Development and validation of a total coronary heart disease risk score in type 2 diabetes mellitus

There are no validated risk scores for predicting coronary heart disease (CHD) in Chinese patients with type 2 diabetes mellitus. This study aimed to validate the UKPDS risk engine and, if indicated, develop CHD risk scores. A total of 7,067 patients without CHD at baseline were analyzed. Data were randomly assigned to a training data set and a test data set. Cox models were used to develop risk scores to predict total CHD in the training data set. Calibration was assessed using the Hosmer-Lemeshow test, and discrimination was examined using the area under the receiver-operating characteristic curve in the test data set. During a median follow-up of 5.40 years, 4.97% of patients (n = 351) developed incident CHD. The UKPDS CHD risk engine overestimated the risk of CHD with suboptimal discrimination, and a new total CHD risk score was developed. The developed total CHD risk score was 0.0267 x age (years) - 0.3536 x sex (1 if female) + 0.4373 x current smoking status (1 if yes) + 0.0403 x duration of diabetes (years) - 0.4808 x Log(10) (estimated glomerular filtration rate [ml/min/1.73 m(2)]) + 0.1232 x Log(10) (1 + spot urinary albumin-creatinine ratio [mg/mmol]) + 0.2644 x non-high-density lipoprotein cholesterol (mmol/L). The 5-year probability of CHD = 1 - 0.9616(EXP(0.9440 x [RISK SCORE - 0.7082])). Predicted CHD probability was not significantly different from observed total CHD probability, and the adjusted area under the receiver-operating characteristic curve was 0.74 during 5 years of follow-up. In conclusion, the UKPDS CHD risk engine overestimated the risk of Chinese patients with type 2 diabetes mellitus and the newly developed total CHD risk score performed well in the test data set. External validations are required in other Chinese populations.     

A voluntary oral ethanol-feeding rat model associated with necroinflammatory liver injury

Background: The intragastric (IG) ethanol infusion model results in fatty liver, necrosis, inflammation and fibrosis. This model was utilized to study the pathogenesis of alcoholic liver disease (ALD). Disadvantages of the IG model include maintenance of the animals and equipment expense. To develop a voluntary feeding model for ALD, we took advantage of two important observations in the IG model: (i) female rats demonstrate greater severity of alcohol‐induced liver injury than males and (ii) rats fed fish oil as a source of fatty acids develop more severe alcoholic liver injury than rats fed other fatty acids with ethanol. Methods: Female Wistar rats (205 to 220 g) were fed for 8 weeks a diet containing 8% ethanol, fish oil (30% of calories), protein, and dextrose. Pair‐fed controls (FD) received dextrose in amounts isocaloric to ethanol. The following measurements were made: liver pathology [fatty liver (0 to 4), necrosis, inflammation and fibrosis by Sirius Red], endotoxin and alanine aminotransferase (ALT) in plasma, urine ethanol, lipid peroxidation, nuclear factor kappa‐B (NF‐κB) and mRNA levels for tumor necrosis factor‐alpha (TNF‐α), cyclooxygenase‐2 (COX‐2), and inducible nitric oxide synthase (iNOS). Protein levels for iNOS and nitrotyrosine were evaluated by immunohistochemistry and Western Blot analysis. Liver proteasome and cytochrome P450 2E1 activity and protein levels of asialoglycoprotein receptor (ASGPR) were also evaluated. In addition, mRNA levels of fibrogenic markers were assessed. Results: All animals lost weight for the initial 2 to 3 weeks but then gained weight until killing at 8 weeks. There was, however, a significant difference (p < 0.05) in weight between the ethanol‐fed (Etoh) and (FD) groups at the end of the experiment. The mean urine ethanol levels ranged between 190 and 240 mg/dl. The severity of pathological changes was greater (p < 0.01) in Etoh vs. FD: fatty liver, 3.0 ± 1.2 vs. 1.2 ± 0.4; necrosis (foci/mm²), 3.9 ± 2.3 vs. 0.4 ± 0.3; inflammation (cells/mm²), 19.0 ± 6.3 vs. 1.8 ± 0.6. Centrilobular collagen deposition (% area), assessed by Sirius Red staining, was greater in Etoh vs. FD. Levels of endotoxin, ALT, CYP2E1 and lipid peroxidation markers were also higher (p < 0.01) in Etoh vs. FD. Levels of NF‐κB and mRNA of pro‐inflammatory mediators (TNF‐α, COX‐2, iNOS) and procollagen‐I were increased (p < 0.05) in ethanol‐fed rats. Immunohistochemical analysis showed more intense staining for both iNOS and nitrotyrosine in the centrilobular areas in the Etoh vs. FD groups. The greater area of positive staining for iNOS and nitrotyrosine in Etoh vs. FD was confirmed by Western Blot analysis. An increase in the expression of mRNA for profibrogenic genes (p < 0.05) was seen in ethanol‐fed rats. Conclusions: A voluntary feeding regimen consisting of fish oil and ethanol in female rats is technically less demanding yet produces pathological and biochemical changes similar to those observed with the IG model. Pathological changes include fatty liver, necrosis and inflammation. Increased NF‐κB and mRNA and protein levels of the pro‐inflammatory mediators TNF‐α, COX‐2 and iNOS, coincided with the presence of necroinflammatory changes. The voluntary feeding regimen is proposed as an alternative to the IG model in the study of alcoholic liver injury.     

Necrotic foci, elevated chemokines and infiltrating neutrophils in the liver of glycogen storage disease type Ia

BACKGROUND/AIMS: Glycogen storage disease type Ia (GSD-Ia) patients manifest the long-term complication of hepatocellular adenoma (HCA) of unknown etiology. We showed previously that GSD-Ia mice exhibit neutrophilia and elevated serum cytokine levels. This study was conducted to evaluate whether human GSD-Ia patients exhibit analogous increases and whether in GSD-Ia mice a correlation exists between immune abnormalities and, biochemical and histological alterations in the liver. METHODS: Differential leukocyte counts and cytokine levels were investigated in GSD-Ia patients. Hepatic chemokine production, neutrophil infiltration, and histological abnormalities were investigated in GSD-Ia mice. RESULTS: We show that GSD-Ia patients exhibit increased peripheral neutrophil counts and serum interleukin-8 (IL-8). Compared to normal subjects, HCA-bearing GSD-Ia patients have a 2.8-fold higher serum IL-8 concentration, while GSD-Ia patients without HCA have a 1.4-fold higher concentration. Hepatic injury in GSD-Ia mice is evidenced by necrotic foci, markedly elevated infiltrating neutrophils, and increased hepatic production of chemokines. CONCLUSIONS: Peripheral neutrophilia and elevated serum chemokines are characteristic of GSD-Ia with HCA-bearing GSD-Ia patients having the highest serum IL-8. In GSD-Ia mice these elevations correlate with elevated hepatic chemokine levels, neutrophil infiltration, and necrosis. Taken together, peripheral neutrophilia and increased serum chemokines may indicate hepatic injuries in GSD-Ia.     

Berberine induces G1 arrest and apoptosis in human glioblastoma T98G cells through mitochondrial/caspases pathway

Berberine is an isoquinoline plant alkaloid with a long history of being used for the treatment of many diseases in Chinese herbal medicine. Berberine has a wide range of biochemical and pharmacological effects, including antitumor activities, but its mechanism of action is not clearly understood. In this study, we investigated that the relationship between the antiproliferative activities of berberine and the apoptotic pathway associated with its molecular mechanism of action in human glioblastoma T98G cells. Berberine treatment of T98G cell lines inhibited cell proliferation and induced cell death in a dose (50-200 microg/ml) dependent manner with an IC50 value of 134 microg/ml, which was associated with an increase in G1 arrest. Western blot analysis showed that the berberine-induced G1 arrest was mediated through the increased expression of P27 and the decreased expression of cyclin-dependent kinase (CDK) 2, CDK4, cyclin D, and cyclin E proteins. Berberine treatment also markedly enhanced apoptosis in T98G cells through the induction of a higher ratio of the Bax/Bcl-2 proteins, the disruption of mitochondrial membrane potential, and the activation of procaspase-9, caspase-9, caspase-3, and poly(ADP-ribose) polymerase (PARP). Berberine can inhibit T98G cell proliferation by inducing G1 arrest and apoptosis. These results demonstrate that the berberine-induced apoptosis of T98G cells is primarily mediated through the mitochondrial/caspases-dependent pathway.     

Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-kappaB activation

N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) is known to inhibit NF-kappaB activation and the expression of inflammation mediators in cultured cells. We measured the potential of TPCK to inhibit the pathogenesis of collagen-induced arthritis by blocking NF-kappaB activation. Arthritis was induced in DBA/1J mice by the injection of bovine type II collagen in adjuvant on days 0 and 14. Mice received either TPCK (3 or 10 mg/kg, i.p.) or vehicle three times a week for 3 weeks starting on day 21. TPCK moderately reduced clinical disease activity scores, whereas it markedly suppressed histological indications of joint destruction. In vitro production of tumor necrosis factor-alpha, interleukin-6, and monocyte chemotactic protein-1 from lipopolysaccharide-stimulated spleen cells was also reduced by in vivo treatment with TPCK. Proliferation of cells isolated from spleen or draining lymph nodes and production of interferon-gamma and interleukin-17 in response to stimulation with type II collagen was decreased by TPCK. Moreover, nuclear NF-kappaB activity induced by collagen immunization was significantly reduced in mice treated with TPCK. Finally, osteoclast differentiation of bone marrow cells induced by macrophage colony-stimulating factor and receptor activator of NF-kappaB ligand was completely inhibited by TPCK. These results indicate that TPCK attenuates collagen-induced arthritis and bone erosion by suppressing NF-kappaB activation and thus expression of inflammatory and osteoclastogenic genes.     

Slow delayed rectifier K+ current block by HMR 1556 increases dispersion of repolarization and promotes Torsades de Pointes in rabbit ventricles

Background and purpose:

The slow delayed rectifier K⁺ current (I_Ks) contributes to ventricular repolarization when the action potential (AP) is prolonged. I_Ks block during drug-induced AP prolongation may promote Torsades de Pointes (TdP), but whether this is due to additional AP prolongation is uncertain.

Experimental approach:

In bradycardic perfused rabbit ventricles, the incidence of spontaneous TdP, monophasic AP duration at 90% repolarization (MAPD₉₀) and ECG interval between the peak and the end of T wave (T_peak−end) (index of dispersion of repolarization) were measured after the administration of veratridine (125 nM, slows Na⁺ channel inactivation), dofetilide (7.5 or 10 nM, a rapid delayed rectifier blocker) and HMR 1556 (HMR, 100 nM, an I_Ks blocker), alone or in combinations (n=6 each).

Key results:

HMR did not prolong MAPD₉₀, whereas veratridine or 7.5 nM dofetilide prolonged MAPD₉₀ (P<0.01) without inducing TdP. Veratridine+7.5 nM dofetilide additively prolonged MAPD₉₀ (P<0.05), induced 4±6 TdP per heart and prolonged T_peak−end by 12±10 ms. Subsequent addition of HMR did not further prolonged MAPD₉₀, but increased the number of TdP to 22±18 per heart and increased T_peak−end by 39±21 ms (P<0.05). Increasing dofetilide concentration from 7.5 to 10 nM (added to veratridine) produced a longer MAPD₉₀, but fewer TdP (5±5 per heart) and less T_peak−end prolongation (17±8 ms) compared to the veratridine+7.5 nM dofetilide+HMR group (P<0.05).

Conclusions and implications:

Adding I_Ks block markedly increases TdP incidence in hearts predisposed to TdP development by increasing the dispersion of repolarization, but without additional AP prolongation.