Functions of E2A-HEB heterodimers in T-cell development revealed by a dominant negative mutation of HEB.

Lymphocyte development and differentiation are regulated by the basic helix-loop-helix (bHLH) transcription factors encoded by the E2A and HEB genes. These bHLH proteins bind to E-box enhancers in the form of homodimers or heterodimers and, consequently, activate transcription of the target genes. E2A homodimers are the predominant bHLH proteins present in B-lineage cells and are shown genetically to play critical roles in B-cell development. E2A-HEB heterodimers, the major bHLH dimers found in thymocyte extracts, are thought to play a similar role in T-cell development. However, disruption of either the E2A or HEB gene led to only partial blocks in T-cell development. The exact role of E2A-HEB heterodimers and possibly the E2A and HEB homodimers in T-cell development cannot be distinguished in simple disruption analysis due to a functional compensation from the residual bHLH homodimers. To further define the function of E2A-HEB heterodimers, we generated and analyzed a dominant negative allele of HEB, which produces a physiological amount of HEB proteins capable of forming nonfunctional heterodimers with E2A proteins. Mice carrying this mutation show a stronger and earlier block in T-cell development than HEB complete knockout mice. The developmental block is specific to the alpha/beta T-cell lineage at a stage before the completion of V(D)J recombination at the TCRbeta gene locus. This defect is intrinsic to the T-cell lineage and cannot be rescued by expression of a functional T-cell receptor transgene. These results indicate that E2A-HEB heterodimers play obligatory roles both before and after TCRbeta gene rearrangement during the alpha/beta lineage T-cell development.

Functional replacement of the mouse E2A gene with a human HEB cDNA.

The mammalian E2A, HEB, and E2-2 genes encode a unique class of basic helix-loop-helix (bHLH) transcription factors that are evolutionarily conserved and essential for embryonic and postnatal development. While the structural and functional similarities among the gene products are well demonstrated, it is not clear why deletion of E2A, but not HEB or E2-2, leads to a complete arrest in B-lymphocyte development. To understand the molecular basis of the functional specificity between E2A and HEB/E2-2 in mammalian development, we generated and tested a panel of E2A knockin mutations including subtle mutations in the E12 and E47 exons and substitution of both E12 and E47 exons with a human HEB cDNA. We find that the alternatively spliced E12 and E47 bHLH proteins of the E2A gene play similar and additive roles in supporting B lymphopoiesis. Further, we find that HEB driven by the endogenous E2A promoter can functionally replace E2A in supporting B-cell commitment and differentiation toward completion. Finally, the postnatal lethality associated with E2A disruption is fully rescued by the addition of HEB. This study suggests that the functional divergence among E12, E47, and HEB in different cell types is partially defined by the context of gene expression.

A new three-dimensional postmortem method to study the topography of atherosclerosis using profilometry.

The examination of high resolution casts of atherosclerotic arteries with a surface profile projector provides a 3-dimensional method for the measurement of atherosclerotic lesions. Lesions recorded on casts are found to predict gross and microscopic pathology. The contrast and magnification provided afford a continuum of atherosclerotic grades from the microscopic to macroscopic range. Lesion size and grade can be related to the geometry of the arterial lumen.

Measurement of atherosclerotic luminal irregularity and obstruction by radiographic densitometry.

This report describes a procedure to quantify stenosis and to detect enface plaques in single plane angiograms. We derived an optical density function proportional to local arterial chord length from arteriographs over a wide range of conditions. Standard radiographs were made of silicone rubber bolus used as the radiopaque medium in cadaver arteries. Replicate arterial casts filled with iodine-bearing contrast medium were radiographed for comparison. Identical atherosclerotic areas in both film sets were scanned with a microdensitometer and vessel chord lengths used to construct arterial cross sections for comparison with vessel casts. Cross-sectional areas derived from cadaver and phantom films were highly correlated with those derived from cast slices (r = 0.997). Thus, luminal cross-sectional areas were accurately estimated independent of plaque location, and accuracy of chord length measurement was +/- 0.28 mm in vessels four to 10 mm in diameter.

Primary hypertension: left ventricular mass and function, sympathetic nervous system activity, and therapy.

Left ventricular (LV) mass is a predictor of morbidity in patients with hypertension. To elucidate the mechanisms of left ventricular hypertrophy (LVH) in primary hypertension, we examined the relationships of LV mass, arterial blood pressure, and plasma norepinephrine--as a marker of sympathetic nervous system tone--in three populations of patients with mild or moderate hypertension. We found linking of neural tone with (a) arterial blood pressure in mild and moderate hypertension, (b) LV mass in moderate hypertension, and (c) diastolic function in mild hypertension, suggesting that neural tone is a factor in both the pathogenesis and the sequelae of hypertension. Additionally, elevated norepinephrine level may reflect subtle loss of diastolic function before hypertrophy is manifest. Both nondrug and drug therapy may regress LV mass after blood pressure reduction with disparate effects on neural tone.

Neurohumoral mechanisms and left ventricular hypertrophy: effects of hygienic therapy.

To determine the effects of hygienic (non-drug) therapy on blood pressure (BP) control and its relationship to sympathetic tone and left ventricular mass (LVM) in primary hypertension, plasma norepinephrine (NE) and renin activity (PRA), LVM, and nutritional and behavioral status were assessed in 76 borderline to mild hypertensives. Pretherapy plasma NE was related to diastolic blood pressure (DBP) and PRA (r = 0.24, P less than .05 and r = 0.37, P less than .01, respectively). Plasma NE of high renin patients (221 +/- 52) (mean +/- SD) was greater than that of normal renin patients (159 +/- 61, ng/l, P less than .01). LVM was related to systolic blood pressure (SBP) (P less than .001), DBP (P less than .01) and urinary sodium (P less than .05), and inversely related to PRA (P less than .01). Septal wall thickness was related to hostility (r = 0.42, P less than .05). After seven weeks of hygienic therapy, DBP was reduced by 6 mmHg (P less than .01). The change in SBP was related to baseline plasma NE (P less than .05) and inversely related to LVM (P less than .05). These results suggest that raised sympathetic tone may be a pathogenic factor in primary hypertension and that hygienic therapy lowers BP more effectively in patients with raised sympathetic tone and low LVM.

Abnormal insulin secretion in adult canines.

Insulin secretion after intravenous administration of glucose was studied in fourteen dogs, twelve of which were four years of age or older. Based on several characteristics of insulin secretion kinetics, normal and impaired insulin responses were defined in the experimental subjects. Among those, five exhibited a marked depression of the initial stage of insulin release which was followed by a stage of relative hyperinsulinemia. Similarities between canine and human insulin secretion patterns were noted, and the potential usefulness of the procedure for the early detection of canine diabetes mellitus discussed.

A novel role for HEB downstream or parallel to the pre-TCR signaling pathway during alpha beta thymopoiesis.

TCR gene rearrangement and expression are central to the development of clonal T lymphocytes. The pre-TCR complex provides the first signal instructing differentiation and proliferation events during the transition from CD4-CD8-TCR- double negative (DN) stage to CD4+CD8+ double positive (DP) stage. How the pre-TCR signal leads to downstream gene expression is not known. HeLa E-box binding protein (HEB), a basic helix-loop-helix transcription factor, is abundantly detected in thymocytes and is thought to regulate E-box sites present in many T cell-specific gene enhancers, including TCR-alpha, TCR-beta, and CD4. Targeted disruption of HEB results in a 5- to 10-fold reduction in thymic cellularity that can be accounted for by a developmental block at the DN to DP stage transition. Specifically, a dramatic increase in the CD4low/-CD8+CD5lowHSA+TCRlow/- immature single positive population and a concomitant decrease in the subsequent DP population are observed. Adoptive transfer test shows that this defect is cell-autonomous and restricted to the alpha beta T cell lineage. Introduction of an alpha beta TCR transgene into the HEBko/ko background is not sufficient to rescue the developmental delay. In vivo CD3 cross-linking analysis of thymocytes indicates that TCR signaling pathway in the HEBko/ko mice appears intact. These findings suggest an essential function of HEB in early T cell development, downstream or parallel to the pre-TCR signaling pathway.

Flow field and mass transport analysis in arteries with longitudinal ridges.

Recent observations have indicated that the earliest lesions of atherosclerosis frequently take the form of longitudinal and helical ridges in arteries of man. Since longitudinal vortices are expected to be present in the troughs between the longitudinal ridges, an analysis was carried out to investigate the three-dimensional flow field and the trasport of lipoproteins and oxygen molecules to arterial walls in the presence of such vortices. The calculations revealed that local hypoxia and lipoprotein accumulation may occur at the ridges, leading to subsequent intimal thickening and ridge growth. Higher shear stresses, calculated in the troughs between ridges, may also partially damage the endothelium and lead to intimal thickening and subsequent merging of the ridges. Meaningful measurements are needed in vivo to determine the strength of the vortices, their time-varying behavior, and the actual transverse variations in shear stress, oxygen transport, and lipoprotein accumulation from trough to ridge regions, in order to appraise the present findings and to learn more about the observed progressive thickening and widening of ridges with increasing degrees of intimal thickening and atherosclerosis.

Surface characteristics of normal and atherosclerotic human arteries including observations suggesting interaction between flow and intimal morphology.

High resolution casts of the femoral and coronary arteries were made at physiologic pressure in 44 cadavers. A magnifying surface and profile analyzer were used for examination of the small surface characteristics of the casts. Corresponding portions of the arterial wall were compared by gross and microscopic examination. In sections without pathologic evidence of atherosclerosis, the cast surface characteristically demonstrated small longitudinal plicae, similar in size to those previously reported in studies with the scanning electron microscope. With increasing degrees of intimal thickening and atherosclerosis, longitudinal ridges were seen. In complete involvement of the intima, the ridge pattern was replaced by a locally smooth, grossly irregular surface. The orientation of ridges at curves and near branches and discrete plaques suggested the direction of secondary surface flow streamlines expected of these areas on the basis of hydrodynamic theory. Therefore, there may be formative interactions between blood flow and wall structure on a more local basis than previously surmised.

Noradrenergic hyperactivity in primary hypertension; central and peripheral markers of both behavioral pathogenesis and efficacy of sympatholytic and relaxation therapy.

The effects of clonidine or relaxation therapy were determined in two separate groups of patients with primary hypertension. Ten patients were treated with clonidine monotherapy for 3 months. There were concurrent reductions of blood pressure, plasma and CSF norepinephrine, all p less than 0.01. The changes of blood pressure and norepinephrine were correlated, p less than 0.05 and 0.01, respectively. Thirty patients received hygienic instructions, and 17 of them had relaxation training in addition. Relaxation lowered blood pressures, p less than 0.01, the reduction of blood pressure was related to baseline plasma norepinephrine, p less than 0.05, and greater in patients with "raised" plasma norepinephrine, p less than 0.02. Plasma norepinephrine was lowered after hygienic therapy, p less than 0.05, the change was not significant after relaxation training. Arterial pressure elevation appears to be related to raised plasma norepinephrine. This noradrenergic hyperactivity is a marker for blood pressure responsiveness to sympatholytic therapy with clonidine or relaxation techniques.

Total serum cholesterol and plasma lipoprotein cholesterol concentrations in cord sera of newborns from hispanic mothers with gestational diabetes.

The objective of this research was to determine if gestational diabetes has an impact on the plasma lipoprotein profiles and/or cholesterol content of the plasma lipoproteins in newborns. Very low plus low density lipoproteins and high density lipoproteins in cord sera of newborns from gestational diabetic and non-diabetic mothers were isolated by heparin-affinity chromatography, and the cholesterol content in these plasma lipoprotein fractions were measured with an enzymatic assay. There were no significant differences in baby weights and total serum cholesterol levels between the two experimental groups. High density lipoprotein cholesterol levels (p less than 0.05) were significantly lower while very low plus low density lipoprotein cholesterol concentrations (p less than 0.05), very low plus low density lipoprotein cholesterol/high density lipoprotein cholesterol ratios (p less than 0.025), and total serum cholesterol/high density lipoprotein cholesterol ratios (p less than 0.025) were significantly elevated in newborns of diabetic mothers. These results suggest that gestational diabetes altered neonatal plasma lipoprotein metabolism.

Central and peripheral noradrenergic tone in primary hypertension.

The contents of norepinephrine (NE), epinephrine (E), dopamine (DA), normetanephrine (NMN), and 4-hydroxy-3-methoxyphenylethylene glycol (MHPG) were measured in the plasma and cerebrospinal fluid (CSF) of 66 patients with primary hypertension and 24 patients with normal blood pressure and minor neurological disorders. Plasma and CSF NE and NMN concentrations were raised in the hypertensive patients. The plasma and CSF NE levels and arterial blood pressure of a small subset of hypertensive patients were normalized after clonidine therapy. In hypertensive patients the content of DA was lower and the ratio of NE/DA was greater; CSF and plasma NE contents were related to the level of arterial blood pressure; and the content of MHPG in CSF was linked strongly with NE content in plasma and CSF and to the level of arterial blood pressure. Thus both central sympathetic nerve tone and peripheral sympathetic nerve tone were enhanced in young patients with uncomplicated hypertension. The elevated levels of neurohormones and their metabolites in some patients with primary hypertension may be related to increased synthesis and release of neural NE and may be pathogenic in the blood pressure elevation.

The rate of atherosclerosis change during treatment of hyperlipoproteinemia.

The rate of change of femoral atherosclerosis has been determined in 25 treated hyperlipoproteinemic patients. There were 13 Type II patients and 12 Type IV patients. Serial angiograms had been performed at an interval averaging 13 months. Film densities were analyzed by digigal image processing to yield a computer estimate of atherosclerosis (CEA). Serial measurements of CEA on each patient were used to determine atherosclerosis percent change per month in that patient. CEA percent change per month was significantly correlated ;ith triglyceride and cholesterol level. Lower lipid levels were associated with more rapid regression. When hyperlipoproteinemic types were considered separately, significant single correlates were confined to Type IV. Triglyceride level and CEA/age significantly predicted atherosclerosis change rate and accounted for 72% of the variability observed in Type IV patients.

Pulsed doppler echocardiographic detection of mitral regurgitation in mitral valve prolapse: correlation with cardiac arrhythmias.

The incidence of mitral regurgitation in mitral valve prolapse was studied retrospectively in 88 consecutive hospital based patients with mitral valve prolapse using pulsed Doppler echocardiography. Pulsed Doppler findings of turbulence in the left atrium were predictive of angiographic findings of mitral regurgitation in 31 patients. On 24-hr Holter monitoring, patients with mitral valve prolapse and mitral insufficiency had a higher prevalence of ventricular arrhythmias than patients without mitral insufficiency (61 vs 22%; P less than 0.02). Detection of mitral insufficiency identified individuals with mitral valve prolapse who are at higher risk of ventricular arrhythmias.

Excess PTH in CRF induces pulmonary calcification, pulmonary hypertension and right ventricular hypertrophy.

Calcification of the lungs occurs in chronic renal failure (CRF) and may adversely affect both pulmonary and right ventricular function. The present study examined the role of excess parathyroid hormone (PTH) in the genesis of pulmonary calcifications in dogs with experimental CRF and evaluated calcium content of lungs, diffusing lung capacity (DCO), mean pulmonary artery pressure (MPAP), right ventricular pressure (RVP), and right ventricular hypertrophy (RVH) in six normal, six with CRF, and six thyroparathyroidectomized (CRF-PTX) dogs. CRF-PTX animals were maintained normocalcemic and euthyroid. The degree and duration of CRF were not different between the two groups with CRF. The mean value of the serum PTH in CRF dogs was 166 +/- 42 microliters Eq/ml, but was undetectable in CRF-PTX animals. Thallium scan provided evidence consistent with RVH in CRF dogs but not in CRF-PTX animals. Calcium content of lungs was markedly elevated in CRF dogs (7656 +/- 1657 mg/kg dry wt) but modestly increased in CRF-PTX (1057 +/- 117 mg/kg dry wt) as compared to normal (673 +/- 34 mg/kg dry wt). RVP and MPAP were significantly (P < 0.01) higher and DCO significantly lower in CRF dogs than in normal or CRF-PTX animals. These parameters were not different in the latter two group of dogs. In three additional dogs with CRF of one year duration which were followed for an additional year after parathyroidectomy, these abnormalities were corrected.(ABSTRACT TRUNCATED AT 250 WORDS)

Regression and progression of early femoral atherosclerosis in treated hyperlipoproteinemic patients.

Femoral angiograms were done to evaluate change in early atherosclerosis in 12 patients with type IV hyperlipoproteinemia and 13 with type II hyperlipoproteinemia. The patients' average age was 48 years; only one had claudication. Elevated blood lipids and blood pressure were treated with drugs and diet. Repeat angiograms after an interval of 13 months showed regression of atherosclerosis in nine patients, no change in three, and progression in 13. Comparison of preangiogram levels with average levels between angiograms showed significant reduction in serum cholesterol, triglyceride, and blood pressure in the group with lesion improvement but not in the group with lesion progression. Sporadic examples of human atherosclerosis regression are known, but most other studies in man indicate only atherosclerosis progression. Our different result appears due to our selection of patients and radiographic method. We have studied patients with earlier atherosclerosis than previous workers, using a radiographic procedure more sensitive to small changes in lesions.