Serum cardiac troponin I concentrations in dogs with systemic inflammatory response syndrome.

BACKGROUND: Myocardial injury can be detected by cardiac troponin I (cTnI) concentration, which appears to be a predictor of short-term death in critically ill patients. It is unknown if the best prognostic indicator of short-term survival is cTnI measurement at admission or at later time points. HYPOTHESIS/OBJECTIVES: Measuring cTnI with a high-sensitivity (HS) test at different time points after admission may be a better short-term prognostic indicator than a single cTnI measurement at admission in dogs with systemic inflammatory response syndrome (SIRS). ANIMALS: Prospective, observational clinical study of 60 dogs with SIRS. METHODS: Cardiac troponin I concentration was measured in 133 serum samples, collected at days 1, 2, 3, and 5. Additionally, the acute patient physiologic and laboratory evaluation (APPLE) fast score was evaluated at admission. Prognostic capabilities of cTnI measurement and APPLE fast score for 28-day mortality were assessed by receiver operating characteristic curve analysis. RESULTS: Forty-one dogs with SIRS that survived 28 days had significantly lower serum cTnI concentrations at admission (median, 0.09 ng/mL; P = .004) and at the peak time point (median, 0.23 ng/mL; P = .01) compared to 19 nonsurvivors (median at admission, 0.63 ng/mL; median at peak, 1.22 ng/mL). Area under the curve to predict survival, using cTnI was similar at admission (0.732) and at peak (0.708), and was 0.754 for the APPLE fast score. CONCLUSIONS AND CLINICAL IMPORTANCE: Increased cTnI concentration in dogs with SIRS is associated with poor outcome. Daily follow-up measurement of cTnI concentration provides no additional prognostic information for short-term mortality.

Association of nucleated red blood cells with†mortality in critically ill dogs.

The occurrence of nucleated red blood cells (NRBC) in the peripheral blood of critically ill human patients is associated with increased mortality. In dogs, the presence of NRBCs in peripheral blood has been used as a sensitive and specific marker of complications and outcome associated with heatstroke. However, no study has investigated their prevalence in critically ill dogs. Thus, the aim of this study was to determine the prevalence of NRBCs in the peripheral blood, and to evaluate their occurrence as a prognostic factor in critically ill dogs. One hundred and one dogs were prospectively included; the presence of NRBCs was studied on a daily basis from the time of admission until day 3 in the intensive care unit (or less if discharged or death occurred earlier). Dogs fulfilled at least two systemic inflammatory response syndrome (SIRS) criteria and suffered from various diseases. Survival was defined as being alive 28 days postdischarge from hospital. In 42 dogs, NRBCs were detected at least once; 59 patients were NRBC negative. Mortality was significantly higher in NRBC-positive than NRBC-negative patients (54.8 v 30.5 per cent) (P=0.014). However, this association was not present when anaemic dogs were excluded from the analysis. Detection of NRBCs in the peripheral blood may be an indicator for regenerative anaemia and may have potential for use as a prognostic tool or in addition to established scoring systems in critically ill dogs.

EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation.

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.

Identification of Rad's effector-binding domain, intracellular localization, and analysis of expression in Pima Indians.

In order to characterize the endogenous gene product for rad (ras-related protein associated with diabetes), we prepared antibodies to synthetic peptides that correspond to amino acids (109-121, 178-195, 254-271) within the protein. These antibodies were used to analyze the expression, structure, and function of rad. Western analysis with these antibodies revealed that rad was a 46 kDa protein which was expressed during myotube formation. Further, immunolocalization studies showed that rad localized to thin filamentous regions in skeletal muscle. Interestingly, when muscle biopsies from diabetic and control Pima Indians were compared, no differences in rad protein or mRNA expression were observed. Similarly, no differences were observed in protein expression in diabetic and control Zucker diabetic fatty (ZDF) rats. Functional analysis of muscle rad revealed that its GTP-binding activity was inhibited by the addition of N-ethylmaliemide, GTP, GTP gamma S, and GDP beta S but not ATP or dithiothreitol. Moreover, cytosol-dependent rad-GTPase activity was stimulated by the peptide corresponding to amino acids 109-121. Antibodies corresponding to this epitope inhibited cytosol-dependent rad-GTPase activity. Taken together, the results indicate that 1) rad is a 46 kDa GTP-binding protein localized to thin filaments in muscle and its expression increases during myoblast fusion, 2) expression of rad in Pima Indians and ZDF rats does not correlate with diabetes, and 3) the amino acids (109-121) may be involved in regulating rad-GTPase activity, perhaps by interacting with a cytosolic factor(s) regulating nucleotide exchange and/or hydrolysis.

Muscle Rad expression and human metabolism: potential role of the novel Ras-related GTPase in energy expenditure and body composition.

Ras associated with diabetes (Rad), a new ras-related GTPase, was recently identified by subtractive cloning as an mRNA in skeletal muscle that is overexpressed in NIDDM. To better understand its metabolic significance, we measured skeletal muscle Rad expression in well-characterized insulin sensitive (IS) and insulin resistant (IR) subjects with normal glucose tolerance and in untreated NIDDM patients. We found no differences in expression of Rad mRNA levels among IS, IR, and NIDDM groups using a ribonuclease protection assay (0.22 +/- 0.06, 0.13 +/- 0.01, and 0.16 +/- 0.02 relative units, respectively; NS) and no differences in Rad protein expression using a specific anti-peptide Rad antibody (1.05 +/- 0.18, 1.14 +/- 0.08, and 1.08 +/- 0.21 units/mg protein, respectively; NS). However, Rad protein levels were positively correlated with BMI (r = 0.43, P = 0.03) and percentage body fat (r = 0.55, P < 0.005), two independent measures of obesity, and negatively correlated with resting metabolic rate (r = 0.49, P = 0.01). In multiple regression analyses, percentage body fat and resting metabolic rate independently accounted for 30 and 10% of individual variability in muscle Rad protein expression. In conclusion, Rad expression in skeletal muscle is not altered as a function of insulin resistance or NIDDM in humans. However, these data, for the first time, implicate a role for Rad in regulating body composition and energy expenditure and provide a framework for studies designed to elucidate Rad's cellular functions.

Phosphorylation of a cAMP-specific phosphodiesterase (HSPDE4B2B) by mitogen-activated protein kinase.

A cAMP-specific phosphodiesterase, HSPDE4B2B, was found to be phosphorylated when expressed in Sf9 cells or yeast. Deletion of amino acids 81-151 and 529-564 had no effect on the phosphorylation of HSPDE4B2B. Mass spectrometric analysis of purified HSPDE4B2B(1-564). HSPDE4B2B(81-564) and HSPDE4B2B(152-528) showed that phosphorylation occurred predominantly on Ser487 and Ser489. The stoicheiometry of phosphorylation was 1.2:1 (Ser487:Ser487, 489). There was no evidence by MS for a non-phosphorylated form of HSPDE4B2B(81-564) or HSPDE4B2B(152-528) when expressed in Sf9 cells. There was no detectable phosphorylation of purified HSPDE4B2B(152-528) expressed in Escherichia coli. Radiolabelling experiments with 32P revealed that phosphorylation of HSPDE4B2B(152-528) expressed in Sf9 cells was abolished when Ser487 and Ser489 were mutated to alanines. Analysis of the amino acid sequence revealed that Ser487 and Ser489 of HSPDE4B2B conform to the consensus motifs for phosphorylation by mitogen-activated protein kinase (MAP kinase) and casein kinase II respectively. Kinetic experiments in vitro showed that MAP kinase-phosphorylated E.coli expressed and purified HSPDE4B2B(151-528) with a K(m) of 63 microM and a Vmax of 3.0 mumol/min per mg. In comparison, MAP kinase phosphorylated myelin basic protein with a Km of 26.0 microM and a Vmax of 5.5 mumol/min per mg under the same conditions. Using MS and mutational analysis we found that MAP kinase-phosphorylated E. coli expressed HSPDE4B2B(152-528) exclusively at Ser487. These results suggest that phosphodiesterases could contribute to the cross-talk between the cAMP and MAP kinase signalling pathways.

Regulation of distinct cyclic AMP-specific phosphodiesterase (phosphodiesterase type 4) isozymes in human monocytic cells.

Many functions of the immune and inflammatory responses are inhibited by agents that increase intracellular levels of cAMP. Recent investigations have revealed that cAMP levels in inflammatory cells are regulated by cyclic nucleotide phosphodiesterases (PDEs) belonging to the PDE4 family (cAMP-specific PDEs). At least four different genes are known to encode PDE4 isozymes, which are characterized by their selectivity for cAMP over cGMP and their sensitivity to the antidepressant drug rolipram. The aim of our studies was to investigate whether monocytic cells could regulate PDE4 activity and whether certain PDE4 isozymes were expressed preferentially over others. Our results showed that treatment of peripheral blood monocytes or closely related Mono Mac 6 cells with dibutyryl-cAMP or other cAMP-elevating agents transiently increased rolipram-sensitive PDE4 activity 2-3-fold, without concomitant increases in cGMP-inhibited PDE (PDE3) activity. PDE4 activity was predominantly cytosolic, whereas PDE3 activity was localized to the particulate fraction. Our Northern and Western blot studies with reagents recognizing three distinct PDE4 gene products (PDE4A, PDE4B, and PDE4D) revealed that their expression is transcriptionally regulated in monocytic cells. Although none of the three isozymes was detectable under normal culture conditions, all of these were up-regulated when Mono Mac 6 cells were exposed to dibutyryl-cAMP. Distinct differences were observed in their temporal patterns of expression. Endotoxin lipopolysaccharide, a potent monocyte stimulus, also enhanced PDE4 activity in monocytic cells. These data indicate that monocytic cells may express different PDE4 isozymes, depending on their state of activation or differentiation. These isozymes could thus regulate intracellular cAMP levels at various stages of monocyte activation and could thereby be important in limiting the inflammatory response.

Recombinant retroviruses encoding human papillomavirus type 18 E6 and E7 genes stimulate proliferation and delay differentiation of human keratinocytes early after infection.

Human papillomavirus (HPV) DNAs are detected in most genital dysplasias and cancers, suggesting that these viruses perturb epithelial growth and differentiation. The E6 and E7 genes of HPV type 18 induce immortality in keratinocytes cultured from genital tract epithelia, and the immortal cell lines display aberrant squamous differentiation. To examine whether the E6 and E7 proteins directly alter keratinocyte growth and differentiation, high-titer recombinant retroviruses were constructed for efficient transfer and expression of HPV-18 genes E6, E6* and E7 in cultures of normal human keratinocytes. Infection with retroviruses encoding E6 and E7 stimulated cell proliferation, reduced the requirement for bovine pituitary extract and induced immortality. E6 and E7 also delayed but did not prevent the onset of terminal squamous differentiation. The magnitude of effects on growth and differentiation of cultured cells was directly related to levels of E7 protein expression. Thus, expression of the HPV-18 E6 and E7 genes stimulates cell proliferation and delays differentiation of keratinocytes in vitro.