Efficacy of VERYL® in the treatment of cattle naturally infected with gastro-intestinal nematodes in Kenya.

Co-infections caused by trypanosomes and gastro-intestinal nematodes (GINs) compromise cattle productivity and their control requires a holistic approach. The effectiveness of trypanocides and anthelmintics is compromised by increasing resistance. Use of combined chemotherapeutic products for synergy, mainly practiced in human medicine, is gaining importance in livestock. A trial to evaluate efficacy of VERYL®, containing diminazene diaceturate (3.5 mg/kg body weight) and levamisole chloride (5 mg/kg body weight) for the control of GINs in cattle, was conducted at KALRO-VSRI Muguga, Kenya, between June and August 2016. Thirty-eight cattle aged between 6 and 12 months, naturally infected with GINs, were randomly allocated into two groups; a treatment group received VERYL® intra-muscularly at 10 mL/100 kg bwt and a control group which received Veriben® (Diminazene aceturate) at 3.5 mg/kg bwt. Faecal egg counts (FECs), coproculture, packed cell volume (PCV) and local tolerance at the injection site were measured during the study. FECs were comparable between the treatment and control groups at day 0. However, treatment of cattle with VERYL significantly (p < 0.001) reduced FECs by day 7 and sustained to day 21 post-treatment. Coproculture results for the treatment and control groups revealed presence of Haemonchus, Cooperia, Ostertagia, Trichostrongylus and Oesophagostomum species. Cattle treated with VERYL® had a significant (p < 0.05) reduction in larval recoveries compared to the control group. VERYL® had minimal adverse effects which cleared after a short while and is thus recommended for controlling GINs in cattle.

Efficacy of cabergoline in a double-blind randomized clinical trial on milk leakage reduction at drying-off and new intramammary infections across the dry period and postcalving.

The abrupt cessation of milking at dry-off may induce milk leakage, which may increase the risk of new intramammary infections (IMI). This study assessed the efficacy of 1 i.m. injection of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France) at drying-off on milk leakage after dry-off and new IMI across the dry period and postcalving compared with a placebo (negative control) and an intramammary antibiotic treatment (positive control) under field conditions. The study was a double-blind, randomized, 3-arm, multicenter, clinical trial performed under Good Clinical Practice conditions. Data from 900 dairy cows of various breeds from 63 farms in France, Germany, and Hungary were analyzed. Only quarters with no bacterial growth at drying-off and a cow somatic cell count ≤200,000 cells/mL were included. Quarters infected with major or minor pathogens or cows with high somatic cell count at time of inclusion were excluded. Cows that qualified for the study were visited 7 times in total before and after drying-off and after calving. Presence (yes/no) of milk leakage was recorded on the day after dry-off. A new infected quarter (new IMI) was defined as one with a major pathogen present in any one of the 2 postcalving samples. Two mixed logistic regression models were fitted to the data to evaluate the efficacy of cabergoline in the reduction of milk leakage and new IMI. One i.m. injection of cabergoline at drying-off significantly reduced the incidence of milk leakage the day after dry-off compared with both placebo and antibiotic treatment. Cabergoline-treated cows significantly reduced the risk of new IMI by major pathogens across the dry period and postcalving by 21% when compared with placebo cows (20.5 vs. 26.0%, respectively). However, when milk leakage was added to the model, the significance of cabergoline was reduced. We interpreted this to show that milk leakage is an intervening variable between treatment with cabergoline and lower risk of new IMI. The antibiotic treatment significantly decreased the odds of new IMI compared with both cabergoline and placebo. However, because several countries are currently disallowing the preventive use of antibiotics at dry-off in noninfected quarters, the dry-off facilitator cabergoline may therefore be of particular value to reduce the risk of new IMI across the dry period.

Inhibiting prolactin by cabergoline accelerates mammary gland remodeling during the early dry period in dairy cows.

The inhibition of prolactin release using cabergoline, a dopamine agonist, is an effective strategy to accelerate the changes in mammary secretion composition after drying-off. The objective of this study was to determine how cabergoline may affect mammary tissue remodeling during early involution. Holstein dairy cows were treated with either a single i.m. administration of 5.6 mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n = 7) or placebo (n = 7) at the time of drying-off. Mammary biopsy samples were collected 1 wk before drying-off (d -6), after 30 h of milk accumulation (d 1), and again 8 d following drying-off (d 8) to determine changes in gene expression, lactoferrin content, and cell turnover. Blood and mammary secretion samples were collected at d -6 and again at d 1, 2, 3, 4, 8, and 14 following the abrupt cessation of lactation to evaluate indicators of blood-milk barrier integrity and other markers of mammary tissue remodeling. Cabergoline induced less SLC2A1, BAX, CAPN2, and IGFBP5 mRNA expression. In contrast, cabergoline did not modify changes in cell proliferation and apoptosis. Following the cessation of lactation, changes in mammary secretion composition (Na+ and K+) and blood lactose concentrations were indicative of a loss in the blood-milk barrier function in both treatment groups. Cabergoline treatment affected only Na+ and K+ concentrations at d 1, suggesting a moderate increase in tight junction permeability. The increase in the activity of MMP9 and in mammary epithelial cell concentration in mammary secretions was greater in cabergoline-treated cows than in control cows, suggesting more mammary tissue remodeling. The increase in lactoferrin immunostaining in the mammary tissue occurred earlier for cabergoline-treated cows than for control cows, and was essentially localized in the stroma. Changes in some key markers of mammary involution suggest that cabergoline accelerates mammary gland remodeling. Thus, a single injection of cabergoline after the last milking would facilitate drying-off by enhancing mammary gland involution.

Effect of a single injection of cabergoline at dry off on udder characteristics in high-yielding dairy cows.

In recent years, relationships between high milk yield at dry off, higher prevalence for new intramammary infections, and stress were evaluated. Considering increasing milk yield, dry off methods need to be refined to ensure udder health and animal welfare, especially in high-yielding dairy cows. The present work evaluated the effect of a single cabergoline injection (Velactis, Ceva Santé Animale, Libourne, France) at dry off on udder pressure, milk leakage, and signs of udder pain after dry off. A total of 234 high-yielding (≥16 kg of milk/d) dairy cows was enrolled 7 d before and followed up until 14 d after dry off. Cows were dried off without preparation (i.e., no feed change or intermittent milking before dry off) and treated with a single i.m. injection of 5.6 mg of cabergoline (n = 115) or placebo (n = 119) after last milking. Udder characteristics were measured 4 d before (i.e., before and after milking) and 1, 2, 3, 7, 10, and 14 d after dry off. Udder pressure was evaluated utilizing a hand-held dynamometer. Milk leakage and signs of udder pain were noted as binary variables. Whereas udder pressure baseline values after last milking did not differ between treatment groups (0.541 ± 0.15 kg), cabergoline significantly reduced udder pressure in primiparous but not in multiparous cows after dry off. Differences between cabergoline- and placebo-treated primiparous cows could be evaluated until 3 d after dry off. The first day after dry off, udder pressure in placebo- and cabergoline-treated cows increased by 115% and 42.3%, respectively. Whereas pressure values in placebo cows were highest on the first day after dry off (1.16 ± 0.61 kg) and slowly decreased afterward, udder pressure in cows treated with cabergoline had a slower increase and peak only 2 d after dry off (0.94 ± 0.44 kg). Furthermore, cabergoline caused a reduction of milk leakage, a known factor for new intramammary infections. Only 11.3% of cows treated with cabergoline showed milk leakage compared with 21.0% placebo-treated cows. Additionally, cows with placebo treatment were 2.8 times as likely to show signs of udder pain compared with cows treated with cabergoline. An effect of cabergoline on udder pressure, milk leakage, and udder pain was limited to the first week after dry off. Our data provide evidence that a single injection of cabergoline reduces risk factors for udder health and animal welfare problems around dry off in high-yielding dairy cows with more than 16 kg of milk/d. Further research is warranted, however, to investigate if cabergoline at dry off can also be used to reduce new intramammary infection rates and improve animal welfare after dry off.

Cabergoline inhibits prolactin secretion and accelerates involution in dairy cows after dry-off.

Dairy cattle require a dry period between successive lactations to ensure optimal milk production. Because prolactin (PRL) is necessary for the initiation and maintenance of milk production, strategies that can inhibit PRL secretion might hasten the involution process. The objective of this study was to determine the effect of the PRL release inhibitor cabergoline on markers of mammary gland involution during the early dry period. To assess the effect of cabergoline treatment on mammary gland involution, 14 Holstein dairy cows in late lactation were treated with either a single i.m. administration of 5.6mg of cabergoline (Velactis, Ceva Santé Animale, Libourne, France, n=7) or placebo (n=7) at the time of dry-off. Blood samples and mammary secretion samples were collected 6d before dry-off and again 1, 2, 3, 4, 8, and 14d following the abrupt cessation of lactation. Blood samples were used to determine plasma PRL concentrations. Mammary secretion samples were used to determine somatic cell count, milk fat, lactose, true protein content, and concentrations of α-lactalbumin, lactoferrin, and citrate. Following the cessation of lactation, changes in mammary secretion composition indicated diminished milk synthesis, including reduced concentrations of α-lactalbumin, citrate, and lactose. In contrast, milk somatic cell count, percent total protein, percent fat content, and lactoferrin concentrations significantly increased as involution progressed. Cabergoline treatment decreased the plasma PRL concentrations during the first week of dry-off, compared with the control treatment. No significant differences in citrate, α-lactalbumin, or protein content were observed between treatment groups. The most dramatic changes in secretion composition as a consequence of cabergoline treatment occurred during the first week of the dry period, when lactose concentrations and the citrate:lactoferrin molar ratio were lower and lactoferrin concentrations higher than in the control cows. Cabergoline treatment also tended to increase fat content and somatic cell count more rapidly following dry-off compared with the control group. These changes in mammary secretion composition following the abrupt cessation of lactation indicate that cabergoline treatment facilitated dry-off and effectively accelerated mammary gland involution.

Paradoxical effect of supplementary progesterone between Day 3 and Day 7 on corpus luteum function and conceptus development in cattle.

The aim of the present study was to investigate the effect of short-term progesterone (P4) supplementation during the early metoestrous period on circulating P4 concentrations and conceptus development in cattle. The oestrous cycles of cross-bred beef heifers were synchronised using a 7-day P4-releasing intravaginal device (PRID® Delta; 1.55 g P4) treatment with administration of a prostaglandin F(2α) analogue (Enzaprost; CEVA Sante Animale) the day before PRID® Delta removal. Only those heifers recorded in standing oestrus (Day 0) were used. In Experiment 1, heifers were randomly assigned to one of five groups: (1) control: no treatment; (2) placebo: insertion of a blank device (no P4) from Day 3 to Day 7; (3) insertion of a PRID® Delta from Day 3 to Day 7; (4) insertion of a PRID® Delta from Day 3 to Day 5; or (5) insertion of a PRID® Delta from Day 5 to Day 7. In vitro-produced blastocysts were transferred to each heifer in Groups 2-5 on Day 7 (n=10 blastocysts per heifer) and conceptuses were recovered when heifers were killed on Day 14. Based on the outcome of Experiment 1, in Experiment 2 heifers were artificially inseminated at oestrus and randomly assigned to one of three treatment groups: (1) placebo; (2) PRID from Day 3 to Day 5; or (3) PRID from Day 3 to Day 7. All heifers were killed on Day 16 and recovered conceptuses were incubated in synthetic oviducal fluid medium for 24 h; spent media and uterine flushes were analysed for interferon-τ (IFNT). In both experiments, daily blood samples were taken to determined serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA). Insertion of a PRID resulted in an increase (P<0.05) in serum P4 that declined following removal. In Experiment 1, P4 supplementation from Day 3 to Day 5 (17.0±1.4 mm) or Day 3 to Day 7 (11.3±2.3 mm) increased conceptus length compared with placebo (2.1±1.8 mm). Serum P4 was significantly lower from Day 9 to Day 14 (P<0.05) and the weight of the Day 14 corpus luteum (CL) was lower in the PRID Day 3-7 group than the placebo or control groups. In Experiment 2, supplementation from Day 3 to Day 5 (94.0±18.8 mm) or Day 3 to Day 7 (143.6±20.6 mm) increased conceptus length on Day 16 compared with placebo (50.3±17.4 mm). Serum P4 was significantly lower in the two supplemented groups following PRID removal compared with placebo (P<0.05) and was associated with a lower CL weight in the Day 3-7 group. Conceptus length was strongly correlated with the IFNT concentration in the uterine flush (r=0.58; P=0.011) and spent culture medium (r=0.68; P<0.002). The findings of the present study highlight the somewhat paradoxical effects of P4 supplementation when given in the early metoestrous period in terms of its positive effect on conceptus development and its potentially negative effects on CL lifespan.

Measurement of fecal glucocorticoid metabolites and evaluation of udder characteristics to estimate stress after sudden dry-off in dairy cows with different milk yields.

Sudden dry-off is an established management practice in the dairy industry. But milk yield has been increasing continuously during the last decades. There is no information whether the dry-off procedure, which often results in swollen and firm udders, causes stress, particularly in high-producing dairy cows. Therefore, we evaluated the effect of a sudden dry-off on extramammary udder pressure and the concentration of fecal glucocorticoid metabolites (i.e., 11,17-dioxoandrostane, 11,17-DOA) as an indirect stress parameter. Measurements were carried out within the last week before dry-off and until 9d after dry-off considering 3 groups of milk yield (i.e., low: <15 kg/d, medium: 15-20 kg/d, and high: >20 kg/d). Udder pressure increased in all yield groups after dry-off, peaked at d 2 after dry-off and decreased afterwards. Pressures were highest in high-yielding cows and lowest in low-yielding cows. But only in high-yielding cows was udder pressure after dry-off higher than before dry-off. Baseline 11,17-DOA concentrations depended on milk yield. They were highest in low-yielding (121.7 ± 33.3 ng/g) and lowest in high-yielding cows (71.1 ± 30.0 ng/g). After dry-off, 11,17-DOA increased in all yield groups and peaked at d 3. Whereas in medium- and high-yielding cows 11,17-DOA levels differed significantly from their respective baseline during the whole 9-d measuring period, low-yielding cows showed elevated 11,17-DOA levels only on d 3 after dry-off. However, especially the increase in 11,17-DOA after dry-off between the 3 yield groups was considerably different. Mean 11,17-DOA increase from baseline to d 3 was highest in high-yielding cows (129.1%) and considerably lower in low-yielding cows (40.1%). The highest fecal 11,17-DOA concentrations were measured on d 3 after dry-off, indicating that the stress was most intense on d 2, which is due to an 18-h time lag; at about the same time, udder pressure peaked. Our results showed a negligible effect of a sudden dry-off on low-yielding cows. High-yielding cows, however, faced high extramammary pressures and increased glucocorticoid production. Considering animal welfare aspects, a review of the current dry-off strategies might be warranted.

Pulse-spray pharmacomechanical thrombolysis for proximal deep vein thrombosis.

OBJECTIVE: The aim of this study was to evaluate the efficacy, safety, and feasibility of pulse-spray pharmacomechanical thrombolysis to treat proximal deep vein thrombosis (DVT) in conjunction with the placement of a non-permanent IVC filter. METHODS: We studied 31 consecutive patients with acute proximal DVT defined as the inferior vena cava (IVC), iliac vein and/or femoral vein, who were diagnosed using duplex ultrasonography and/or contrast venography. All were treated with pulse-spray urokinase. Early success was assessed by comparing the pre- and post-treatment venographic severity score. Non-permanent IVC filters were used to reduce the risk of pulmonary thromboembolism. RESULTS: The average total urokinase dose was 1.71 million IU (range: 0.72-3.6 million IU) and the average duration of therapy was 2.4 days. The average percentage of thrombus lysed was 85% (range: 22-100%). A large thrombus trapped by the filter was detected using cavography before extraction of the filter in one patient. There was no major treatment-related adverse event. CONCLUSION: The combination of pulse-spray pharmacomechanical thrombolysis and the prophylactic use of a non-permanent IVC filter was a safe and effective approach for treating acute proximal DVT.

Epidemiological characteristics of acute pulmonary thromboembolism in Japan.

AIM: In Japan, acute pulmonary thromboembolism (APTE) is still rare, but the number of patients with APTE has been steadily increasing. It is important for early diagnosis and early management of APTE to recognize epidemiological characteristics of this condition. METHODS: We investigated the epidemiological characteristics of 252 patients with APTE who were admitted to our institutions between 1975 and 2001. APTE was more prevalent in women that in men. It was observed the most in the age group between 50s to 70s, especially in women. Many patients had prolonged immobilization, recent major operation, obesity, or cancer, as risk factors for venous thromboembolism. One hundred and thirty-eight patients developed APTE in hospital; 60 patients were in Department of Internal Medicine, 28 in General Surgery, 15 in Orthopedics, 15 in Obstetrics and Gynecology, and 20 in other services. RESULTS: Among 58 patients with malignancy, 43% had cancers in digestive organs, 21% in gynecological, and 17% in urological. Among 61 patients who were examined for the presence of thrombophilia, 13 patients had inherited thrombophilia (8 protein C deficiency, 4 protein S deficiency, and 1 antithrombin III deficiency) 11 had antiphospholipid antibodies which indicated thrombophilia. Five out of the above 61 patients (8%) had no obvious risk factors including thrombophilia. CONCLUSION: The findings in our patients were almost the same as those in Western patients, except for some points. These results might be useful to establish a preventive approach for APTE in Japan.

Circadian rhythm of blood pressure is transformed from a dipper to a non-dipper pattern in shift workers with hypertension.

Shift workers make great use of health care services because they are associated with increased cardiovascular morbidity and mortality. Whether the circadian rhythm of blood pressure rapidly adapts to shift work is controversial. It is unknown if shift work has adverse effects on blood pressure in patients with hypertension. To evaluate the effects of shift work, we examined 12 male shift workers with untreated hypertension aged 53.6 +/- 2.5 years. Twenty-four hour ambulatory blood pressure monitoring was performed three times as follows: the last day of a 4-day period of day shifts (09.00 to 21.00), the first day of a 4-day period of night shifts (21.00 to 09.00), and the fourth day of night shifts (21.00 to 09.00). Blood pressure at night-time dropped significantly in the day-shift workers, showing a dipper pattern. Average differences in blood pressure in the sleep-wake cycle were decreased by 8.5% at the beginning of night shift work showing a non-dipper pattern. After 4 days the pattern was completely reversed to a dipper pattern. The results indicate that the circadian blood pressure pattern is changed from a dipper to a non-dipper pattern on the first day of the night shift and reverses to a dipper pattern within a few days. We suggest that night shift work may have unfavourable effects on blood pressure in patients with hypertension.

Over-expression of vascular endothelial growth factor after preoperative radiation therapy for rectal cancer.

Vascular endothelial growth factor expression before and after preoperative radiation therapy was examined in 16 stage III rectal carcinoma patients. The biopsied tissues before preoperative radiation therapy and the tissues at operation were immunohistochemically stained. Four cases were negative for VEGF expression before radiation, but the other 12 cases were positive, with 4 cases showing strong immunoreactivity. After radiation, all except 1 case showed VEGF positive patterns, in which 14 cases demonstrated strong staining. In 12 cases, VEGF expression became higher after radiation therapy than before, as compared to the only 1 case that showed lower expression than before.

Effects of therapeutic doses of human atrial natriuretic peptide on load and myocardial performance in patients with congestive heart failure.

The benefits of atrial natriuretic peptide (ANP) in patients with congestive heart failure (CHF) have been demonstrated. However, the myocardial actions of ANP remain unclear. Using relatively load-insensitive left ventricular pressure-volume analysis, the myocardial and load-altering actions of ANP in patients with moderate CHF were studied. After obtaining steady-state data using micromanometers and conductance catheters, ANP was infused in 9 patients with CHF at 0.01 and 0.1 microg/kg/min for 30 minutes, respectively. Hemodynamic variables, plasma ANP, and cyclic guanosine monophosphate (cGMP) levels were determined before and 30 minutes after each ANP infusion. ANP at 0.01 microg/kg/min increased plasma ANP and cGMP levels from 73 +/- 34 to 139 +/- 34 pg/ml and from 4 +/- 1 to 8 +/- 2 pmol/ml, respectively. ANP infusion caused a significant decrease in end-systolic pressure without any changes in heart rate. End-diastolic pressure was significantly decreased but there was no significant change in left ventricular end-diastolic volume. The time constant for isovolumetric relaxation was decreased. ANP infusion at 0.1microg/kg/min caused further decreases in end-systolic pressure, end-diastolic pressure and volume, and the time constant for isovolumetric relaxation (p <0.05) without any changes in heart rate. The slope of the end-systolic pressure-volume relation was increased from 1.3 +/- 0.2 to 1.6 +/- 0.3 mm Hg/ml (p <0.05), indicating increased contractility. Plasma ANP and cGMP levels were increased to 422 +/- 44 pg/ml and 16 +/- 3 pmol/ml, respectively. Thus, ANP infusion increased cGMP generation, decreased afterload and preload, and improved left ventricular systolic and diastolic function.

Multiple pulmonary emboli with pulmonary hypertension caused by effort thrombosis and effective balloon venoplasty of the subclavian vein.

A 36-year-old woman with effort thrombosis of the subclavian vein associated with multiple pulmonary emboli was successfully treated with local thrombolysis of the subclavian vein using a pulse-spray catheter and systemic anticoagulation. Balloon venoplasty of the residual stenosis of subclavian vein was carried out and in follow-up venography 6 months later, there was no restenosis, and the patient has been asymptomatic for 12 months. Pulmonary embolism is not a rare complication of upper extremity deep vein thrombosis and should be managed as aggressively as lower extremity deep vein thrombosis.

Functional role of endogenous endothelin-1 in congestive heart failure treated with angiotensin II receptor antagonist.

Interactions between angiotensin (ANG) II and endothelin (ET)-1 receptor transduction pathways have been unclear in congestive heart failure (CHF). Therefore the objects of this study are, in CHF, whether production of ET-1 is modulated by ANG II and/or whether hemodynamic effects of endogenous ET-1 are modulated by ANG II. Twelve dogs were randomly assigned to two groups: untreated (n = 6) and treated with ANG II type 1 (AT1) receptor antagonist (TCV116, 1.5 mg/kg/d) (n = 6). After rapid ventricular pacing (240 bpm) for 4 weeks, plasma and cardiac ET-1 levels were compared between the two groups. Acute hemodynamic effects of a nonspecific ET(A&B) receptor antagonist, TAK044 (3 mg/kg plus 3 mg/kg/h i.v.) were examined in both groups by a conductance catheter and a micromanometer. After 4 weeks of pacing, plasma and cardiac tissue ET-1 levels were elevated in both groups to a similar degree. In the group treated with TCV116, TAK044 produced an increase in stroke volume and a decrease in total systemic resistance; heart rate was unchanged. The time constant of left ventricular (LV) relaxation was significantly decreased. The slope of LV end-systolic pressure-volume relation (E(ES)) was increased (p < 0.05), indicating an increased LV contractility. Thus endogenous ET-1 produces an arterial vasoconstriction and impairs LV contractility and relaxation in CHF with AT1 receptor antagonism. These hemodynamic responses to TAK044 in CHF treated with TCV116 were similar in untreated CHF. These results suggest that the production of ET-1 and the cardiac effects of endogenous ET-1 in CHF may be unaffected by ANG II acting through AT1 receptors.

Serial extracellular matrix changes in neointimal lesions of human coronary artery after percutaneous transluminal coronary angioplasty: clinical significance of early tenascin-C expression.

It has become clear that deposition of extracellular matrix(ECM) proteins is a major cause of human restenosis after percutaneous coronary angioplasty (PTCA). To define the composition and organization of the involved ECM in human restenotic tissue, we morphologically and semiquantitatively analyzed specimens obtained by means of directional coronary atherectomy at various stages after PTCA with anti-fibronectin, tenascin-C, collagens I and III, and PG-M/versican antibodies. Tenascin-C deposition transiently increased within 1 month after PTCA, when smooth muscle cell migration and proliferation was active. Following the disappearance of tenascin-C, PG-M/versican accumulation increased and peaked between 1 month and 3 months when clinical restenosis was most actively progressing. At later stages, the PG-M/versican was replaced by a more mature ECM consisting of collagens I and III. The volume ratio of elastin remained at a low level throughout. Our results demonstrate that the matrix proteins of human restenotic lesions sequentially change after angioplasty and that tenascin-C could be a key molecule in the early stages.

Inhibition by combined therapy with ticlopidine and aspirin of enhanced platelet aggregation during physical exercise in patients with coronary artery disease.

BACKGROUND: Strenuous exercise can be a major trigger for coronary thrombosis and it enhances platelet aggregation. METHODS: We evaluated the effect of antiplatelet therapy on shear stress-induced platelet aggregation (SIPA), in addition to agonist-induced aggregation, before and immediately after ergometer exercise in patients with stable coronary artery diseases (CAD). Forty-eight patients with stable CAD were randomly distributed into 3 groups: no antiplatelet drug (patient control, n = 16), aspirin (ASA) monotherapy (n = 16), and combined therapy with ticlopidine (TIC) and ASA (n = 16). RESULTS: There were significant increases in not only adenosine phosphate (ADP)- and collagen-induced platelet aggregation but also in SIPA during exercise by the patient control group. ASA monotherapy did not attenuate the enhanced ADP-induced aggregation nor SIPA. Combined ASA + TIC therapy significantly inhibited SIPA as well as ADP-induced aggregation both before and after exercise. Significant increases in levels of plasma von Willebrand factor (vWF) occurred during exercise, and these antiplatelet therapies had no apparent effect on increased vWF levels during exercise. Exercise induced a significant increase in the plasma thrombin-antithrombin III complex level with no significant changes in the level of plasmin-plasmin inhibitor complex level in all 3 groups. CONCLUSIONS: Combined therapy with ASA + TIC effectively inhibited increased platelet aggregability in response to acute exercise, with no effects on coagulant or fibrinolytic potentials in patients with CAD. The data suggest that TIC combined with ASA may be superior to ASA alone in preventing acute coronary events during exercise in patients with coronary atherosclerotic disease.

Identification of human CPI-17, an inhibitory phosphoprotein for myosin phosphatase.

CPI-17 is a phosphorylation-dependent inhibitor of myosin phosphatase. cDNA clones encoding CPI-17 were isolated from a human aorta library. Overlapping clones indicated two isoforms: CPI-17alpha was 147 residues and mass of 16.7 kDa; CPI-17beta (120 residues, mass 13.5 kDa) resulted from a deletion in the alpha-isoform of 27 residues, sequence 68-94. N-terminal 67 residues of all CPI-17 isoforms (human, porcine, rat and mouse) were highly conserved (for the human and porcine isoforms the identity was 91%). The presence of the two human isoforms was detected from cDNA sequences amplified by RT-PCR and by Western blots on human aorta. The cloned human CPI-17 gene indicated 4 coding exons and CPI-17beta was an alternative splice variant due to deletion of the second exon. FISH analysis located the human CPI-17 gene on chromosome 19q13.1.

Molecular cloning and analysis of the 5'-flanking region of the human MYPT1 gene.

We have cloned and sequenced a 5 kb genomic fragment in the 5'-flanking region of the human myosin phosphatase target subunit 1. The transcription initiation site (+1) was 268 bp upstream from the translation start site. In this promoter there are no canonical TATA or CAAT box elements but there is a high GC-rich sequence. Basal promoter activity was due to the GC-rich region that contained one Sp1 transcription factor binding site, thus demonstrating that the MYPT1 gene is a housekeeping gene. Luciferase reporter assays showed the presence of two regions for positive elements and one for a negative element.

Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase.

Arachidonic acid activates isolated Rho-kinase and contracts permeabilized smooth muscle fibres. Various assays were carried out to examine the mechanism of this activation. Native Rho-kinase was activated 5-6 times by arachidonic acid but an N-terminal, constitutively-active fragment of Rho-kinase, expressed as a glutathione-S-transferase (GST) fusion protein and including the catalytic subunit (GST-Rho-kinase-CAT), was not. GST-Rho-kinase-CAT was inhibited by a C-terminal fragment of Rho-kinase and arachidonic acid removed this inhibition. These results suggest that the C-terminal part of Rho-kinase, containing the RhoA binding site and the pleckstrin homology domain, acts as an autoinhibitor. It is suggested further that activation by arachidonic acid is due to its binding to the autoinhibitory region and subsequent release from the catalytic site. Arachidonic acid, at concentrations greater than 30 microM, increases force in alpha-toxin-permeabilized femoral artery but not in Triton X-100-skinned fibres. The content of Rho-kinase in the latter was lower than in alpha-toxin-treated or intact fibres. The arachidonic acid-induced contraction was not observed at a pCa above 8.0 and was inhibited by Y-27632 and wortmannin, inhibitors of Rho-kinase and myosin light-chain kinase (MLCK), respectively. The activation of Rho-kinase and subsequent phosphorylation of the myosin phosphatase target subunit inhibits myosin phosphatase and increases myosin phosphorylation.

Assessment of coronary flow velocity reserve using fast velocity-encoded cine MRI for noninvasive detection of restenosis after coronary stent implantation.

PURPOSE: Serial change of the coronary flow velocity reserve was evaluated with fast velocity-encoded cine magnetic resonance imaging (MRI) for noninvasive detection of restenosis after coronary stent implantation. METHOD: In total, 60 MRI flow studies were performed in 10 patients with coronary artery disease who undersvent elective successful stent implantation to the lesion in the proximal left anterior descending artery. Flow velocities in the segment that was distal to the stent were measured before and after intravenous injection of dipyridamole. MRI measurements of coronary flow velocity reserve were repeated every 4 weeks for 6 months, and follow-up angiography was performed 6 months after the procedure. RESULTS: In patients without restenosis (n = 7, % diameter stenosis: 27.8%+/-7.1) at follow-up angiography, the coronary flow velocity reserve remained normal during the 6-month follow-up time. The flow velocity reserve was 2.31+/-0.30 at 1 month and 2.52+/-0.25 at 6 months after stent implantation (p = NS). In contrast, the coronary flow velocity reserve showed a significant decrease after 4 months in patients with restenosis (n = 3, % diameter stenosis: 66.3%+/-8.1) at follow-up angiography. The flow velocity reserve was 2.26+/-0.49 at 1 month and 1.52+/-0.09 at 6 months after stent implantation (p < 0.05). CONCLUSION: Fast velocity-encoded cine MRI is a technique that shows promise in providing non-invasive detection of restenosis of coronary stent implantation.