The influence of experimental anterior knee pain during running on electromyography and articular cartilage metabolism.

OBJECTIVE: To determine whether anterior knee pain (AKP), during running, acutely affects lower-extremity electromyography (EMG) and articular cartilage metabolism. METHODS: A within-subjects design was used. Each of 12 able-bodied subjects ran on a treadmill for 30 min for three different sessions: control (no infusion), sham (0.9% NaCl infusion into the involved-leg infrapatellar fat pad), and pain (5.0% NaCl infusion into the involved-leg infrapatellar fat pad). Bilateral surface EMG was monitored for the vastus medialis (VM), vastus lateralis (VL), and gastrocnemius (GA). Serum cartilage oligomeric matrix protein (COMP) concentration was determined before, after, and 60 min after the run. A functional analysis approach was used to compare EMG amplitude, across the entire stance phase, between sessions and legs. Mixed-model analysis of covariance was used to compare serum COMP concentration between sessions, across time. RESULTS: Relative to the uninvolved leg, greater involved-leg VL and GA EMG amplitude existed during midstance for the sham and control sessions (P < 0.01). During the painful session, however, involved-leg VM, VL, and GA EMG amplitude was 5-10% less than for the uninvolved leg. COMP concentration immediately post-run was 14% and 21% greater than pre-run (P = 0.01) and 60 min post-run concentrations (P < 0.01), respectively. Session, however, did not significantly influence COMP. CONCLUSION: During a 30-min run, AKP acutely alters midstance VM, VL, and GA EMG amplitude. AKP during a 30-min run does not, however, acutely influence articular cartilage metabolism.

Transgenic avian-derived recombinant human interferon-alpha2b (AVI-005) in healthy subjects: an open-label, single-dose, controlled study.

BACKGROUND/AIMS: This study characterized the safety and pharmacological properties of AVI-005, a novel glycosylated recombinant human interferon-alpha2b produced from the egg whites of chickens transfected with human cDNA. METHODS: 18 healthy volunteers received single subcutaneous rising doses (0.5, 1.66 or 5 million international units, MIU) of AVI-005. A randomized parallel comparator group of 10 subjects received 5 MIU of unglycosylated IFN-alpha2b (Intron A). The pharmacokinetic parameters t1/2, tmax, Cmax, AUC0-24h, Vd, and clearance were compared between AVI-005 and unglycosylated IFN-alpa2b. RESULTS: At equipotent doses, AVI-005 had a larger AUC0-24h than the control interferon. Pharmacodynamic markers ofneopterin and beta2-microglobulin for the two treatments were similar. These markers were increased by AVI-005 in a dose-dependent manner. Pharmacodynamic responses to treatment with AVI-005 were shown by the change in mRNA expression for interferon inducible protein kinase and 2'5'-oligoadenylate synthetase. Adverse events in the two groups were qualitatively and quantitatively similar. CONCLUSION: AVI-005 demonstrates biological activity and pharmaco-kinetic properties in humans that support further development.

Mutagenesis of a highly conserved lysine 340 of the PRD1 DNA polymerase.

All known family B DNA polymerases contain a conserved region of amino acids, KX6-7YG, which appears to be correspond to the 'finger' alpha helix O of the Klenow fragment of E. coli DNA polymerase I, a family A DNA polymerase. Toward the goal of establishing the evolutionary relationship between the family A and B DNA polymerases, we have employed site-directed mutagenesis to access the functional role of the invariant amino acid lysine-340 of the PRD1 DNA polymerase. We have replaced the lysine-340 with three amino acids: histidine, asparagine and glutamic acid, respectively. Mutant DNA polymerases were overexpressed and purified to near homogeneity. Our results showed that the modification of the lysine-340 of the PRD1 DNA polymerase abolishes the polymerase activity without affecting the 3' to 5' exonuclease activity. These results support the proposal that the KX6-7YG motif of the family B DNA polymerases may be analogous to the KX7YG motif of the family A DNA polymerases, suggesting that two family DNA polymerases share a common ancestor.

Clinical correlates and reference intervals for pulmonary artery systolic pressure among echocardiographically normal subjects.

BACKGROUND: Data in normal human subjects on the factors affecting pulmonary artery systolic pressure (PASP) are limited. We determined the correlates of and established a reference range for PASP as determined by Doppler transthoracic echocardiography (TTE) from a clinical echocardiographic database of 102 818 patients, of whom 15 596 (15%) had a normal Doppler TTE study. METHODS AND RESULTS: A normal TTE was based on normal cardiac structure and function during complete Doppler TTE studies. The PASP was calculated by use of the modified Bernoulli equation, with right atrial pressure assumed to be 10 mm Hg. Among TTE normal subjects, 3790 subjects (2432 women, 1358 men) from 1 to 89 years old had a measured PASP. The mean PASP was 28.3+/-4.9 mm Hg (range 15 to 57 mm Hg). PASP was independently associated with age, body mass index (BMI), male sex, left ventricular posterior wall thickness, and left ventricular ejection fraction (P<0.001). The estimated upper 95% limit for PASP among lower-risk subjects was 37.2 mm Hg. A PASP >40 mm Hg was found in 6% of those >50 years old and 5% of those with a BMI >30 kg/m(2). CONCLUSIONS: Among 3790 echocardiographically normal subjects, PASP was associated with age, BMI, sex, wall thickness, and ejection fraction. Of these subjects, 28% had a PASP >30 mm Hg, and the expected upper limit of PASP may include 40 mm Hg in older or obese subjects. These findings support the use of age- and BMI-corrected values in establishing the expected normal range for PASP.

Acute reduction in functional infarct expansion with late coronary reperfusion: assessment with quantitative two-dimensional echocardiography.

Reperfusion performed too late to salvage myocardium decreases chronic infarct expansion in experimental animals. However, the acute effects of delayed reperfusion are not known. Twenty-two dogs underwent 3 (n = 8), 4 (n = 8) or 6 h (n = 6) of circumflex artery occlusion followed by 3 h of reperfusion. Effects of reperfusion on diastolic expansion were assessed in two ways: 1) change in mean radius of curvature of the infarct segment, and 2) change in the ratio of the length of the diameter from the center of the infarct zone to the opposite wall (septal-lateral diameter) to the length of the diameter perpendicular to this (anteroposterior diameter). Effects on systolic expansion were examined with quantitative two-dimensional echocardiographic systolic thickening analysis. Delayed reperfusion produced an immediate decrease in diastolic infarct expansion. The ratio of septal-lateral/anteroposterior diameters, which had increased with occlusion from a preocclusion baseline of 0.98 +/- 0.06 to 1.13 +/- 0.08 (p less than 0.001), decreased with reperfusion to 1.02 +/- 0.07 at 15 min and 1.03 +/- 0.08 at 3 h of reperfusion (p = 0.001). This was due solely to a decrease in the septal-lateral diameter. The radius of curvature of the infarcted segment increased from 2.1 +/- 0.5 cm before reperfusion to 2.74 +/- 0.8 cm at 15 min and 2.6 +/- 0.85 cm at 3 h of reperfusion (p = 0.009). This occurred despite a significant (13.6%) decline in end-diastolic cavity area and is compatible with flattening of the reperfused infarct region. Systolic infarct expansion also improved slightly.(ABSTRACT TRUNCATED AT 250 WORDS)

Superiority of quantitative exercise thallium-201 variables in determining long-term prognosis in ambulatory patients with chest pain: a comparison with cardiac catheterization.

The purpose of this study was to determine the prognostic utility of quantitative exercise thallium-201 imaging and compare it with that of cardiac catheterization in ambulatory patients. Accordingly, long-term (4 to 9 years) follow-up was obtained in 293 patients who underwent both tests for the evaluation of chest pain: 89 had undergone coronary artery bypass graft surgery within 3 months of testing and were excluded from analysis, 119 experienced no cardiac events and 91 had an event (death in 20, nonfatal myocardial infarction in 21 and coronary artery bypass operations performed greater than 3 months after cardiac catheterization in 50). When all variables were analyzed using Cox regression analysis, the quantitatively assessed lung/heart ratio of thallium-201 activity was the most important predictor of a future cardiac event (chi 2 = 40.21). Other significant predictors were the number of diseased vessels (chi 2 = 17.11), patient gender (chi 2 = 9.43) and change in heart rate from rest to exercise (chi 2 = 4.19). Whereas the number of diseased vessels was an important independent predictor of cardiac events, it did not add significantly to the overall ability of the exercise thallium-201 test to predict events. Furthermore, information obtained from thallium-201 imaging alone was marginally superior to that obtained from cardiac catheterization alone (p = 0.04) and significantly superior to that obtained from exercise testing alone (p = 0.02) in determining the occurrence of events. In addition, unlike the exercise thallium-201 test, which could predict the occurrence of all categories of events, catheterization data were not able to predict the occurrence of nonfatal myocardial infarction. The exclusion of bypass surgery and previous myocardial infarction did not alter the results. In conclusion, data from this study demonstrate that exercise thallium-201 imaging may be superior to data from both exercise testing alone and cardiac catheterization data alone for predicting future events in ambulatory patients who have undergone both exercise thallium-201 imaging and catheterization for the evaluation of chest pain.

Doppler echocardiographic assessment of long-term progression of mitral stenosis in 103 patients: valve area and right heart disease.

OBJECTIVES: The purpose of this study was to determine, in a large referral population, the rate of echocardiographic change in mitral valve area (MVA) without interim intervention, to determine which factors influence progression of narrowing and to examine associated changes in the right side of the heart. BACKGROUND: Little information is currently available on the echocardiographic progression of mitral stenosis, particularly on progressive changes in the right side of the heart and the ability of a previously proposed algorithm to predict progression. METHODS: We studied 103 patients (mean age 61 years; 74% female) with serial two-dimensional and Doppler echocardiography. The average interval between entry and most recent follow-up study was 3.3 +/- 2 years (range 1 to 11). RESULTS: During the follow-up period, MVA decreased at a mean rate of 0.09 cm2/year. In 28 patients there was no decrease, in 40 there was only relatively little change (< 0.1 cm2/year) and in 35 the rate of progression of mitral valve narrowing was more rapid (> or = 0.1 cm2/year). The rate of progression was significantly greater among patients with a larger initial MVA and milder mitral stenosis (0.12 vs. 0.06 vs. 0.03 cm2/year for mild, moderate and severe stenosis, p < 0.01). Although the rate of mitral valve narrowing was a weak function of initial MVA and echocardiographic score by multivariate analysis, no set of individual values or cutoff points of these variables or pressure gradients could predict this rate in individual patients. There was a significant increase in right ventricular diastolic area (17 to 18.7 cm2) and tricuspid regurgitation grade (2 + to 3 +; p < 0.0001 between entry and follow-up studies). Progression in right heart disease occurred even in patients with minimal or no change in MVA. Patients with associated aortic regurgitation had a higher rate of decrease in MVA than did those with trace or no aortic regurgitation (0.19 vs. 0.086 cm2/year, p < 0.05). CONCLUSIONS: The rate of mitral valve narrowing in individual patients is variable and cannot be predicted by initial MVA, mitral valve score or transmitral gradient, alone or in combination. Right heart disease can progress independent of mitral valve narrowing.

Quantitation of acute myocardial infarct size by nuclear magnetic resonance imaging.

Nuclear magnetic resonance (NMR) imaging has shown potential in the detection and characterization of acute myocardial infarction in humans. This study was performed to evaluate the capability of NMR imaging in the measurement of infarct size in patients with recent myocardial infarction. Electrocardiographic (ECG)-gated spin-echo NMR imaging was performed in 26 patients a mean of 9 +/- 3 days (range 5 to 20) after infarction. The imaging technique used provided single-slice, spin-echo (time to echo [TE] = 60 ms) images of the left ventricle in its true short axis, allowing direct correlation of NMR infarct location and size with the region of severe hypokinesia on left ventriculography. In all 20 patients with complete NMR studies, infarct location was correctly identified by using specific, objective criteria. The correlation between the mean infarct volume (29 +/- 11 ml) and the quantitated left ventricular hypokinetic segment (7.5 +/- 4.0 cm) was good (r = 0.84, p = 0.0002), suggesting that NMR imaging of the heart may have a role in the noninvasive assessment of myocardial infarct size in patients.

Restricted diastolic opening of the mitral leaflets in patients with left ventricular dysfunction: evidence for increased valve tethering.

OBJECTIVES: We tested the hypothesis that patients with incomplete systolic mitral leaflet closure (IMLC: apically displaced coaptation) also have restricted diastolic leaflet opening that is independent of mitral inflow volume and provides evidence supporting increased leaflet tethering. BACKGROUND: Competing hypotheses for functional mitral regurgitation (MR) with IMLC include global left ventricular (LV) dysfunction per se (reduced leaflet closing force) versus geometric distortion of the mitral apparatus by LV dilation (augmented leaflet tethering). These are inseparable in systole, but restricted leaflet motion has also been observed in diastole, and attributed to reduced mitral inflow. METHODS: Diastolic mitral leaflet excursion and orifice area were measured by two-dimensional echocardiography in 58 patients with global LV dysfunction, 36 with and 22 without IMLC, compared with 21 normal subjects. The biplane Simpson's method was used to calculate LV ejection volume, which equals mitral inflow volume in the absence of aortic regurgitation. RESULTS: The diastolic mitral leaflet excursion angle was markedly reduced in patients with IMLC compared with those without IMLC, whose ventricles were smaller, and normal subjects (17 +/- 10 degrees vs. 58 +/- 13 degrees vs. 67 +/- 8 degrees, p < 0.0001). Excursion angle was dissociated from mitral inflow volume (r2 = 0.04); excursion was reduced in patients with IMLC despite a normal inflow volume in the larger ventricles with MR (60 +/- 25 vs. 61 +/- 12 ml in normal subjects, p = NS), and excursion was nearly normal in patients without IMLC despite reduced inflow volume (40 +/- 10 ml, p < 0.001 vs. normal subjects). The anterior leaflet when maximally open coincided well with the line connecting its attachments to the anterior annulus and papillary muscle tip (angular difference = 3 +/- 7 degrees vs. 25 +/- 9 degrees vs. 32 +/- 10 degrees in patients with and without IMLC vs. normal subjects, p < 0.0001). In patients with IMLC, the leaflet tip orifice was smaller in an anteroposterior direction but wider than in the other groups, giving a normal total area (6.8 +/- 1.8 vs. 7.1 +/- 1.2 vs. 6.9 +/- 0.8 cm2, p = NS). CONCLUSIONS: Patients with LV dysfunction and systolic IMLC also have restricted diastolic leaflet excursion that is independent of inflow volume, coincides with the tethering line connecting the annulus and papillary muscle and reflects limitation of anterior motion relative to the posteriorly placed papillary muscles without a decrease in total orifice area. These observations are consistent with increased tethering by displaced mitral leaflet attachments in the dilated ventricles of patients with IMLC that can restrict both diastolic opening and systolic closure.

Transfer of an anti-HIV-1 ribozyme gene into primary human lymphocytes.

We reported previously that human CD4+ T cell lines stably expressing a hairpin ribozyme targeted to the human immunodeficiency virus type 1 (HIV-1) U5 leader sequence were resistant to challenge with diverse HIV-1 viral clones and clinical isolates (Yamada et al., 1994). To simulate more closely the in vivo infection process for investigations of anti-HIV-1 ribozyme gene therapy, we developed a system to transfer this ribozyme gene into freshly isolated human peripheral blood lymphocytes (PBLs) using a murine retrovirus vector. Following transduction and G418 selection, human PBLs from multiple donors expressed the ribozyme and resisted challenge by HIV-1 viral clones and clinical isolates, while control vector-transduced PBLs remained fully permissive for HIV-1 infection. No inhibition of an HIV-2 clone lacking the target was seen in ribozyme-expressing PBLs. Ribozyme expression had no effect on viability or proliferation kinetics of the primary lymphocytes. This study is the first demonstration in primary human T cells of resistance to HIV-1 infection conferred by gene transfer. A human clinical trial is in development to test further the safety and efficacy of this ribozyme in PBLs of HIV-1-infected patients in vivo.

Inhibition of fetal adrenal adrenocorticotropin receptor messenger ribonucleic acid expression by betamethasone administration to the baboon fetus in late gestation.

Throughout the majority of intrauterine development, the primate fetal adrenal gland is comprised primarily of fetal zone cells and only late in gestation do definitive zone cells, which express the enzyme delta5-3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) emerge to produce cortisol. The present study was designed to determine whether the induction of definitive zone ACTH receptor messenger RNA (mRNA) levels and components of the steroidogenic pathway known to be expressed specifically in the definitive zone, e.g. the 3beta-HSD enzyme, are dependent upon fetal pituitary ACTH. Fetal pituitaries and adrenal glands were obtained on day 165 (term = day 184) from untreated controls (n = 7) and from baboons in which betamethasone was administered im to the fetus (0.6 mg/100 microl; n = 4) or to the fetus (0.6 mg) and mother (6 mg/ml; n = 4) every other day between days 150 and 164 of gestation. Although fetal pituitary weight was not altered by betamethasone, POMC mRNA levels determined by in situ hybridization were lower (P < 0.05) in betamethasone-treated (0.34 +/- 0.07 arbitrary densitometric units) than in untreated controls (0.63 +/- 0.04). Associated with this decline in pituitary POMC, levels of the major 3.4-kb mRNA transcript for the ACTH receptor expressed as a ratio of beta-actin were approximately 80% lower (P < 0.05) in fetal adrenals of betamethasone-treated baboons (0.12 +/- 0.02) than in untreated controls (0.84 +/- 0.05). In situ hybridization indicated that ACTH receptor mRNA expression in the definitive zone exceeded that in the fetal zone and was reduced by betamethasone. Associated with the decrease in ACTH receptor expression, fetal adrenal weight was suppressed (P < 0.05) by 50% and reflected a marked reduction (P < 0.05) in the size of the cells of the definitive and fetal zones. Betamethasone treatment also induced a decrease (P < 0.05) in the width (microm) of the definitive zone (183 +/- 14 vs. 128 +/- 7; determined by immunohistochemical expression of 3beta-HSD), as well as the levels of the mRNA and protein for 3beta-HSD. Levels of the mRNA for the LDL-receptor and the enzymes 17alpha-hydroxylase-C(17,20) lyase and P450 cholesterol side chain cleavage were also suppressed in adrenals of betamethasone-treated baboons. These findings indicate that treatment of the baboon fetus with betamethasone in late gestation suppressed fetal pituitary POMC mRNA expression and ACTH receptor mRNA levels in the fetal adrenal gland, as well as the hypertrophy and ACTH receptor mRNA and 3beta-HSD mRNA/protein levels in the cells comprising the newly emerging definitive zone. We conclude that ACTH is necessary for the up-regulation of the mRNAs for the ACTH receptor and steroidogenic enzymes in the definitive zone of the primate fetal adrenal gland in late gestation.

Developmental increase in expression of the messenger ribonucleic acid and protein levels of 11beta-hydroxysteroid dehydrogenase types 1 and 2 in the baboon placenta.

Cortisol-cortisone interconversion is catalyzed by the NADP/NADPH-dependent oxido-reductase, 11beta-hydroxysteroid dehydrogenase-1 (11betaHSD-1) and the NAD-dependent oxidase, 11betaHSD-2. Because of the importance of placental corticosteroid metabolism in dictating the amount of cortisol arriving in the fetus to regulate fetal pituitary-adrenocortical function, the present study determined whether there was a developmental change in the expression of 11betaHSD-1 and/or -2 in placental syncytiotrophoblast, the site of maternal:fetal exchange. A syncytiotrophoblast-enriched (>95%) cell fraction was isolated from baboon placentas obtained at early (day 60), mid (day 100), and late (day 165) gestation (term = day 184), and 11betaHSD-1 and -2 messenger RNA (mRNA) and protein levels were determined by Northern and Western blots. The levels (mean +/- SE) of the single 1.6-kilobase (kb) mRNA for 11betaHSD-1, expressed as a ratio to beta-actin, increased (P < 0.05) between early (0.36 +/- 0.16; n = 4) and mid (0.95 +/- 0.21; n = 11) gestation and further increased (P < 0.05) by late gestation (1.82 +/- 0.29; n = 13). Similarly, the levels of the single 1.9-kb mRNA for 11betaHSD-2 in late gestation (2.46 +/- 0.35; n = 8) were greater (P < 0.05) than respective values at mid (1.36 +/- 0.22; n = 8) and early (0.64; n = 2) gestation. The levels of 11betaHSD-1 (arbitrary densitometric units), detected as a dominant band of 34 kDa, were greater (P < 0.05) in late gestation (2.6 +/- 0.2; n = 4) than at early (1.2 +/- 0.1; n = 4) or mid (1.9 +/- 0.3; n = 4) gestation. In contrast, 11betaHSD-2 was not detected by Western blot in syncytiotrophoblast isolated by collagenase dispersion. However, immunocytochemistry revealed that 11betaHSD-2 was present in and localized to the syncytiotrophoblast layer of the baboon placenta and that expression in late gestation (n = 4) appeared to exceed that in placentas of early (n = 4) and mid (n = 4) gestation. These results indicate that both 11betaHSD-1 and 11betaHSD-2 were expressed in syncytiotrophoblasts of the baboon placenta and that the mRNA and protein levels of these two 11betaHSD enzymes increased with advancing gestation. However, because 11betaHSD-2 was not detected in syncytiotrophoblast isolated by collagenase dispersion, we suggest that the 11betaHSD-1 and -2 reside in different membrane fractions of the syncytiotrophoblast. Consequently, the estrogen-regulated change in transplacental cortisol metabolism with advancing gestation may result in a developmental change in the expression and location of the two 11betaHSD enzymes controlling cortisol-cortisone metabolism and transfer into the fetus, resulting in activation of the fetal pituitary adrenocortical system.

Identification and developmental expression of the estrogen receptor alpha and beta in the baboon fetal adrenal gland.

We have previously shown that estrogen regulates the development and function of the fetal and definitive/transitional zones of the primate fetal adrenal gland. Thus, during baboon pregnancy estrogen acts directly on the fetal zone to suppress ACTH-stimulated dehydroepiandrosterone (DHA) formation, potentially to modulate C19-steroid production and consequently placental estrogen synthesis. It is proposed that this action of estrogen is mediated by the estrogen receptor. Therefore, in the present study a developmental approach was used to determine whether the messenger RNA (mRNA) and protein for the estrogen receptor were expressed in the fetal and definitive/transitional zones ofthe baboon fetal adrenal gland at mid (day 100) and late (day 170) gestation (term = 184 days). Estrogen receptor alpha mRNA levels, determined by competitive RT-PCR, were approximately 7-fold greater (P < 0.02) in the fetal adrenal of late (187.8+/-40.3 attomoles/microg RNA) compared with mid (27.4+/-5.4 attomoles/microg RNA) gestation. Moreover, estrogen receptor alpha mRNA expression, determined by quantitative in situ hybridization, was approximately 2.5-fold greater (P < 0.05) in the definitive/transitional zones (21.6+/-0.5 silver grains/0.025 mm2) than in the fetal zone (8.3+/-1.5 grains/0.025 mm2) late in gestation. The mRNA for the beta-isoform of the estrogen receptor was also expressed in the baboon fetal adrenal cortex. There was a gradient of immunocytochemical staining for the estrogen receptor alpha and beta proteins, with extensive immunoreactivity for both isoforms in the definitive zone and lower staining in the transitional zone and the fetal zone. In summary, the results of the present study show that estrogen receptor alpha and beta were expressed in the fetal and definitive/transitional zones of the baboon fetal adrenal cortex at mid and late gestation. The presence of the estrogen receptor provides a mechanism for mediating the action of estrogen in modulating ACTH-dependent and cortical zone-specific development and function of the primate fetal adrenal gland.

Development of the baboon fetal adrenal gland: regulation of the ontogenesis of the definitive and transitional zones by adrenocorticotropin.

Throughout gestation, the primate fetal adrenal gland is comprised of the fetal zone, which expresses the P-450 17alpha-hydroxylase-C17,20 lyase (P-450c17) enzyme that catalyzes the synthesis of C19 steroids used for placental estrogen production. The development of the transitional zone comprised of cortical cells that express the P-450c17 and the 3beta-hydroxysteroid dehydrogenase-isomerase (3betaHSD) enzymes for cortisol production, and the definitive zone, which expresses 3betaHSD, but not P-450c17, for mineralocorticoid synthesis, does not occur until relatively late in gestation. Although ACTH is considered essential to fetal adrenal growth and function, the role that ACTH has in the development of the transitional and definitive zones, is less clear. To answer this question, the width of these zones was determined by immunocytochemical expression of P-450c17 and/or 3betaHSD in fetal adrenal glands obtained on day 100 (mid) of gestation (term = day 184) from baboons in which ACTH was administered to the fetus on days 95-99 of gestation or on day 165 (late) of gestation from baboons in which fetal ACTH was suppressed by treatment of the mother and fetus with betamethasone on days 150-164 of gestation. At midgestation, the fetal adrenal was comprised almost exclusively of fetal zone cells and a small definitive zone (38 +/- 2 microm in width), but was essentially devoid of a transitional zone (7 +/- 2 microm). Treatment with ACTH enhanced (P < 0.05) the width of the transitional zone (67 +/- 4 microm), but not the size of the definitive zone (10 +/- 4 microm). In late gestation, the width of the definitive zone, although 2-fold greater than that on day 100, was smaller (P < 0.05) than that of the transitional zone (120 +/- 15 microm), which greatly exceeded that at midgestation. Treatment with betamethasone in late gestation eliminated the transitional zone, but had no effect on the size of the definitive zone (120 +/- 8 microm). These findings indicate that the development of the baboon fetal adrenal transitional zone late in gestation is dependent on fetal pituitary ACTH. In contrast, the ontogenesis of the definitive zone at midgestation and its growth in late gestation occur in the relative absence of ACTH.

Suppression of stimulated plasma renin by clonidine in the dog.

The mechanism by which clonidine suppresses plasma renin activity (PRA) was investigated in dogs anesthetized with pentobarbital. Injection of clonidine (1 micrograms/kg) into the cisterna magna decreased PRA from levels stimulated by prior hemorrhage into a blood reservoir to reduce mean blood pressure by 25% (21.7 ng/ml/hr +/- 6.6 SE leads to 11.1 ng/ml/hr +/- 2.4 SE; p less than 0.05). Clonidine reduced heart rate but mean arterial pressure remained constant due to fluid movement between the reservoir and the arterial circulation of the dog. These effects could not be attributed to leakage of clonidine from the cerebrospinal fluid since intravenous administration of the same dose had no effect on PRA. In animals bilaterally splanchnicotomized at the level of the diaphragm, elevated PRA was not reduced by intracisternal clonidine. When return of reservoir fluid was prevented, animals became hypotensive after central clonidine and renin tended to increase. These results indicate that clonidine reduces stimulated renin by a central mechanism that is dependent upon the integrity of the sympathetic innervation of the kidney. Other stimuli for renin release may override the inhibitory effect of central clonidine.

Effect of maternal betamethasone administration at midgestation on baboon fetal adrenal gland development and adrenocorticotropin receptor messenger ribonucleic acid expression.

Although fetal pituitary ACTH is important to fetal adrenal growth and steroidogenesis in the second half of primate pregnancy, its role in adrenal development and function has not been established in vivo in the first half of gestation. In the present study, therefore, baboons were treated at midgestation with betamethasone to determine the effect of fetal pituitary ACTH on fetal adrenal growth, development, and ACTH receptor and P-450 enzyme messenger ribonucleic acid (mRNA) levels. The administration of betamethasone to baboon mothers on days 60-99 of gestation (term = 184 days) decreased fetal pituitary POMC mRNA levels by 54% (P < 0.01) and fetal serum ACTH levels to undetectable values (P < 0.05). The decline in ACTH was associated with decreases in fetal adrenal weight (P < 0.001), cortical cell size (P < 0.05), appearance of apoptosis and cellular disorganization, and a loss of immunocytochemically demonstrable definitive zone-specific delta5-3beta-hydroxysteroid dehydrogenase expression. The concomitant administration of ACTH and betamethasone restored these aspects of adrenal integrity to normal. Moreover, there was approximately a 95% decrease (P < 0.01) in fetal adrenal expression of ACTH receptor, P-450 cholesterol side-chain cleavage, and P-450 17alpha-hydroxylase 17/20-lyase mRNA levels after betamethasone administration. We conclude that fetal pituitary ACTH is necessary for the growth and development of fetal and definitive cortical zones and the marked coordinated increase in ACTH receptor and maintenance of P-450 cholesterol side-chain cleavage/P-450 17alpha-hydroxylase 17/20-lyase expression in the baboon fetal adrenal gland during the first half of gestation.

Characterization of a molecular clone of HIV type 2 infectious for Macaca nemestrina.

A lambda phage clone containing a full-length HIV-2 provirus, designated HIV-2KR, was obtained from the genomic DNA of Molt4 clone 8 (Molt4/8) lymphoblastic cells infected with the HIV-2PEI2 strain. HIV-2KR is genetically distinct from known HIV-2 isolates, possessing both a unique deletion in the LTR promoter region, and a long rev reading frame. It is replication competent in vitro after transfection into Molt4/8 cells, replicates in a variety of established human T lymphoblastic (Molt-3, Molt4/8, SupT1, H9, C8166) and myelomonocytic (U937) cell lines, and displays prominent cytopathic effects on infection of Molt4/8 cells, reflecting usage of both CCR5 and CXCR4 coreceptors. In addition, HIV-2KR was found to be infectious for human and Macaca nemestrina peripheral blood lymphocytes, and primary human monocyte-macrophage cultures. Intravenous inoculation of cell-free virus into M. nemestrina resulted in infection characterized by transient, low-level viremia and modest temporary decline in CD4 lymphocyte numbers, making HIV-2KR the first HIV-2 molecular clone reported to be infectious for this primate species.

Significant tricuspid regurgitation does not resolve after percutaneous balloon mitral valvotomy.

A total of 318 consecutive patients with mitral stenosis underwent percutaneous mitral valvotomy at our institution from 1987 to 1993. Of those, 98 patients had color Doppler echocardiographic studies performed before, 24 hours after, and late after the intervention. On the basis of color Doppler echocardiographic grading of tricuspid regurgitation, 32 patients (32%; mean age 57 +/- 15 years) had significant (moderate or severe) tricuspid regurgitation before the intervention and were the subject of this study. The follow-up study was performed 18.4 +/- 13 months after the procedure. Successful percutaneous mitral valvotomy (> or = 1.5 cm2 valve area or > or = 50% increase after valvotomy) with no restenosis at follow-up was achieved in 20 patients. Tricuspid regurgitation decreased by one grade (from severe to moderate) in only four subjects in this group and in none of the 12 patients who did not meet the criteria for successful percutaneous mitral valvotomy or who had restenosis. Thus tricuspid regurgitation did not improve in 88% of all patients studied. On average, no significant change was observed in the ratio of maximal tricuspid regurgitant jet area to right atrial area 24 hours after percutaneous mitral valvotomy and at late follow-up (37% vs .33% vs 34%, respectively) or in any of the right heart dimensions, even in patients who underwent successful percutaneous mitral valvotomy. Right ventricular systolic pressure also did not change significantly on average in those patients (46 +/- 15 versus 42 +/- 14 versus 48 +/- 18 mm Hg, respectively). However, right ventricular dimensions did not decrease and tricuspid regurgitation did not resolve even in a subgroup of patients in whom right ventricular systolic pressure fell by more than 10 mm Hg (up to 41 mm Hg).

Ultraprofound hypothermia with complete blood substitution in a canine model.

The potential for hypothermia to prevent or ameliorate ischemic injury to the central nervous system is well known. To determine if a more prolonged period of metabolic suppression with blood substitution is possible, a method was developed to lower body temperature to near the freezing point. Eight adult mongrel dogs underwent closed-chest extracorporeal circulation with both external and internal body cooling. As they were cooled, progressive hemodilution was employed until complete exsanguination and blood substitution with an aqueous solution was accomplished. Continuous circulation and a core temperature at a mean of 1.7 degrees C were maintained from 2 1/2 to 3 hours. After rewarming to 20 degrees C, the animals were autotransfused and allowed to recover. Of the eight animals, two died due to technical factors related to cardiac defibrillation. Of the six surviving animals, five survived over a long period and one died on the 10th postoperative day with hepatorenal failure resulting from a presumed blood transfusion incompatability reaction. All six showed normal neurological function and kennel behavior, except one dog with mild weakness of a hindlimb. When the dogs were sacrificed 1 to 2 months postoperatively, all organs were histologically normal. Specifically, there was no gross or microscopic evidence of ischemic or hypoxic injury to any central nervous system structures. This pilot study demonstrates that it is possible to successfully achieve complete exsanguination, blood substitution, and ultraprofound body temperature, while continuous circulation of the blood substitute is maintained. With the capability of controlling and repeatedly performing washout of the extracellular environment and by reaching lower temperatures, it may be possible to attain greater cellular metabolic suppression. This perhaps will extend the allowable times for circulatory arrest procedures. In addition, "bloodless ischemia" may be beneficial in removing both blood substances and formed elements which may mediate organ ischemia. With replacement of blood at warm temperatures, coagulopathy is avoided. This preliminary evidence demonstrates potential in the combination of ultraprofound hypothermia and complete blood component substitution. However, further study is required to confirm the potential of achieving circulatory arrest of longer duration.

A priori information in image analysis: assessment of intensity distribution for definition of shape and size of small vessels.

A method for incorporating prior information in computer-based image analysis is described and critically evaluated. The specific application improves pixel resolution (spot size) and shape estimation of small vessel cross sections in exchange for dynamic range information. The potential subpixel spot size sensitivity for a 16-bit gray scale is better than one part in 32000. The performance of shape recovery is assessed in relation to signal-to-noise ratio, acquired image resolution, vessel shape complexity and aspect ratio. The process is shown to be effective and stable when the signal-to-noise ratio exceeds 10, when the number of pixels across the vessel is two or more, when the vessel contour has as many as six lobes, and when the aspect ratio is in the range 0.2-5.0.