Total but not resting energy expenditure is increased in infants with ventricular septal defects

OBJECTIVE: The purpose of this study was to determine the effect of left-to-right shunting on the resting energy expenditure (REE), total energy expenditure (TEE), and energy intake in a group of 3- to 5-month-old infants with moderate to large unrepaired ventricular septal defects (VSDs) compared with age-matched, healthy infants. METHODS: Eight infants with VSDs and 10 healthy controls between 3 to 5 months of age participated in the study. Indirect calorimetry was used to measure REE and the doubly-labeled water method was used to measure TEE and energy intake. An echocardiogram and anthropometric measurements were performed on all study participants. Daily urine samples were collected at home for 7 days. Samples were analyzed by isotope ratio mass spectrometry. Data were compared using analysis of variance. RESULTS: No significant differences were found in REE (VSD, 42.2 +/- 8.7 kcal/kg/d; control, 43.9 +/- 14.1 kcal/kg/d) or energy intake (VSD, 90.8 +/- 19.9 kcal/kg/d; control, 87.1 +/- 11.7 kcal/kg/d) between the groups. The percent total body water was significantly higher in the VSD infants and the percent fat mass was significantly lower. TEE was 40% higher in the VSD group (VSD, 87.6 +/- 10.8 kcal/kg/d; control, 61.9 +/- 10.3 kcal/kg/d). The difference between TEE and REE, reflecting the energy of activity, was 2.5 times greater in the VSD group. CONCLUSIONS: REE and energy intake are virtually identical between the two groups. Despite this, infants with VSDs have substantially higher TEE than age-matched healthy infants. The large difference between TEE and REE in VSD infants suggests a substantially elevated energy cost of physical activity in these infants. These results demonstrate that, although infants with VSDs may match the energy intake of healthy infants, they are unable to meet their increased energy demands, resulting in growth retardation.     

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.      

STRESS FRACTURES: EFFECT OF PRIOR PHYSICAL ACTIVITY,SPORTS PARTICIPATION AND MILITARY TRAINING

A study was carried out to find out the effects of prior physical activity, sports participation and prior military training on the incidence of stress fractures among Gentlemen Cadets (GC''s) undergoing military training at Indian Military Academy (IMA). One thousand and fourteen GC''s were followed up for a period of 12 weeks. Thirty-seven GC''s developed stress fractures during the study period. The incidence of stress fractures was significantly higher in GC''s without any prior military training (p=0.0009). They were compared with 100 healthy controls drawn from the study population to study the influence of the other mentioned factors. There was no significant association between prior physical activity and stress fractures (OR=0.74, 95% CL=0.26 to 2.05, p=0.688). There was also no significant relationship between sports participation and stress fractures (OR=0.79. 95% CCL=0.35 to 1.81, p=0.684).KEY WORDS: Risk factors, Stress fractures     

Ultrasound evaluation of the multifetal gestation

With the increasing incidence of multifetal gestations, it is essential for the clinician to appreciate the benefit of ultrasound evaluation in these pregnancies. Multifetal gestation pregnancies are at increased risk for a range of both antepartum and intrapartum complications, such as intrauterine growth restriction, premature delivery, congenital anomalies, cord accidents, malpresentations, placenta previa, and abruptio placentae. First-trimester ultrasonic evaluation, amniotic fluid assessment, monitoring of fetal growth, diagnosing the twin-to-twin transfusion syndrome, and assessment of fetal anomalies will be reviewed. An early and accurate assessment of amnionicity and chorionicity are paramount in these gestations. The impact of chorionicity, in particular, can have profound consequences in the management of multifetal gestations. The determination of chorionicity onultrasonic evaluation in a multifetal gestations. The determination of chorionicity on ultrasonic evaluation in a multifetal pregnancy should be determined in a systematic way between 10-14 weeks' gestation. The amniotic fluid volume should be routinely assessed when performing an ultrasonic evaluation in a multifetal pregnancy. Amniotic fluid changes may serve as the only useful indicator to a potential pathological condition. In addition, evaluation of fetal growth in twins is essential, because these pregnancies are at increased risk for growth restriction and increased perinatal mortality rates compared to singletons. Finally, a careful anatomical evaluation in mulitifetal gestation is important because twin pregnancies have higher rates of anomalies than their singleton counterparts.     

Cytogenetic and histologic correlations in malignant lymphoma

Although a number of studies have indicated correlations between histologic subtypes of tumors and certain nonrandom chromosome changes, cytogenetic studies of lymphoma are in an early stage compared to those of leukemia. No comprehensive analysis of available data has so far been attempted in the literature either. Here we present an analysis of chromosome changes and their correlation with subtypes of lymphoma studied by conventional histology and cell surface markers, as observed in two sets of data: a group of 65 karyotypically abnormal tumors sequentially ascertained and studied by us during the period January 1, 1984 to April 30, 1985, and a larger data set derived by combining our data with those from two published series from the University of Minnesota that are comparable to our data. These combined data, which comprise the largest data set on the cytogenetics of lymphomas assembled so far, enabled a comprehensive analysis of correlation between chromosome change and tumor histology and the patterns of chromosome instability in these tumors. We found several significant associations, some previously described and others now recognized, between nonrandom chromosome gains, breaks, translocations, and deletions and histologic subtypes of tumors that characterize lymphomas. The data indicate that finding of chromosome breaks at certain sites (eg, 8q24, 14q32, 18q21) is of diagnostic value in dealing with cases of unusual lymphoma. Furthermore, nonrandom chromosome breakage exhibited three distinct patterns that reflected three levels of etiologically relevant genetic change.     

Quantitative evaluation of liver-specific promoters from retroviral vectors after in vivo transduction of hepatocytes

Hepatic gene therapy could be used to treat a number of inherited blood diseases such as hemophilia or thrombophilia. Although liver-directed retroviral transduction can result in long-term gene expression in vivo, the low level of protein production has limited its clinical application. We reasoned that the insertion of liver-specific promoters into retroviral vectors would increase gene expression in vivo. The 347- bp human alpha 1-antitrypsin (hAAT), the 810-bp murine albumin (mAIb), the 490-bp rat phosphoenolpyruvate carboxykinase (rPECK), and the 596- bp rat liver fatty acid binding protein promoters were inserted into a Moloney murine leukemia retroviral backbone containing the hAAT reporter gene. Vectors that produced appropriately sized RNA and hAAT protein in vitro were tested in vivo by transducing regenerating rat livers. Long-term serum expression of the hAAT reporter gene was normalized to retroviral transduction efficiency as determined by using a polymerase chain reaction-based assay of genomic DNA from transduced rat livers. The hAAT, mAIb, and rPEPCK promoters were, respectively, 35- , 8-, and 0.02-fold as strong as the previously studied constitutive Pol-II promoter. We conclude that the hAAT promoter resulted in the highest expression from a retroviral vector and may result in therapeutically significant expression of other clinically significant blood proteins.     

Molecular basis of altered red blood cell membrane properties in Southeast Asian ovalocytosis: role of the mutant band 3 protein in band 3 oligomerization and retention by the membrane skeleton

Southeast Asian ovalocytosis (SAO) is an asymptomatic trait characterized by rigid, poorly deformable red cells that resist invasion by several strains of malaria parasites. The underlying molecular genetic defect involves simple heterozygous state for a mutant band 3 protein, which contains a deletion of amino acids 400 through 408, linked with a Lys 56-to-Glu substitution (band 3-Memphis polymorphism). To elucidate the contribution of the mutant SAO band 3 protein to increased SAO red blood cell (RBC) rigidity, we examined the participation of the mutant SAO band 3 protein in increased band 3 attachment to the skeleton and band 3 oligomerization. We found first that SAO RBC skeletons retained more band 3 than normal cells and that this increased retention preferentially involved the mutant SAO band 3 protein. Second, SAO RBCs contained a higher percentage of band 3 oligomer-ankyrin complexes than normal cells, and these oligomers were preferentially enriched by the mutant SAO protein. At the ultrastructural level, the increased oligomer formation of SAO RBCs was reflected by stacking of band 3-containing intramembrane particles (IMP) into longitudinal strands. The IMP stacking was not reversed by treating SAO RBCs in alkaline pH (pH 11), which is known to weaken ankyrin-band 3 interactions, or by removing the cytoplasmic domain of band 3 from SAO membranes with trypsin. Finally, we found that band 3 protein in intact SAO RBCs exhibited a markedly decreased rotational mobility, presumably reflecting the increased oligomerization and the membrane skeletal association of the SAO band 3 protein. We propose that the mutant SAO band 3 has an increased propensity to form oligomers, which appear as longitudinal strands of IMP and exhibit increased association with membrane skeleton. This band 3 oligomerization underlies the increase in membrane rigidity by precluding membrane skeletal extension, which is necessary for membrane deformation.     

Effects of interleukin-11 on the proliferation and cell cycle status of myeloid leukemic cells

Interleukin-11 (IL-11) is a pleiotropic cytokine with effects on many different targets. Within the hematopoietic system, the effects of IL- 11 are largely manifest only through combination with other cytokines, including IL-3 and Steel factor (SF). In the present study, we addressed the question of IL-11 responsiveness within the different types of human leukemic cells, as well as the mechanism of action of IL- 11 at the cellular level. Analysis of a panel of samples from different patients with acute myeloblastic leukemia (AML) and myeloid leukemic cell lines indicated that IL-11 alone was ineffective in supporting myeloid leukemic cell growth but frequently enhanced growth supported by IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), or SF. In contrast, three acute pre-B lymphocytic leukemia (pre-B-ALL) and two acute T lymphocytic leukemia (T-ALL) lines failed to respond to IL- 11 alone or when combined with other cytokines. The growth enhancement of IL-11 among the AML patient samples was dose dependent and remarkably constant with half-efficient concentrations in the range of 0.3 to 0.4 ng/mL. The thymidine suicide studies with the patient samples revealed that 40% to 50% of the blast cells were in S-phase when exposed for 16 hours to IL-3 and this level was increased to 70% to 90% in response to either IL-11 or IL-6. Our data suggest that the latter two interleukins act synergistically with the direct mitogenic factor, IL-3, in triggering AML blast-cell proliferation. Detailed analysis with several patient samples further revealed that SF and IL- 11 both enhance IL-3-supported clonogenic growth of AML blasts and the combination of all three growth factors yields optimal growth. In contrast, IL-6 does not further enhance the effect of IL-11. These results indicate that SF and IL-11 enhance IL-3-dependent clonogenic growth through two distinct pathways, whereas IL-6 and IL-11 may trigger the same pathway.     

Amplification of genes encoding human myeloid membrane antigens after DNA-mediated gene transfer

Spontaneous amplification of genes encoding two different human myeloid surface antigens was observed after DNA-mediated gene transfer of cellular DNA from the human myeloid cell line HL-60 into NIH-3T3 mouse fibroblasts. Transformed recipient cells with highly amplified expression of either of two donor membrane polypeptides, gp150 or p67, were isolated with a fluorescence-activated cell sorter (FACS), using monoclonal antibodies specific for human myeloid cells. Immunoprecipitation of enzymatically radioiodinated polypeptides from the surface of transformed NIH-3T3 cells confirmed that expression of these proteins was amplified tenfold to 20-fold in comparison to their expression on human myeloid cell lines. The cellular DNA of cloned secondary and tertiary transformants expressing high levels of gp150 and p67 contained amplified sets of DNA restriction fragments that hybridized with human repetitive DNA sequences. Cytogenetic analysis of subclones overexpressing gp150 revealed extrachromosomal double minutes (DMs), whose presence correlated with the unstable expression of the membrane polypeptide. Human sequences in gp150-positive clones did not localize to chromosomes, consistent with their association with extrachromosomal DMs. By contrast, p67-positive subclones stably expressed the antigen, and in situ hybridization to metaphase spreads demonstrated that amplified human DNA sequences were integrated into a specific marker chromosome. Cytogenetic analysis of the parental NIH- 3T3 subclone used in these studies disclosed DMs in a low percentage of metaphases, suggesting that the recipient cells have a propensity for amplifying donor DNA.