Induction of expression and phosphorylation of heat shock protein B5 (CRYAB) in rat myometrium during pregnancy and labour.

During pregnancy the myometrium undergoes a programme of differentiation induced by endocrine, cellular, and biophysical inputs. Small heat shock proteins (HSPs) are a family of ten (B1-B10) small-molecular-weight proteins that not only act as chaperones, but also assist in processes such as cytoskeleton rearrangements and immune system activation. Thus, it was hypothesized that HSPB5 (CRYAB) would be highly expressed in the rat myometrium during the contractile and labour phases of myometrial differentiation when such processes are prominent. Immunoblot analysis revealed that myometrial CRYAB protein expression significantly increased from day (D) 15 to D23 (labour; P<0.05). In correlation with these findings, serine 59-phosphorylated (pSer59) CRYAB protein expression significantly increased from D15 to D23, and was also elevated 1-day post-partum (P<0.05). pSer59-CRYAB was detected in the cytoplasm of myocytes within both uterine muscle layers mid- to late-pregnancy. In unilaterally pregnant rats, pSer59-CRYAB protein expression was significantly elevated in the gravid uterine horns at both D19 and D23 of gestation compared with non-gravid horns. Co-immunolocalization experiments using the hTERT-human myometrial cell line and confocal microscopy demonstrated that pSer59-CRYAB co-localized with the focal adhesion protein FERMT2 at the ends of actin filaments as well as with the exosomal marker CD63. Overall, pSer59-CRYAB is highly expressed in myometrium during late pregnancy and labour and its expression appears to be regulated by uterine distension. CRYAB may be involved in the regulation of actin filament dynamics at focal adhesions and could be secreted by exosomes as a prelude to involvement in immune activation in the myometrium.

Distension of the uterus induces HspB1 expression in rat uterine smooth muscle.

The uterine musculature, or myometrium, demonstrates tremendous plasticity during pregnancy under the influences of the endocrine environment and mechanical stresses. Expression of the small stress protein heat shock protein B1 (HspB1) has been reported to increase dramatically during late pregnancy, a period marked by myometrial hypertrophy caused by fetal growth-induced uterine distension. Thus, using unilaterally pregnant rat models and ovariectomized nonpregnant rats with uteri containing laminaria tents to induce uterine distension, we examined the effect of uterine distension on myometrial HspB1 expression. In unilaterally pregnant rats, HspB1 mRNA and Ser(15)-phosphorylated HspB1 (pSer(15) HspB1) protein expression were significantly elevated in distended gravid uterine horns at days 19 and 23 (labor) of gestation compared with nongravid horns. Similarly, pSer(15) HspB1 protein in situ was only readily detectable in the distended horns compared with the nongravid horns at days 19 and 23; however, pSer(15) HspB1 was primarily detectable in situ at day 19 in membrane-associated regions, while it had primarily a cytoplasmic localization in myometrial cells at day 23. HspB1 mRNA and pSer(15) HspB1 protein expression were also markedly increased in ovariectomized nonpregnant rat myometrium distended for 24 h with laminaria tents compared with empty horns. Therefore, uterine distension plays a major role in the stimulation of myometrial HspB1 expression, and increased expression of this small stress protein could be a mechanoadaptive response to the increasing uterine distension that occurs during pregnancy.

Natural variability of circulating levels of cytokines and cytokine receptors in patients with heart failure: implications for clinical trials.

OBJECTIVES: The purpose of this study was to examine the variability in cytokines and cytokine receptors in patients with heart failure in comparison with a group of healthy control subjects who were free of cardiovascular disease. BACKGROUND: Despite increasing interest in cytokines as mediators of disease progression in heart failure and the recent interest in suppressing cytokines in clinical studies, the extent of variability in cytokines and cytokine receptors is largely unknown. This information is important for interpreting the results of studies in which changes in cytokine levels are measured in response to a specific form of therapy. METHODS: Circulating levels of tumor necrosis factor-alpha (TNF-alpha), and soluble TNF receptors (types 1 and 2), as well as interleukin (IL)-6 and IL-6 receptor were measured on a daily, weekly and monthly basis in heart failure patients (New York Heart Association class IIIa and IIIb; n = 10) and healthy volunteer subjects (n = 10). Measurements of cytokines and cytokine receptors were performed on plasma samples by enzyme-linked immunoassay. The daily, weekly and monthly degree of variability in cytokine and cytokine receptor levels was assessed by determining the coefficient of variation each point in time. RESULTS: The coefficient of variation for TNF-alpha and IL-6 levels increased over time in patients with heart failure; moreover, the coefficient of variation in heart failure subjects was significantly greater for IL-6 than for TNF-alpha. The coefficient of variation in cytokine receptor levels was minimal, and did not differ significantly between heart failure and control subjects. CONCLUSIONS: In patients with heart failure the degree of natural variability in circulating cytokine levels increases with time, and is greater for IL-6 than for TNF-alpha. Accordingly, the results of the present study suggest that the sample size needed to show a statistically significant change in the circulating level of a given cytokine will vary depending on the specific cytokine that is being measured, as well as the time period over which that cytokine is being assayed.

Estrogen is associated with improved survival in aging women with congestive heart failure: analysis of the vesnarinone studies.

OBJECTIVES: This study sought to evaluate the effects of postmenopausal estrogen use on mortality in aging women with congestive heart failure (CHF). BACKGROUND: The age-related increase in CHF mortality in women may be related to a menopause-associated increased incidence of coronary artery disease. In addition to inhibiting coronary atherosclerosis, estrogen may also have protective effects on cardiac myocytes independent of the coronary vasculature. We hypothesized that estrogen use is associated with improved survival in elderly women with CHF. METHODS: Associations between survival, estrogen use and patient characteristics were assessed in 1,134 women who were at least 50 years of age, had CHF and left ventricular ejection fraction (EF) < or =30% and were enrolled in one of three clinical trials of vesnarinone. RESULTS: All-cause 12-month mortality was 15.0% among the 237 estrogen users versus 27.1% among the 897 estrogen nonusers (p = 0.004 for unadjusted comparison of survival). Similar results were observed for cardiac mortality. Regression analysis demonstrated that estrogen use was independently associated with improved survival (relative risk of mortality = 0.68, 95% confidence interval 0.48 to 0.96, p = 0.03). Advanced age, low EF, New York Heart Association class IV CHF, Caucasian race and abnormal serum creatinine, sodium, potassium and transaminase were independently associated with increased mortality. CONCLUSIONS: Estrogen use among older women with CHF is associated with decreased overall and cardiac mortality.

The efficacy of carbamazepine combinations in epilepsy.

The efficacy and bioavailability, and tolerance to carbamazepinee when administered with phenobarbital or phenytoin or with both drugs were evaluated in a prospective, double-blind study of patients whose seizures were not completely controlled by currently available antiepileptic drugs in usually therapeutic dosages as determined by serum levels. During each of four 21-day treatment periods, one fourth of the patients received daily doses of: (1) carbamazepine (1,200 mg) and phenytoin (300 mg); (2) carbamazepine (1,200 mg) and phenobarbital (300 mg); (3) phenytoin (300 mg) and phenobarbital (300 mg); or (4) carbamazepine (1,200 mg), with phenytoin (300 mg) and phenobarbital (300 mg). The treatment periods were separated by 2 wk of each patient's usual prestudy medication. Treatment with all three drugs was the most efficacious for seizure control. Serum carbamazepine concentration was significantly decreased when the drug was administered with either phenytoin or phenobarbital or both.

Interlaboratory variability in determination of plasma antiepileptic drug concentrations.

The usefulness of plasma antiepileptic drug concentrations in treatment of epilepsy has been established, and many laboratories provide this service. A "blind" survey utilizing pooled patient plasma samples was conducted among 197 laboratories in the United States and Canada to establish the interlaboratory reproducibility. Three "patient specimens" containing different amounts of phenobarbital, phenytoin (diphenylhydantoin), primidone, and ethosuximide were employed; 112 laboratories reported results within five weeks. The average cost for analyzing four drugs in a single sample was $43.27. Half of the laboratories reported results outside +/- 1 standard deviation of the mean of five reference laboratories. Wide interlaboratory variability must be considered by the practicing physician. Until certified antiepileptic drug standards in a biologic matrix are available from the National Bureau of Standards, a volunteer quality control program among laboratories is needed.

Clinical and EEG estimates of absence seizure frequency.

Absence seizure frequency was estimated in 20 patients (5 to 15 years old) before and after treatment with ethosuximide. Estimates were obtained from mothers' histories, observations by nurses, intensive observation by trained observers, physical and neurological examinations, routine EEG, and 12-hour telemetered EEG. Both before treatment (high seizure frequency) and after treatment (low frequency), telemetered EEG was the most reliable method of estimation, and intensive observation was the next best method. After treatment, the mothers' and nurses' estimates of seizure frequency were significantly less than the telemetered EEG estimates. The neurological examination and routine EEG were sufficient to diagnose absence attacks in all 20 patients and to determine if the attacks were completely controlled by therapy in all but two patients.

The hospital experience and seizure control.

We studied 30 patients who were admitted to the hospital because of intractable seizures. Twenty-three had fewer seizures during one or both of the first 2 hospital weeks than before admission, although medication was not changed. The role of environment in seizure control is difficult to measure, but hospital admission itself is a form of environmental manipulation. When seizure control is achieved in the hospital, the hospital experience itself must be considered in addition to other therapeutic interventions.

Ethosuximide in the treatment of absence (peptit mal) seizures.

Thirty-seven patients with previously untreated absence seizures were treated with ethosuximide. Seizures were completely controlled in 7 patients (19 percent); 90 to 100 percent control was achieved in 18 patients (49 percent) and 50 to 100 percent control in 35 (95 percent). Plasma ethosuximide concentration increased with dose, but variability in the plasma concentration produced by a given ethosuximide dose made it impossible to predict a patient's plasma concentration from the dose. The therapeutic range of plasma ethosuximide concentration was 40 to 100 mug per milliliter. Patients with evidence of structural central nervous system abnormalities responded as well or better to the drug as patients without such evidence. Ethosuximide did not impair psychometric performance, but rather resulted in improved performance in 17 cases. The side effects of ethosuximide were minor, and rarely required withdrawal of the drug.

Effects of vesnarinone on peripheral circulating levels of cytokines and cytokine receptors in patients with heart failure: a report from the Vesnarinone Trial.

STUDY OBJECTIVES: Proinflammatory cytokines may contribute to disease progression in heart failure by virtue of the direct toxic effects that these molecules exert on the heart and the circulation. Accordingly, there is interest in developing therapeutic agents with anticytokine properties that might be used as adjunctive therapy to modulate proinflammatory cytokine levels in patients with heart failure. Previous experimental studies suggested that vesnarinone has potent anticytokine properties in vitro. Therefore, we examined the effects of vesnarinone on circulating levels of cytokines and cytokine receptors in a large-scale, multicenter, clinical trial of patients with moderate-to-advanced heart failure: the Vesnarinone Trial (VEST). METHODS: Circulating levels of tumor necrosis factor (TNF)-alpha, soluble TNF-receptor type 1, soluble TNF-receptor type 2, as well as interleukin (IL)-6 and soluble IL-6 receptor (sIL-6R) were measured on plasma samples by enzyme-linked immunosorbent assay at baseline and at 24 weeks in patients who were receiving placebo (n = 352), 30 mg of vesnarinone (n = 367), and 60 mg of vesnarinone (n = 327). RESULTS: Treatment with 30 mg and 60 mg of vesnarinone had no effect on circulating levels of cytokines or cytokine receptors in patients with advanced heart failure over a 24-week period. CONCLUSIONS: In contrast to the potent anticytokine effects observed with vesnarinone in experimental studies in vitro, the results of this clinical study suggest that vesnarinone does not have any measurable anticytokine effects in vivo in patients with moderate-to-advanced heart failure.

A double-masked comparison of carteolol and timolol in ocular hypertension.

We performed a double-masked study in which 98 patients with ocular hypertension who had been previously treated with timolol received either timolol 0.25% or carteolol 1%, a beta-blocker with intrinsic sympathomimetic activity. The drugs were administered topically twice daily for one month after a one-week washout period. Intraocular pressure was measured at baseline and after one and four weeks of treatment. The appearance of the fundus, external eye, visual fields, tear secretion, blood pressure, and pulse were recorded. Adverse symptoms were elicited using a menu-type questionnaire and an overall judgment of therapy was recorded. Carteolol was as effective as timolol in reducing intraocular pressure. There were significantly fewer patients reporting adverse events overall (P = .019), and eye irritation specifically (P = .02), in the group treated with carteolol.

A quarter-century of experience with career change education: an option for turning specialists into generalists.

The Physician Refresher program, now 25 years old, at the Medical College of Pennsylvania offers training to clinically inactive physicians. The authors examined the characteristics of program participants to determine whether specialists who took the program to prepare for a change to a primary care career made the intended change. Application data from all registrants between 1982 and 1993 were compared with data on previously reported groups from 1968-1975 and 1976-1981. Specialist registrants' subsequent practice activities were documented from the AMA Medical Directory and the American Board of Medical Specialties Directory; a telephone survey elicited their reasons for making or not making the career change to primary care. During the last decade of the program women registrants constituted 36% of the total; international medical graduates (IMGs) 43%; and clinically inactive physicians 38%. Specialists outnumbered those in primary care by two to one. Although 65% of specialists planned to switch to primary care, ultimately only 27% of the total did so; many, especially the IMGs, were serving the disadvantaged. Those who did not switch cited a variety of disincentives, including individual educational needs not met by a general refresher course. If the medical profession accepts the need to devise programs that "retool" specialists to provide primary care, those programs should be specifically designed to address the individual needs of the specialists involved. Concomitantly, the incentives to make the switch need to be enhanced.

Expression of alpha5 integrin (Itga5) is elevated in the rat myometrium during late pregnancy and labor: implications for development of a mechanical syncytium.

The underlying mechanisms controlling uterine contractions during labor are still poorly understood. Integrins are heterodimeric, transmembrane receptors composed of alpha and beta subunits that can be found in focal adhesions. Because these structures play an important role in the regulation of smooth muscle contractility and cell adhesion, we hypothesized that alpha5 integrin mRNA (Itga5) and protein (ITGA5) expression would be induced in the rat myometrium during late pregnancy and labor. Itga5 mRNA expression was significantly increased (P < 0.05) from Day 17 to labor, noticeably decreasing 1 day postpartum (PP). Immunoblot analysis illustrated a continual increase in ITGA5 levels during pregnancy, labor, and PP, with levels reaching significance at labor (P < 0.05). Analysis of ITGA5 expression by immunocytochemistry demonstrated that it is primarily localized to myometrial cell membranes in the longitudinal muscle layer of the myometrium from before pregnancy to Day 6, and in both the longitudinal and circular muscle layers from Day 15 to PP. Treatment of late-pregnant rats with progesterone blocked labor and resulted in sustained expression of Itga5 mRNA expression to Day 24. In addition, immunocytochemistry experiments showed ITGA5 was detectable at higher levels in cell membranes of both myometrial layers in progesterone-treated animals on Days 23 and 24, compared with vehicle controls. We propose that ITGA5, with its sole known partner, ITGB1, may be important in promoting cellular cohesion during late pregnancy. This process may aid the development of a mechanical syncytium for efficient force transduction during the sustained, coordinated, and powerful contractions of labor.

Small heat shock protein 27 (Hsp27) expression is highly induced in rat myometrium during late pregnancy and labour.

The underlying mechanisms that regulate uterine contractions during labour are still poorly understood. A candidate regulatory protein is heat shock protein 27 (Hsp27). It belongs to the small heat shock protein family and can regulate actin cytoskeleton dynamics, act as a chaperone, and may regulate contractile protein activation. As a result, we hypothesized that Hsp27 expression would be highly induced during late pregnancy and labour. Hsp27 mRNA expression was significantly elevated (P <0.05) on days 17 to 22 of gestation. In addition, immunoblot analysis demonstrated that detection of total Hsp27 increased (P <0.05) between day 21 and 1 day post-partum (PP) inclusive. Since phosphorylation of Hsp27 has been reported to be a prerequisite for smooth muscle contraction, we examined the temporal and spatial expression of Ser-15 phosphorylated Hsp27. Immunoblot analysis showed that the detection of Ser-15 phosphorylated Hsp27 significantly increased (P <0.05) between days 19 and 23 (active labour) inclusive, in parallel with detection of total Hsp27. Immunocytochemical analysis of Ser-15 phosphorylated Hsp27 expression in situ demonstrated that phosphorylated Hsp27 in circular muscle became detectable in peri-nuclear and membrane regions on days 19 to 22, but was primarily restricted to the cytoplasm on days 23 to PP. In contrast, phosphorylated Hsp27 in longitudinal muscle was primarily detected in myocyte membranes on days 15 to 22, and then also became detectable in the cytoplasm of myocytes on days 23 and PP. Our results demonstrate that Hsp27 expression is highly upregulated during late pregnancy and labour and suggest that Hsp27 is a potential candidate contraction-associated protein.

The Antiepileptic Drug Development Program: an example of government-industry collaboration.

By collaborating with the pharmaceutical industry in key areas of drug development, the ADD Program of the Epilepsy Branch, National Institute of Neurological and Communicative Disorders and Stroke, has responded to the need for more effective and less toxic antiepileptic drugs than those currently available. The program screens large numbers of compounds for anticonvulsant activity, conducts toxicology studies, and sponsors clinical trials of promising new drugs for the treatment of epilepsy. This collaboration with the pharmaceutical industry is providing a valuable model for a shared drug development program.

Visual evoked potentials and eye dominance.

The amplitudes of pattern-reversal VEPs in 25 healthy volunteers were significantly higher from the dominant eye than the non-dominant eye in right eye dominant subjects. The difference was present over both hemispheres and over the midline. Handedness did not appear to influence the amplitude asymmetry. A similar trend was noted in left eye dominant subjects, but the difference was significantly only at O2. The mean latency of the P100 peak was significantly shorter with stimulation of the dominant eye. These amplitude and latency disparities between dominant and non-dominant eyes provide electrophysiological evidence of lateralization in the nervous system.