A role for histone acetylation in the developmental regulation of VDJ recombination.

VDJ recombination is developmentally regulated in vivo by enhancer-dependent changes in the accessibility of chromosomal recombination signal sequences to the recombinase, but the molecular nature of these changes is unknown. Here histone H3 acetylation was measured along versions of a transgenic VDJ recombination reporter and the endogenous T cell receptor alpha/delta locus. Enhancer activity was shown to impart long-range, developmentally regulated changes in H3 acetylation, and H3 acetylation status was tightly linked to VDJ recombination. H3 hyperacetylation is proposed as a molecular mechanism coupling enhancer activity to accessibility for VDJ recombination.

Accessibility control of T cell receptor gene rearrangement in developing thymocytes. The TCR alpha/delta locus.

The joining of T cell receptor (TCR) and immunoglobulin (Ig) gene segments through the process of V(D)J recombination occurs in a lineage-specific and developmental-stage-specific way during the early stages of lymphocyte development. Such developmental regulation is thought to be mediated through the control of gene segment accessibility to the recombinase. We have studied the regulation of V(D)J recombination at the TCR alpha/delta locus, because this locus provides a fascinating model in which distinct sets of gene segments are activated at different stages of T cell development. The transcriptional enhancers Edelta and Ealpha have been implicated as critical regulators that, in conjunction with other cis-acting elements, confer region-specific and developmental-stage-specific changes in gene segment accessibility within TCR alpha/delta locus chromatin. Current work suggests that they may do so by functioning as regional modulators of histone acetylation.

Developmental regulation of V(D)J recombination at the TCR alpha/delta locus.

The T-cell receptor (TCR) alpha/delta locus includes a large number of V, D, J and C gene segments that are used to produce functional TCR delta and TCR alpha chains expressed by distinct subsets of T lymphocytes. V(D)J recombination events within the locus are regulated as a function of developmental stage and cell lineage during T-lymphocyte differentiation in the thymus. The process of V(D)J recombination is regulated by cis-acting elements that modulate the accessibility of chromosomal substrates to the recombinase. Here we evaluate how the assembly of transcription factor complexes onto enhancers, promoters and other regulatory elements within the TCR alpha/delta locus imparts developmental control to VDJ delta and VJ alpha rearrangement events. Furthermore, we develop the notion that within a complex locus such as the TCR alpha/delta locus, highly localized and region-specific control is likely to require an interplay between positive regulatory elements and blocking or boundary elements that restrict the influence of the positive elements to defined regions of the locus.

Effects of total parental nutrition (TPN) during high-dose interleukin-2 treatment for metastatic cancer.

Patients treated with high doses of interleukin-2 (IL-2) develop profound anorexia, malaise, loss of energy, mucositis, nausea, and vomiting, which may contribute to poor nutrition. We hypothesized that total parenteral nutrition (TPN) administration would ameliorate these changes and could improve fluid and electrolyte balance. A retrospective analysis of protein and energy intake was performed in 21 sequential patients who received a normal diet (controls) and 16 subsequent patients who received TPN during IL-2 treatment. The effect of TPN on laboratory abnormalities induced by IL-2 was also evaluated. Within 24 h of starting IL-2, mean energy intake declined to 2.5-2.8 kcal/kg in controls in contrast to the energy intake of 25-29 kcal/kg in patients receiving TPN. Protein nutrition was affected in a similar fashion, with a markedly lower protein intake in controls (0.08-0.12 g/kg) than in the TPN group (1.02-1.10 g/kg). TPN improved serum calcium and potassium concentrations, particularly during spontaneous diuresis after completion of IL-2 treatment. Unexpectedly, TPN decreased the frequency and severity of cholestatic jaundice caused by IL-2. Patients receiving TPN had an increased propensity for hyperglycemia and hypophosphatemia. High-dose intravenous bolus IL-2 therapy resulted in a markedly negative nutritional balance in control patients. A brief period of TPN during IL-2 treatment was well tolerated and corrected calorie and protein malnutrition. TPN administration also improved control of serum electrolytes. TPN did not adversely affect tumor progression or patient survival.

Enhancer control of local accessibility to V(D)J recombinase.

We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delta gene minilocus. This transgene is composed of unrearranged variable (Vdelta and Vdelta2), diversity (Ddelta3), joining (Jdelta1 and Jdelta3), and constant (Cdelta) gene segments. Previous data indicated that with the TCR delta enhancer (Edelta) present in the Jdelta3-Cdelta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that Edelta is necessary for J segment but not D segment accessibility and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype. In this study, we tested this notion by using ligation-mediated PCR to assess the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of Ddelta3 and 5' of Jdelta1. In five lines of mice carrying multicopy integrants of constructs that either lacked Edelta or carried an inactivated Edelta, the frequency of DSBs 5' of Jdelta1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of Ddelta3 was unaffected. We interpret these results to indicate that Edelta is required for Jdelta1 but not Ddelta3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than Edelta must maintain Ddelta3 in an accessible state in the absence of Edelta. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3' of Ddelta3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.

Translation of the mRNA for the sporulation gene spoIIID of Bacillus subtilis is dependent upon translation of a small upstream open reading frame.

We report the existence of a small open reading frame (usd) that is located between the promoter and coding sequence for the sporulation gene spoIIID in Bacillus subtilis. The mRNA from the usd-spoIIID operon contains an inverted repeat sequence that is predicted to form a stem-loop structure that would sequester the ribosome binding site for spoIIID. A mutation eliminating the ribosome binding site for the upstream open reading frame caused an oligosporogenous phenotype and interfered with the translation, but not the transcription, of the downstream gene spoIIID. We propose that efficient synthesis of SpoIIID requires that the putative stem-loop structure be disrupted by translation through the upstream open reading frame.

Evidence for sex steroid inhibition of lipoprotein lipase in men: comparison of abdominal and femoral adipose tissue.

Plasma estradiol has been suggested to suppress adipose tissue lipoprotein lipase (LPL) activity in women. The present study explores the regulation of LPL by sex steroids in sedentary obese men (N = 24) at their usual weight. Femoral adipose tissue LPL activity, eluted with serum and heparin or extracted with detergent, showed significant inverse correlations with plasma levels of testosterone, bioavailable testosterone, dihydrotestosterone, and estradiol. Both measures of femoral LPL activity were also correlated with the weight change occurring despite efforts to maintain a constant weight. Abdominal LPL activity showed significant but weaker inverse correlations with bioavailable testosterone only. Multivariate analysis of potential predictors for eluted femoral LPL activity showed that plasma testosterone, dihydrotestosterone, and estradiol were interdependent, whereas the rate of weight change was an independent variable. In the regression equation, only bioavailable testosterone and weight change were retained, explaining 63% of the variability (R = .79, P = .0002). These results suggest that sex steroids suppress adipose tissue LPL activity in men, and more so in the thigh than in the abdomen, thereby possibly contributing to a central fat accumulation. The data are compatible with a model from male animals suggesting that testosterone effects on adipose tissue LPL are mediated by estradiol formed locally.

Effects of dairy products on bone and body composition in pubertal girls.

OBJECTIVE: To study the effect of calcium supplementation with dairy products on the bone and body composition of pubertal girls. DESIGN: Randomized control study with 12-month follow-up. SETTING: General community. SUBJECTS: Forty-eight white girls whose mean age was 11 years and sexual development at Tanner stage 2. INTERVENTION: One group's diet was supplemented with dairy products to the recommended dietary allowance of 1200 mg calcium daily. The other group ate their usual diet. MAIN OUTCOME MEASURES: Bone mineral content and density were measured at the radius, femoral neck, lumbar spine, and total body bone mineral by single-photon and dual-energy x-ray absorptiometry at the start of the study and after 3, 6, 9, and 12 months. Body composition (lean body mass and body fat) was measured by dual-energy x-ray absorptiometry at the same intervals. Serum calcium, phosphate, 25-hydroxyvitamin D, 1,25-dihydroxyvitamin D, alkaline phosphatase, magnesium, and albumin concentrations were determined at the start and end of the study. The urinary calcium/creatinine ratio and hydroxyproline concentration were also determined. RESULTS: The dairy group had higher intakes of calcium, phosphate, vitamin D, and protein than control subjects. The dairy group had significantly greater increases during the 1-year study in bone mineral density at the lumbar spine bones (22.8% +/- 6.9% vs 12.9% +/- 8.3%) and in total body bone mineral (14.2% +/- 7.0% vs 7.6% +/- 6.0%) than control subjects. Dietary calcium, phosphate, vitamin D, and protein intakes were associated with the lumbar bone density and total body bone calcium. There were no differences in serum or urinary biochemical values between the two groups at the start or end of the study. CONCLUSIONS: Young girls whose dietary calcium intake was provided primarily by dairy products at or above the recommended dietary allowances had an increased rate of bone mineralization. Increased intake of dairy foods did not increase overall total or saturated fat intake and was not associated with excessive weight gain or increased body fat.

Health care professionals' accuracy in predicting patients' preferred code status.

BACKGROUND: In spite of the emphasis on physician and patient communication in the new guidelines for the use of do-not-resuscitate orders published by the American Medical Association, informal information indicates that physicians and other health care professionals often formulate code status decisions without formal knowledge of the patient's wishes. The purpose of this study was to determine how accurately health care professionals are able to predict a patient's desired code status given a profile of the patient's medical history. METHODS: A consecutive sample of physicians and other health care professionals attending on-site primary care and long-term rehabilitation staff meetings were asked to participate in the study. Subjects read profiles of actual patients and attempted to predict the patients' desired code status. Subjects also highlighted factors of the patient profile that they deemed important in predicting each patient's desired code status. RESULTS: For the 12 patient profiles examined, the respondents accurately estimated patients' desired code status an average of only 6.5 times. Patient ability to perform the basic activities of daily living was the patient profile factor cited most frequently as influential in determining code status. CONCLUSIONS: Given only clinical and demographic data, health care professionals are only slightly better than chance in determining patients' desired code status. Health care professionals working with long-term care patients should become familiar with individual patient's values and desires for code status decisions.

Bone mineral status in children with phenylketonuria--relationship to nutritional intake and phenylalanine control.

The mineral status in phenylketonuria (PKU) was measured by single-photon densitometry of the distal radius and plasma concentrations in 26 subjects. Bone mineral content increased normally with age in the younger children despite strict dietary restrictions. Subjects aged greater than 8 y, however, were frequently below the normal curve for bone mineral content. Blood phenylalanine concentrations were significantly higher in the older group of subjects and this correlated with decreased compliance with dietary prescriptions. PKU children had significantly decreased plasma concentrations of alkaline phosphatase, magnesium, and parathyroid hormone. Subnormal concentrations of plasma zinc and plasma and red blood cell (RBC) copper were common, but RBC zinc was normal. We conclude that compliance with dietary therapy for PKU is associated with normal bone mineral development in young children. Older patients with PKU who follow the diet less carefully are at risk for low bone mineral content.

Evidence for cytokine-inducible nitric oxide synthesis from L-arginine in patients receiving interleukin-2 therapy.

An interferon-gamma, tumor necrosis factor, and interleukin-1-inducible, high-output pathway synthesizing nitric oxide (NO) from L-arginine was recently identified in rodents. High-dose interleukin-2 (IL-2) therapy is known to induce the same cytokines in patients with advanced cancer. Therefore, we examined renal cell carcinoma (RCC; n = 5) and malignant melanoma (MM; n = 7) patients for evidence of cytokine-inducible NO synthesis. Activity of this pathway was evaluated by measuring serum and urine nitrate (the stable degradation product of NO) during IL-2 therapy. IL-2 administration caused a striking increase in NO generation as reflected by serum nitrate levels (10- and 8-fold increase [P less than 0.001, P less than 0.003] for RCC and MM patients, respectively) and 24-h urinary nitrate excretion (6.5- and 9-fold increase [both P less than 0.001] for RCC and MM patients, respectively). IL-2-induced renal dysfunction made only a minor contribution to increased serum nitrate levels. Metabolic tracer studies using L-[guanidino-15N2]arginine demonstrated that the increased nitrate production was derived from a terminal guanidino nitrogen atom of L-arginine. Our results showing increased endogenous nitrate synthesis in patients receiving IL-2 demonstrate for the first time that a cytokine-inducible, high-output L-arginine/NO pathway exists in humans.

Changes in lipid and lipoprotein levels and body weight in Tarahumara Indians after consumption of an affluent diet.

BACKGROUND: Major new public health problems occur in developing countries as they become more affluent and change their traditional dietary patterns. To study this phenomenon in microcosm, we substituted an "affluent" diet for the traditional diet of a group of Tarahumara Indians, a Mexican people known to consume a low-fat, high-fiber diet and to have a very low incidence of risk factors for coronary heart disease. METHODS: Thirteen Tarahumara Indians (five women and eight men [including one adolescent]) consumed their traditional diet (2700 kcal per day) for one week, and were then fed a diet typical of affluent societies, which contained excessive calories (4100 kcal per day), total fat, saturated fat, and cholesterol, for five weeks. RESULTS: After five weeks of consuming the affluent diet, the subjects' mean (+/- SE) plasma cholesterol level increased by 31 percent, from 121 +/- 5 to 159 +/- 6 mg per deciliter (3.13 +/- 0.13 to 4.11 +/- 0.16 mmol per liter, P less than 0.001). The increase in the plasma cholesterol level was primarily in the low-density lipoprotein (LDL) fraction, which rose 39 percent, from 72 +/- 3 to 100 +/- 4 mg per deciliter (1.86 +/- 0.08 to 2.59 +/- 0.10 mmol per liter, P less than 0.001). High-density lipoprotein (HDL) cholesterol, usually low in this population, increased by 31 percent, from 32 +/- 2 to 42 +/- 3 mg per deciliter (0.83 +/- 0.05 to 1.09 +/- 0.08 mmol per liter). Consequently, the ratio of LDL to HDL levels changed little (2.25 with the base-line diet and 2.38 with the affluent diet). Plasma triglyceride levels increased by 18 percent, from 91 +/- 8 to 108 +/- 11 mg per deciliter (1.03 +/- 0.09 to 1.22 +/- 0.12 mmol per liter, P less than 0.05), with a significant increase in the very-low-density lipoprotein triglyceride fraction. All the subjects gained weight, with a mean increase of 3.8 kg (7 percent). CONCLUSIONS: When Tarahumara Indians from a population with virtually no coronary risk factors consumed for a short time a hypercaloric diet typical of a more affluent society, they had dramatic increases in plasma lipid and lipoprotein levels and body weight. If sustained, such changes might increase their risk of coronary heart disease.

Sluggish sitosterol turnover and hepatic failure to excrete sitosterol into bile cause expansion of body pool of sitosterol in patients with sitosterolemia and xanthomatosis.

Sitosterolemia and xanthomatosis are characterized by the development of tendon and tuberous xanthomas at an early age and premature atherosclerosis despite normal plasma cholesterol concentrations. The reason(s) for the xanthoma formation and premature atherosclerosis are not clearly understood. The accumulation of sitosterol in the tissues of these patients could be due to increased uptake of low density lipoprotein (LDL) via LDL receptors because of an expanded sitosterol pool caused by sluggish turnover and decreased excretion of sitosterol into bile and feces coupled with the hyperabsorption of sitosterol. We have studied sitosterol and cholesterol turnovers, the biliary and fecal excretion of neutral and acidic steroids, and the response of plasma sterol (sitosterol and cholesterol) levels to either a sterol-free formula or low plant sterol diet in three patients. The average half-life of the first exponential (tA1/2) for sitosterol was 9.2 +/- 3.3 (mean +/- SD) days, which was more than twice that in normal humans. The second exponential (tB1/2) was 156 +/- 108 days, which was nearly 10 times longer than that for normal humans. The average cholesterol production rate in pool A was 0.87 g/day, which is about 40% of that in normal humans. Cholesterol synthesis measured by the sterol balance technique was also found to be about 70% lower than that for normal humans. In two patients fed a sterol-free formula diet, by 25 days their plasma sitosterol and cholesterol levels had decreased by 42% and 36%, respectively. However, in one patient plasma sitosterol and cholesterol concentrations remained unchanged while on the low plant sterol-mixed food diet.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of an elemental diet with and without gluten on disease activity in dermatitis herpetiformis.

Elemental diets are reported to decrease activity of patients with dermatitis herpetiformis. We tested the hypothesis that gluten, given in addition to an elemental diet, is responsible for the intestinal abnormalities, cutaneous immunoreactant deposition, and skin disease activity in dermatitis herpetiformis. At entry eight patients with dermatitis herpetiformis, who were consuming unrestricted diets, were stabilized on their suppressive medications at dosage levels that allowed individual lesions to erupt. Six patients were then given an elemental diet plus 30 of gluten for 2 weeks, followed by the elemental diet alone for 2 weeks. Conversely, two patients received an elemental diet alone for 2 weeks followed by an elemental diet plus gluten during the final 2 weeks. Small bowel biopsies, skin biopsies, and clinical assessments were done at 0, 2, and 4 weeks. Suppressive medication dose requirement decreased over the 4 weeks by a mean of 66%. Six of eight subjects significantly improved clinically during the gluten-challenge phase of the elemental diet and all were improved at the end of the study. The amount of IgA in perilesional skin did not change significantly, but the amount of C3 increased in five of seven evaluable subjects after gluten challenge. Circulating anti-gluten and anti-endomysial antibodies were not significantly affected by the diets. All subjects completing evaluable small bowel biopsies (seven of seven) demonstrated worsening of their villus architecture (by scanning electron microscopy and intraepithelial lymphocyte counts) during gluten challenge and improvement (six of six subjects) after 2 weeks of elemental dietary intake. We conclude that 1) there is a significant improvement in clinical disease activity on an elemental diet, independent of gluten administration, 2) small bowel morphology improves rapidly on an elemental diet, and 3) complement deposition but neither IgA deposition nor circulating antibody levels correlate with gluten intake. It seems likely that dietary factors other than gluten are important in the pathogenesis of the skin lesions in dermatitis herpetiformis.

Family-oriented nutrition intervention for a lipid clinic population.

We have developed a unique, family-oriented approach to lowering plasma cholesterol concentrations in persons with familial hyperlipidemias. The approach includes individual clinic visits and group nutrition classes and uses dietary goals outlined in The New American Diet. A series of 13 nutrition classes is presented to small groups, usually composed of relatives from pedigrees with familial hypercholesterolemia or other familial hyperlipidemias. Dietary action goals, cooking demonstrations, food tasting, and finger-stick plasma cholesterol determinations are important components of the classes. Problem-solving discussion is encouraged in the group. Over the past 4 years, 143 hyperlipidemic individuals, along with at least 94 unaffected family members, have participated in 31 groups, which have met for at least six classes. Many clinic participants lower plasma cholesterol by 20% or more. Keys to the success of this program include emphasizing dietary therapy, using the family setting for nutrition intervention, providing hands-on experience with food and recipes, promoting problem solving for dietary action goals, measuring blood cholesterol during classes, and encouraging long-term follow-up for participants with physicians and dietitians.

Effects of a fat-containing meal on sex hormones in men.

The effect of a fat-containing meal on plasma sex steroid concentrations was investigated in normal men. After an overnight fast on two separate occasions, subjects ingested a liquid meal containing either a nonnutritive sweetener (control), or isocaloric meals of mixed calorie sources with either high-fat content or mixed carbohydrate and protein with minimal fat. The order of the meals was alternated. Blood samples were collected at 15-minute intervals and pooled each hour. Sampling began at 7:00 AM and the test meal was ingested at 8:00 AM. Sex steroids, including estrone, estradiol, testosterone, and dihydrotestosterone (DHT), sex hormone-binding globulin (SHBG) capacity, free testosterone concentration, and luteinizing hormone (LH) were determined by either specific radioimmunoassay or dialysis. The fat-containing meal, but not the nonnutritive or mixed carbohydrate and protein meal, resulted in a significant (P less than .01) reduction in total and free testosterone. Estrogens and luteinizing hormone were unaffected by either meal. This is the first documentation, to our knowledge, of the acute effect of a fat-containing meal on sex steroid concentrations in blood. Our observations suggest that a fat-containing meal reduces testosterone concentrations without affecting luteinizing hormone. This might indicate that fatty acids modulate testosterone production by the testes.

Effects of increased dietary calcium intake upon the calcium and bone mineral status of lactating adolescent and adult women.

We studied the effectiveness of an increased calcium (Ca) diet in preventing bone mineral loss in lactating adolescent mothers. Three groups of lactating women were studied: 15 control adolescents consuming their usual Ca diet (900 mg/d), 21 experimental adolescents consuming a high-Ca diet (greater than 1600 mg/d), and 12 adults. At 2 and 16 wk postpartum, serum calcium, phosphate, magnesium, albumin, alkaline phosphatase, vitamin D, parathyroid hormone (PTH), and calcitonin (CT) were determined. Bone mineral analyses were performed by photon absorptiometry. By 16 wk the control adolescent group had a 10% decrease in bone mineral content (BMC) and increased PTH and CT. The experimental adolescent and adult groups had no significant change in BMC during the study. There was a positive correlation (r = 0.45, p less than 0.01) between dietary Ca intake and BMC in all adolescents. Data suggest that bone loss during lactation in adolescents may be prevented with adequate dietary Ca intakes.

The absorption of cholesterol and the sterol balance in the Tarahumara Indians of Mexico fed cholesterol-free and high cholesterol diets.

The Tarahumara Indians of Mexico are habituated to a very low cholesterol, low fat diet and have lifelong low plasma cholesterol concentrations. To study cholesterol metabolism in these unusual people, 8 Tarahumara men were fed sequentially a cholesterol-free diet and then a diet containing 900 mg cholesterol under controlled conditions. The intestinal absorption of cholesterol, fecal steroid excretion and sterol balance were determined. During the high cholesterol diet period, the plasma cholesterol level increased from 113 +/- 8 mg/dl to 147 +/- 11 mg/dl (means +/- SD). Cholesterol biosynthesis decreased from 14.0 +/- 0.7 to 7.1 +/- 1.0 mg/kg/day (means +/- SE). The intestinal absorption of cholesterol was 27.7 +/- 6.7% (means +/- SE) during both dietary periods. Compared to other cultures, Tarahumaras had a reduced ability to absorb dietary cholesterol and higher total sterol turnover primarily because of an increased bile acid output. The total sterol disposition over three weeks of the high cholesterol diet accounted for all the absorbed dietary cholesterol.

Hypercholesterolemia associated with alpha-1 antitrypsin deficiency and hepatitis: lipoprotein and apoprotein determinations, sterol balance and treatment.

Lipid metabolism was investigated in a 4-year-old boy with alpha-1 antitrypsin deficiency (ZZ phenotype) and liver disease. Plasma cholesterol and triglyceride levels were 604 mg/dl and 336 mg/dl respectively. Both parents had normal plasma lipids. Lipoprotein X was present at a concentration of 855 mg/dl and levels of apoproteins A-I, A-II, B and C-III were elevated. The plasma free fatty acid pattern was normal. Plasma cholesterol esterification was greatly depressed. Cholesterol absorption on two occasions was reduced about 13% compared with adult controls. Neutral and total steroid excretion was normal with increased excretion of bile acids. A low-cholesterol, low-fat diet reduced plasma cholesterol to 374 mg/dl and triglyceride to 236 mg/dl in two months. Cholesterol and lipoprotein X concentrations were elevated far out of proportion to the severity of the liver disease (total bilirubin 3.7 mg/dl, SGOT 280 IU/L). This suggests that lipoprotein metabolism in patients with this disorder is unusual and may differ from the derangements seen in other forms of liver disease.