Immunomodulator FTY720 improves glucose homeostasis and diabetic complications by rejuvenation of ß-cell function in nonhuman primate model of diabetes.

Inadequate ß-cell mass is essential for the pathogenesis of type 2 diabetes (T2D). Previous report showed that an immunomodulator FTY720, a sphingosine 1-phosphate (S1P) receptor modulator, sustainably normalized hyperglycemia by stimulating ß-cell in vivo regeneration in db/db mice. We further examined the effects of FTY720 on glucose homeostasis and diabetic complications in a translational nonhuman primate (NHP) model of spontaneously developed diabetes. The male diabetic cynomolgus macaques of 18-19 year old were randomly divided into Vehicle (Purified water, n = 5) and FTY720 (5 mg/kg, n = 7) groups with oral gavage once daily for 10 weeks followed by 10 weeks drug free period. Compared with the Vehicle group, FTY720 effectively lowered HbA1c, blood concentrations of fasting glucose (FBG) and insulin, hence, decreased homeostatic model assessment of insulin resistance (HOMA-IR); ameliorated glucose intolerance and restored glucose-stimulated insulin release, indicating rejuvenation of ß-cell function in diabetic NHPs. Importantly, after withdrawal of FTY720, FBG, and HbA1c remained at low level in the drug free period. Echocardiography revealed that FTY720 significantly reduced proteinuria and improved cardiac left ventricular systolic function measured by increased ejection fraction and fractional shortening in the diabetic NHPs. Finally, flow cytometry analysis (FACS) detected that FTY720 significantly reduced CD4 + and CD8 + T lymphocytes as well as increased DC cells in the circulation. Immunomodulator FTY720 improves glucose homeostasis via rejuvenation of ß-cell function, which can be mediated by suppression of cytotoxic CD8 + T lymphocytes to ß-cells, thus, may be a novel immunotherapy to reverse T2D progression and ameliorate the diabetic complications.

Effects of mixed meal tolerance test on gastric emptying, glucose and lipid homeostasis in obese nonhuman primates.

Meal ingestion elicits a variety of neuronal, physiological and hormonal responses that differ in healthy, obese or diabetic individuals. The mixed meal tolerance test (MMTT) is a well-established method to evaluate pancreatic ß-cell reserve and glucose homeostasis in both preclinical and clinical research in response to calorically defined meal. Nonhuman primates (NHPs) are highly valuable for diabetic research as they can naturally develop type 2 diabetes mellitus (T2DM) in a way similar to the onset and progression of human T2DM. The purpose of this study was to investigate the reproducibility and effects of a MMTT containing acetaminophen on plasma glucose, insulin, C-peptide, incretin hormones, lipids, acetaminophen appearance (a surrogate marker for gastric emptying) in 16 conscious obese cynomolgus monkeys (Macaca fascicularis). Plasma insulin, C-peptide, TG, aGLP-1, tGIP, PYY and acetaminophen significantly increased after meal/acetaminophen administration. A subsequent study in 6 animals showed that the changes of plasma glucose, insulin, C-peptide, lipids and acetaminophen were reproducible. There were no significant differences in responses to the MMTT among the obese NHPs with (n = 11) or without (n = 5) hyperglycemia. Our results demonstrate that mixed meal administration induces significant secretion of several incretins which are critical for maintaining glucose homeostasis. In addition, the responses to the MMTTs are reproducible in NHPs, which is important when the MMTT is used for evaluating post-meal glucose homeostasis in research.

Diastolic dysfunction and impaired cardiac output reserve in dysmetabolic nonhuman primate with proteinuria.

BACKGROUND: Cardiorenal complications are common in patients with dysmetabolism and diabetes. The present study aimed to examine if a nonhuman primate (NHP) model with spontaneously developed metabolic disorder and diabetes develops similar complications to humans, such as proteinuria and cardiac dysfunction at resting condition or diminished cardiac functional reserve following dobutamine stress echocardiography (DSE). METHODS AND RESULTS: A total of 66 dysmetabolic and diabetic cynomolgus (Macaca fascicularis) NHPs were enrolled to select 19 NHPs (MetS) with marked metabolic disorders and diabetes (fasting blood glucose: 178 ±â€¯18 vs. 61 ±â€¯3 mg/dL) accompanied by proteinuria (ACR: 134 ±â€¯34 vs. 1.5 ±â€¯0.4 mg/mmol) compared to 8 normal NHPs (CTRL). Under resting condition, MetS NHPs showed mild left ventricular (LV) diastolic dysfunction (E/A: 1 ±â€¯0.06 vs. 1.5 ±â€¯0.13), but with preserved ejection fraction (EF: 65 ±â€¯2 vs. 71 ±â€¯3%) compared to CTRL. DSE with an intravenous infusion of dobutamine at ascending doses (5, 10, 20, 30 and 40 µg/kg/min, 7 min for each dose) resulted in a dose-dependent increase in cardiac function, however, with a significantly diminished magnitude at the highest dose of dobutamine infusion (40 µg/kg/min) in both diastole (E/A: -12 ±â€¯3 vs. -38 ±â€¯5%) and systole (EF: 25 ±â€¯3 vs. 33 ±â€¯5%) as well as ~42% reduced cardiac output reserve (COR: 63 ±â€¯8 vs. 105 ±â€¯18%, p < 0.02) in the MetS compared to CTRL NHPs. CONCLUSION: These data demonstrate that MetS NHPs with cardiorenal complications: proteinuria, LV diastolic dysfunction and preserved LV systolic function under resting conditions displayed compromised cardiac functional reserve under dobutamine stress. Based on these phenotypes, this NHP model of diabetes with cardiorenal complications can be used as a highly translational model mimic human disease for pharmaceutical research.

Neural network method for localization of radioactive sources within a partially coded field-of-view in coded-aperture imaging.

Coded-aperture imagers typically have a smaller field-of-view (FOV) than in un-collimated gamma imaging systems. However, sources out of the fully coded field-of-view (FCFOV) can cause pseudo hotspots on the wrong side of an image reconstructed using the cross-correlation method. In this work, we propose a neural network method to identify and localize the sources within the partially coded field-of-view (PCFOV). The model was trained using Monte Carlo simulation data and evaluated with both simulation and experimental data. The results showed that the proposed model can identify and localize sources with good classification accuracy, low positioning error, and strong robustness to the statistical noise.

Carbon tetrachloride (CCl4) accelerated development of non-alcoholic fatty liver disease (NAFLD)/steatohepatitis (NASH) in MS-NASH mice fed western diet supplemented with fructose (WDF).

BACKGROUND: Multiple murine models of nonalcoholic fatty liver disease/steatohepatitis (NAFLD/NASH) have been established by using obesogenic diets and/or chemical induction. MS-NASH mouse (formally FATZO) is a spontaneously developed dysmetabolic strain that can progress from hepatosteatosis to moderate fibrosis when fed a western diet supplemented with 5% fructose (WDF). This study aimed to use carbon tetrachloride (CCl4) to accelerate and aggravate progression of NAFLD/NASH in MS-NASH mouse. METHODS: Male MS-NASH mice at 8 weeks of age were fed WDF for the entire study. Starting at 16 weeks of age, CCl4 was intraperitoneally administered twice weekly at a dose of 0.2 mL/kg for 3 weeks or 0.08 mL/kg for 8 weeks. Obeticholic acid (OCA, 30 mg/kg, QD) was administered in both MS-NASH and C57Bl/6 mice fed WDF and treated with CCl4 (0.08 mL/kg). RESULTS: WDF enhanced obesity and hepatosteatosis, as well as induced moderate fibrosis in MS-NASH mice similar to previous reports. Administration of CCl4 accelerated liver fibrosis with increased bridging and liver hydroxyproline contents, but had no significant impact on liver steatosis and lipid contents. High dose CCl4 caused high mortality and dramatic elevation of ALT and ASL, while low dose CCl4 resulted in a moderate elevation of ALT and AST with low mortality. Compared to C57BI/6 mice with WDF and CCl4 (0.08 mL/kg), MS-NASH mice had more prominent hepatosteatosis and fibrosis. OCA treatment significantly lowered liver triglycerides, steatosis and fibrosis in both MS-NASH and C57Bl/6 mice fed WDF with CCl4 treatment. CONCLUSIONS: CCl4 reduced induction time and exacerbated liver fibrosis in MS-NASH mice on WDF, proving a superior NASH model with more prominent liver pathology, which has been used favorably in pharmaceutical industry for testing novel NASH therapeutics.

Comparison of Continuous Glucose Monitoring between Dexcom G4 Platinum and HD-XG Systems in Nonhuman Primates (Macaca Fascicularis).

Timely knowing glucose level helps diabetic patients to manage the disease, including decisions about food, physical activity and medication. This study compared two continuous glucose monitoring systems in conscious and moving-free nonhuman primates (NHPs, Macaca fascicularis). Each normoglycemic or diabetic monkey was implanted with one Dexcom G4 Platinum subcutaneously or one HD-XG glucose sensor arterially for glucose monitoring. The glucose levels measured by both telemetry devices significantly correlated with the glucometer readings. The data of oral glucose tolerance test (oGTT) showed that the glucose levels measured by either Dexcom G4 Platinum or HD-XG transmitter were very similar to glucometer readings. However, compared to HD-XG transmitter or glucometer, Dexcom G4 Platinum detected a decreased glucose peak of ivGTT with approximately 10 min delay due to interstitial glucose far behind blood glucose change. Our data showed the advantages of the telemetry systems are: (1) consecutive data collection (day and night); (2) no bleeding; (3) no anesthesia (moving freely); (4) recording natural response without physical restriction and stress; (5) less labor intensity during ivGTT and other tests; (6) quick outcomes without lab tests. This article summarized and compared the differences of the general characteristics of two continuous glucose monitoring systems in diabetic research.

Effects of a purified krill oil phospholipid rich in long-chain omega-3 fatty acids on cardiovascular disease risk factors in non-human primates with naturally occurring diabetes type-2 and dyslipidemia.

BACKGROUND: High serum levels of cholesterol, in particular low-density lipoprotein cholesterol, are considered a significant risk factor for development of cardiovascular disease. Therefore, rigorous treatment regimens with statins and other pharmaceuticals have been used extensively to reduce elevated cholesterol levels. Literature data have not clearly concluded whether long-chain omega-3 fatty acids reduce, increase or leave circulating cholesterol unaffected. In the present study a novel krill-oil derived preparation of omega-3 rich phospholipids, mainly phosphatidylcholine, was administered orally at increasing doses for 12 weeks to dyslipidemic non-human primates, and cholesterols and several other risk factors for cardiovascular disease were measured before, during and after treatment. METHODS: Six dyslipidemic non-human primates suffering from naturally occurring diabetes type-2 were included, three in a vehicle control group and three being treated with the omega-3 rich phospholipid preparation. The control and test items were given daily by gavage and the doses of the test item were 50, 150 and 450 mg phospholipids/kg/day. Each dose level was given for 4 weeks. Plasma concentrations of the omega-3 fatty acids were measured in connection with change in dose and the omega-3 index in erythrocytes was determined bi-weekly. Blood lipids, apolipoproteins and diabetes, inflammatory and safety biomarkers were determined either weekly, biweekly or every 4 weeks. For the blood lipids and apolipoproteins, control-adjusted mean values are presented while absolute values are presented for the other parameters. Due to the low number of animals in each group, no statistical analyses were done. RESULTS: The only detectable effects measured during dosing with the lowest dose were an increase in HDL-cholesterol and apolipoprotein A1. The intermediate and high doses decreased total cholesterol, LDL-cholesterol, apolipoprotein B100 and triglycerides and increased HDL-cholesterol and apolipoprotein A1. No effects were seen on the diabetes and inflammatory markers and on safety biomarkers. CONCLUSIONS: The results indicate that the omega-3 rich phospholipid preparation had a positive impact on cardiovascular disease risk factors by reducing total cholesterol, LDL-cholesterol and triglycerides and increasing HDL-cholesterol. These findings justify further investigations of this preparation in animal models of dyslipidemia and, provided the current findings are confirmed, in human trials.

The time course of erythrocyte membrane fatty acid concentrations during and after treatment of non-human primates with increasing doses of an omega-3 rich phospholipid preparation derived from krill-oil.

BACKGROUND: A commonly used measure to reflect the intake of the long-chain omega-3 fatty acids EPA and DHA is the omega-3 index, defined as the sum of EPA + DHA as % of total fatty acids in erythrocyte membrane. When the omega-3 index changes it follows that the relative fractions of other fatty acids in the membrane are also changed. In the present study, increasing doses of a preparation of omega-3 rich phospholipids extracted from krill oil were administered orally to non-human primates for 12 weeks and the time course of EPA, DHA and 22 other fatty acids in erythrocytes was determined bi-weekly during treatment and for 8 weeks after cessation of treatment. Plasma concentrations of six endocannabinoid-type mediators being downstream metabolites of some fatty acids analyzed in erythrocytes were also determined. METHODS: Six diabetic, dyslipidemic non-human primates were included, three in a vehicle control group and three being treated with the omega-3 rich phospholipid preparation. The vehicle control and test items were given daily by gavage and the test item doses were 50, 150 and 450 mg phospholipids/kg/day. Each dose level was given for four weeks. Blood was sampled at baseline and thereafter bi-weekly. Fatty acids were determined in erythrocytes by methylation followed by gas-chromatography. Endocannabinoids and endocannabinoid-like mediators were analyzed in plasma by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. RESULTS: The treatment resulted in a dose-related increase in the fraction of EPA and DHA in erythrocyte membranes and a dose-related decrease of other poly-unsaturated fatty acids, in particular omega-6 polyunsaturated fatty acids. Erythrocyte concentrations of saturated fatty acids remained unchanged throughout the experiment. Plasma concentrations of endocannabinoids and endocannabinoid-like mediators changed accordingly as those being downstream arachidonic acid decreased, downstream of the saturated palmitic and oleic acids remained unchanged while a downstream EPA metabolite increased. CONCLUSION: Increasing the omega-3 index by administering an omega-3 rich phospholipid extracted from krill oil did not alter the ratio of unsaturated vs. saturated fatty acids in the erythrocyte membranes but only the relative concentrations of unsaturated fatty acids, in particular unsaturated omega-6 fatty acids. Concentrations of saturated fatty acids remained unchanged.

Left ventricular diastolic dysfunction in nonhuman primate model of dysmetabolism and diabetes.

BACKGROUND: Diabetes is one of the major risk factors for cardiomyopathy and heart failure with reduced ejection fraction (EF) and highly associated with left ventricular (LV) dysfunction in human. This study aimed 1) to noninvasively assess cardiac function using echocardiography; 2) to test the hypothesis that like diabetic human, cardiac function may also be compromised; in spontaneously developed obese, dysmetabolic and diabetic nonhuman primates (NHPs). METHODS: Cardiovascular functions were measured by noninvasive echocardiography in 28 control, 20 dysmetabolic/pre-diabetic and 41 diabetic cynomolgus monkeys based on fasting blood glucose and other metabolic status. RESULTS: The LV end-systolic volume (ESV) was higher while end-diastolic volume (EDV, 12 ± 5.7 mL) and EF (63 ± 12.8 %) significantly lower in the diabetic compared to control (14 ± 7 mL and 68 ± 9.8 %) group, respectively. The E/A ratio of LV trans-mitral peak flow rate during early (E) over late (A) diastole was significantly lower in the diabetic (1.19 ± 0.45) than control (1.44 ± 0.48) group. E-wave deceleration time (E DT) was prolonged in the diabetic (89 ± 41 ms) compared to control (78 ± 26 ms) group. Left atrial (LA) maximal dimension (LADmax) was significantly greater in the diabetic (1.3 ± 0.17 cm) than control (1.1 ± 0.16 cm) group. Biochemical tests showed that total cholesterol and LDL were significant higher in the diabetic (167 ± 63 and 69 ± 37 mg/dL) than both pre-diabetic (113 ± 37 and 41 ± 23 mg/dL) and control (120 ± 28 and 41 ± 17 mg/dL) groups, respectively. Multivariable logistic regression analysis demonstrated that LV systolic (reduced EF) and diastolic (abnormal E/A ratio) dysfunctions are significantly correlated with aging and hyperglycemia. Histopathology examination of the necropsy heart revealed inflammatory infiltration, cardiomyocyte hypertrophy and fragmentation, indicating the myocardial ischemia and remodeling which is consistent with the LV dysfunction phenotype. CONCLUSIONS: Using noninvasive echocardiography, the present study demonstrated for the first time that dysmetabolic and diabetic NHPs are associated with LV systolic (increased ESV, decreased EF, etc.) and diastolic (decreased EDV and E/A ratio, prolonged E DT, etc.) dysfunctions, accompanied by LA hypertrophic remodeling (increased LADmax), the phenotypes similarly to those found in diabetic patients. Thus, spontaneously developed dysmetabolic and diabetic NHPs is a highly translatable model to human diseases not only in the pathogenic mechanisms but also can be used for testing novel therapies for cardiometabolic disorders.

Xylazine-induced reduction of tissue sensitivity to insulin leads to acute hyperglycemia in diabetic and normoglycemic monkeys.

BACKGROUND: The α2-adrenoceptor agonist xylazine as an anesthetic has been widely used either alone or in combination with other anesthetics, such as ketamine, in veterinary clinic and research. In the last decade xylazine has been used in drug abusers in certain geographic area. This study investigated the effects of xylazine on blood glucose level and insulin secretion in normoglycemic and insulin-dependent diabetic monkeys. METHODS: Both adult cynomolgus (n = 10) and rhesus (n = 8) monkeys with either sex were used in the study. Xylazine (1-2 mg/kg) was administrated intramuscularly. Blood glucose, insulin, glucagon and glucagon-like peptide 1 in overnight-fasted monkeys were measured immediately before and after xylazine administration. The hyperinsulinemic-euglycemic clamp method was used in the study for assessing the potential mechanism of xylazine-induced hyperglycemia. RESULTS: Xylazine administration increased the blood glucose levels from 58 ± 3 to 108 ± 12 mg/dL in normoglycemic (n = 5, p < 0.01) and from 158 ± 9 to 221 ± 13 mg/dL in insulin-dependent diabetic (n = 5, p < 0.01) monkeys and was not accompanied by any significant changes in blood insulin, glucagon, and glucagon-like peptide-1. Xylazine-induced hyperglycemia occurred within 10 min and reached the peak at 35 min after injection. Xylazine-induced hyperglycemia declined slowly in diabetic animals. The α2-adrenoceptor antagonist yohimbine was administrated to bring down the elevated glucose level to the pre-xylazine one in 4 out of 5 diabetic animals. To assess the potential mechanism, the hyperinsulinemic-euglycemic clamp was used to maintain a nearly saturated and constant insulin level for minimizing endogenous insulin glucoregulation. Xylazine administration decreased glucose infusion rate, from 14.3 ± 1.4 to 8.3 ± 0.8 mg/min/kg (n = 6, p < 0.01) in normoglycemic rhesus monkeys, which indicates that the glucose metabolic rate (M rate) was decreased by xylazine. In addition, after clamping blood glucose level in a range of 55 to 75 mg/dL for 40 min with constant glucose infusion, xylazine administration still increased blood glucose concentration. CONCLUSIONS: We conclude that xylazine administration induces hyperglycemia in normoglycemic and insulin-dependent diabetic monkeys potentially via stimulation of α2-adrenoceptors and then reducing tissue sensitivity to insulin and glucose uptake.

Glucagon-like peptide-1 enhances cardiac L-type Ca2+ currents via activation of the cAMP-dependent protein kinase A pathway.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) is a hormone predominately synthesized and secreted by intestinal L-cells. GLP-1 modulates multiple cellular functions and its receptor agonists are now used clinically for diabetic treatment. Interestingly, preclinical and clinical evidence suggests that GLP-1 agonists produce beneficial effects on dysfunctional hearts via acting on myocardial GLP-1 receptors. As the effects of GLP-1 on myocyte electrophysiology are largely unknown, this study was to assess if GLP-1 could affect the cardiac voltage-gated L-type Ca2+ current (I(Ca)). METHODS: The whole-cell patch clamp method was used to record I(Ca) and action potentials in enzymatically isolated cardiomyocytes from adult canine left ventricles. RESULTS: Extracellular perfusion of GLP-1 (7-36 amide) at 5 nM increased I(Ca) by 23 ± 8% (p < 0.05, n = 7). Simultaneous bath perfusion of 5 nM GLP-1 plus 100 nM Exendin (9-39), a GLP-1 receptor antagonist, was unable to block the GLP-1-induced increase in I(Ca); however, the increase in I(Ca) was abolished if Exendin (9-39) was pre-applied 5 min prior to GLP-1 administration. Intracellular dialysis with a protein kinase A inhibitor also blocked the GLP-1-enhanced I(Ca). In addition, GLP-1 at 5 nM prolonged the durations of the action potentials by 128 ± 36 ms (p < 0.01) and 199 ± 76 ms (p < 0.05) at 50% and 90% repolarization (n = 6), respectively. CONCLUSIONS: Our data demonstrate that GLP-1 enhances I(Ca) in canine cardiomyocytes. The enhancement of I(Ca) is likely via the cAMP-dependent protein kinase A mechanism and may contribute, at least partially, to the prolongation of the action potential duration.

Electrophysiological mechanisms of the anti-arrhythmic effects of omega-3 fatty acids.

Heart rhythm disorders, or arrhythmias, are a leading cause of morbidity and mortality worldwide. Omega-3 polyunsaturated fatty acids (ω3PUFAs), commonly found in fish oils and plant seeds, have recently emerged as potential anti-arrhythmic agents. The purpose of this review is to summarize the electrophysiological basis of the anti-arrhythmic properties of ω3PUFAs from clinical, animal, and cellular research. Evidence of the anti-arrhythmic effects of ω3PUFAs originated from epidemiological studies that correlated a low incidence of sudden cardiac death with high dietary ω3PUFA intake. Subsequently, multiple clinical trials have confirmed the therapeutic effects of ω3PUFAs in preventing sudden cardiac death and multiple other arrhythmia-related disorders. This has led basic scientists to investigate the effects of ω3PUFAs on several ion channels including sodium, potassium, and calcium channels, as well as Na/Ca exchangers. Therefore, ω3PUFAs may hold promise as safe and effective anti-arrhythmic agents. Nevertheless, further research is needed in areas such as: (1) identifying which form(s) of ω3PUFAs (i.e., phospholipid, triglyceride, or free) is (are) responsible for anti-arrhythmic actions; and (2) developing reproducible methods for delivery so that the appropriate form and concentration may be present at the target site to prevent and treat arrhythmias.

Cell and gene therapy for arrhythmias: Repair of cardiac conduction damage.

Action potentials generated in the sinoatrial node (SAN) dominate the rhythm and rate of a healthy human heart. Subsequently, these action potentials propagate to the whole heart via its conduction system. Abnormalities of impulse generation and/or propagation in a heart can cause arrhythmias. For example, SAN dysfunction or conduction block of the atrioventricular node can lead to serious bradycardia which is currently treated with an implanted electronic pacemaker. On the other hand, conduction damage may cause reentrant tachyarrhythmias which are primarily treated pharmacologically or by medical device-based therapies, including defibrillation and tissue ablation. However, drug therapies sometimes may not be effective or are associated with serious side effects. Device-based therapies for cardiac arrhythmias, even with well developed technology, still face inadequacies, limitations, hardware complications, and other challenges. Therefore, scientists are actively seeking other alternatives for antiarrhythmic therapy. In particular, cells and genes used for repairing cardiac conduction damage/defect have been investigated in various studies both in vitro and in vivo. Despite the complexities of the excitation and conduction systems of the heart, cell and gene-based strategies provide novel alternatives for treatment or cure of cardiac arrhythmias. This review summarizes some highlights of recent research progress in this field.

Grass height and transmission ecology of Echinococcus multilocularis in Tibetan communities, China.

BACKGROUND: Alveolar echinococcosis is a major zoonosis of public health significance in western China. Overgrazing was recently assumed as a potential risk factor for transmission of alveolar echinococcosis. The research was designed to further test the overgrazing hypothesis by investigating how overgrazing influenced the burrow density of intermediate host small mammals and how the burrow density of small mammals was associated with dog Echinococcus multilocularis infection. METHODS: The study sites were chosen by previous studies which found areas where the alveolar echinococcosis was prevalent. The data, including grass height, burrow density of intermediate host small mammals, dog and fox fecal samples as well as Global Positioning System (GPS) position, were collected from field investigations in Shiqu County, Sichuan Province, China. The fecal samples were analyzed using copro-PCR. The worms, teeth, bones and hairs in the fecal samples were visually examined. Single factor and multifactor analyses tools including chi square and generalized linear models were applied to these data. RESULTS: By using grass height as a proxy of grazing pressure in the homogenous pasture, this study found that taller grass in the pasture led to lower small mammals' burrow density (chi(2) = 4.670, P = 0.031, coefficient = -1.570). The Echinococcus multilocularis worm burden in dogs was statistically significantly related to the maximum density of the intermediate host Ochotona spp. (chi(2) = 5.250, P = 0.022, coefficient = 0.028). The prevalence in owned dogs was positively correlated to the number of stray dogs seen within a 200 meter radius (Wald chi(2) = 8.375, P = 0.004, odds ratio = 1.198). CONCLUSIONS: Our findings support the hypothesis that overgrazing promotes transmission of alveolar echinococcosis and confirm the role of stray dogs in the transmission of alveolar echinococcosis.

Hysteresis in human HCN4 channels: a crucial feature potentially affecting sinoatrial node pacemaking.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels modulate and regulate cardiac rhythm and rate. It has been suggested that, unlike the HCN1 and HCN2 channels, the slower HCN4 channel may not exhibit voltage-dependent hysteresis. We studied the electrophysiological properties of human HCN4 (hHCN4) channels and its modulation by cAMP to determine whether hHCN4 exhibits hysteresis, by using single-cell patch-clamp in HEK293 cells stably transfected with hHCN4. Quantitative real-time RT-PCR was also used to determine levels of expression of HCNs in human cardiac tissue. Voltage-clamp analysis revealed that hHCN4 current (I(h)) activation shifted in the depolarizing direction with more hyperpolarized holding potentials. Triangular ramp and action potential clamp protocols also revealed hHCN4 hysteresis. cAMP enhanced I(h) and shifted activation in the depolarizing direction, thus modifying the intrinsic hHCN4 hysteresis behavior. Quantitative PCR analysis of human sinoatrial node (SAN) tissue showed that HCN4 accounts for 75% of the HCNs in human SAN while HCN1 (21%), HCN2 (3%), and HCN3 (0.7%) constitute the remainder. Our data suggest that HCN4 is the predominant HCN subtype in the human SAN and that I(h) exhibits voltage-dependent hysteresis behavior that can be modified by cAMP. Therefore, hHCN4 hysteresis potentially plays a crucial role in human SAN pacemaking activity.

Pericardial delivery of omega-3 fatty acid: a novel approach to reducing myocardial infarct sizes and arrhythmias.

Basic and clinical evidence suggests that omega-3 (n-3) polyunsaturated fatty acids (PUFAs) decrease fatal arrhythmias and infarct sizes. This study investigated if pericardial delivery of n-3 PUFAs would protect the myocardium from ischemic damages and arrhythmias. Acute myocardial infarctions were induced in 23 pigs with either 45 min balloon inflations or clamp occlusions of the left anterior descending coronary arteries and 180 min reperfusion. Docosahexaenoic acid (C22:6n-3, DHA, 45 mg), one of the main n-3 PUFAs in fish oil, was infused within the pericardial space only during the 40-min stabilizing phase, 45 min ischemia and initial 5 min reperfusion. Hemodynamics and cardiac functions were very similar between the DHA-treated and control groups. However, DHA therapy significantly reduced infarct sizes from 56.8 +/- 4.9% for controls (n = 12) to 28.8 +/- 7.9% (P < 0.01) for DHA-treated animals (n = 11). Compared with controls, DHA-treated animals significantly decreased heart rates and reduced ventricular arrhythmia scores during ischemia. Furthermore, three (25%) control animals experienced eight episodes of ventricular fibrillation (VF), and two died subsequent to unsuccessful defibrillation. In contrast, only 1 (9%) of 11 DHA-treated pigs elicited one episode of VF that was successfully converted via defibrillation to normal rhythm; thus, mortality was reduced from 17% in the controls to 0% in the DHA-treated animals. These data demonstrate that pericardial infusion of n-3 PUFA DHA can significantly reduce both malignant arrhythmias and infarct sizes in a porcine infarct model. Pericardial administration of n-3 PUFAs could represent a novel approach to treating or preventing myocardial infarctions.

Fish oil fatty acids as cardiovascular drugs.

Starting in the 1970s the hypothesis that the low mortality from coronary heart disease among the Greenland Eskimos was due to their high consumption of n-3 fish oil fatty acids, initiated many studies to find if the n-3 polyunsaturated fatty acids in fish oils (PUFAs) could prevent cardiac atherosclerosis. To date this possibility has not achieved clinical recognition. The recent literature shows an increase of intervention studies to learn if the fish oil fatty acids can reduce mortality from sudden cardiac death, and the mechanism(s) of such a protective effect. Indeed the most definite beneficial cardiac action of these n-3 PUFAs seems now to be their ability in the short term to prevent sudden cardiac death. It is apparent that over long periods of time the n-3 fish oil fatty acids also prevent atherosclerosis. Definition of the fatty acids to which I will be referring in the text: n-6 (omega-6) polyunsaturated fatty acids; linoleic acid (18:2n-6, LA); arachidonic acid (C20:4n-6, AA). n-3 (omega-3) fatty acids; alpha-linolenic acid (18:3n-3, ALA); eicosapentaenoic acid (20:5n-3, EPA); docosahexaenoic acid (C22:6n-3, DHA). The bold, underlined abbreviation will appear in the text to identify the fatty acid being discussed.

Epidemiology and risk factor analysis for canine echinococcosis in a Tibetan pastoral area of Sichuan.

OBJECTIVE: To determine the prevalence and evaluate the risk factors of canine echinococcosis based on a field survey of dog infections with Echinococcus granulosus and E. multilocularis in Chalong, Kalong, Dade and Chazha Townships in a district of Ganzi County, Sichuan Province, China. METHOD: Questionnaire associated with the acquisition of canine echinococcosis was administered to dog owners. Stray dogs were examined post-mortem and rectal faeces at necropsy were collected to validate a copro-antigen ELISA. Owned dogs were screened for Echinococcus spp. infection in faeces using the genus specific copro-ELISA and the effectiveness of dog treatment was assessed. Chi-square and one-way ANOVA were used for statistical analysis. RESULTS: The prevalence of Echinococcus spp. infection at necropsy in stray dogs was 60.9% (14/23) in 2000; E. multilocularis infection accounted for 34.8% (8/23) and E. granulosus for 26.1% (6/23). The specificity of the copro-ELISA was 80.0% and the sensitivity was 92.3%, compared with the results at necropsy. Fifty percent of owned dogs (290/580) tested was copro-antigen positive at the beginning of the project in 2000, which decreased to 17% (99/580) in the same cohort of owned dogs after praziquantel treatment (5 mg/ kg) at 6-monthly period from 2003 to 2005. Analysis for risk factors associated with copro-antigen positive dogs showed that the never tethered dogs had a higher rate (40.4%, 65/161) than dogs tethered during the day (32.3%, 109/337), or tethered at night [29.2% (21/72)], or those always tethered [20%(2/10)](P<0.01). Dogs that their owners lacked hydatid transmission knowledge [38.1% (121/318)] and did not have de-worming practice [47.7% (92/193)] had significantly higher copro-antigen positive rate than those dogs that their owners knew relevant knowledge [28.6% (75/262)] and were dewormed regularly [20.4% (79/387)](P<0.05 and P<0.01). There was no correlation between the prevalence and dog sex or age or the varieties of livestock that the owner raised. CONCLUSION: Local dogs show high prevalence with both E. granulosus and E. multilocularis. The copro-ELISA can be used to detect infection of Echinococcus in dogs. Allowing dogs to roam, lack of the basic knowledge of hydatid disease transmission and no de-worming practice for dogs are significant factors for the transmission of canine echinococcosis.

Biological approaches to generating cardiac biopacemaker for bradycardia.

Normal rhythm in a healthy human heart originates from the natural biological pacemaker, the sinoatrial (SA) node which locates in the right atrium. SA node dysfunction or atrial-ventricular (AV) conduction block causes improper heart rate (bradycardia). Such dysfunction, if severe enough, is currently treated by implanting an electronic pacemaker which has been well established technically, but there are some limitations and inadequacies. Recently, progress in developing engineered cardiac biopacemakers with use of genes or cells has been made in experimental animal models. The hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel (pacemaker channel) modulates cardiac automaticity via the hyperpolarization-activated cation current (I(f)). HCN genes have been delivered to animal myocardium via viral vectors or HCN-transferred cells for recreating biological pacemakers. Approaches with non-HCN genes or transplantation of beating cells are also novel and have been investigated for generating cardiac biopacers. This article summarizes the progresses in research on recreation of cardiac biopacemakers. Genetically engineered biological pacemaker holds great promise to potentially cure severe bradycardia if critical issues, such as their stability and longevity, are properly solved.