deficient transferrin as a marker of chronic alcohol abuse: a critical review of preanalysis, analysis, and interpretation.

BACKGROUND: Carbohydrate-deficient transferrin (CDT) is used for diagnosis of chronic alcohol abuse. Some 200-300 reports on CDT have been published in impact factor-listed journals. The aims of this review were to condense the current knowledge and to resolve remaining issues on CDT. APPROACH: The literature (1976-2000) was searched using MEDLINE and Knowledge Server with "alcohol and CDT" as the search items. The data were reviewed systematically, checked for redundancy, and organized in sequence based on the steps involved in CDT analysis. CONTENT: The review is divided into sections based on microheterogeneity of human serum transferrin (Tf), definition of CDT, structure of human serum CDT, pathomechanisms of ethanol-induced CDT increase, preanalysis, analysis, and medical interpretation (postanalysis). Test-specific cutoff values for serum CDT and causes of false positives and negatives for chronic alcohol abuse are discussed and summarized. SUMMARY: Asialo- and disialo-Fe(2)-Tf, which lack one or two complete N-glycans, and monosialo-Fe(2)-Tf (structure remains unclear) are collectively referred to as CDT. Diminished mRNA concentration and glycoprotein glycosyltransferase activities involved in Tf N-glycan synthesis and increased sialidase activity most likely account for alcohol-induced increases in CDT. Knowledge about in vivo and in vitro effects on serum CDT is poor. Reliable CDT and non-CDT fractionation is needed for CDT measurement. Analysis methods with different analytical specificities and recoveries decreased the comparability of values and statistical parameters of the diagnostic efficiency of CDT. CDT is the most specific marker of chronic alcohol abuse to date. Efforts should concentrate on the pathomechanisms (in vivo), preanalysis, and standardization of CDT analysis.

Assessment of cold induced alterations in catecholamine turnover of lean and glutamate-treated obese rats.

Excretion of norepinephrine (NE) and vanillylmandelic acid (VMA) in urine as well as NE-turnover in tissues from lean and glutamate-treated obese rats were determined in warm and cold environment. NE-and VMA-excretion in urine was elevated by cold exposure, indicating an activation of the sympathetic nervous system in animals of both groups. Organspecific NE-turnover responds with higher sensitivity to cold in obese rats but without complete compensation in brown adipose tissue. Urinary NE- and VMA- excretion as well as NE-turnover in organs confirmed that cold exposure activates the sympathetic nervous system. Measurement of NE-turnover in tissues gives organspecific information regarding alterations in sympathetic activity during cold exposure, whereas excretion of NE and VMA in urine is a summarizing measure for the whole body turnover only.

Echinococcal cyst causing bladder outlet obstruction. A case report.

The case of a 36-year-old black man with bladder outlet obstruction caused by a retrovesical echinococcal cyst is described. The diagnosis was made by conventional radiography, pelvic ultrasonography and computed tomography. The cyst was removed and the patient made an uneventful recovery.