Body composition changes in patients with human immunodeficiency virus infection

Malnutrition characterized by weight loss and often extreme wasting generally develops when patients progress from infection with human immunodeficiency virus (HIV) to AIDS. There is evidence that before the development of AIDS, HIV-infected patients without weight loss show early signs of malnutrition, defined as an increase in the ratio of extracellular mass (ECM) to body cell mass (BCM). As part of a dietary intervention study, body composition measurement were obtained at baseline and after 6 wk in 18 patients with HIV infection and CD4 counts between 140 and 740 cells/mm3. Only one patient had a prior weight loss (3.7 kg); patients gained 2 pounds after 3 wk of dietary supplementation of 500 kcal daily. Bioelectrical impedance was used to measured body compartments. The average ECM/BCM ratio (0.77 +/- 0.13) was within the normal range (0.83 +/- 0.16) indicating the absence of malnutrition by this criterion. Most measurements of BCM (kg) approximated normal values, while several for BCM (kg) exceeded normal. BCM (kg) correlated poorly with the ECM/BCM ratio (r2 = 0.08; P = 0.11) in contrast to ECM (kg), which was well correlated (r2 = 0.82; P = 0.00). In addition, there was a significant correlation of body mass index (BMI) with the ECM/BCM ratio (r2 = 0.38; P = 0.00) and with ECM (r2 = 0.244; P = 0.003) indicating that overweight patients may be more likely to be considered malnourished than normal weight patients using this ratio. Without use of bioelectrical impedance, these subtle changes might be missed. Once significant weight loss has occurred coupled with decreases in BCM (kg), the ECM/BCM ratio may be more reflective of malnutrition. These conjectures will require prospective evaluation, but for now it seems reasonable to include bioelectrical impedance as a potentially useful tool in the evaluation of malnutrition in this population.     

Genomic instability and catalase gene amplification induced by chronic exposure to oxidative stress

Chronic exposure (>200 days) of HA1 fibroblasts to increasing concentrations of H2O2 or O2 results in the development of a stable oxidative stress-resistant phenotype characterized by increased cellular antioxidant levels, particularly catalase (D. R. Spitz et al, Arch. Biochem. Biophys., 279: 249-260, 1990; D. R. Spitz et al., Arch. Biochem. Biophys., 292: 221-227, 1992; S. J. Sullivan et al., Am. J. Physiol. (Lung Cell. Mol. Physiol.), 262: L748-L756, 1992). Acutely stressed cells failed to develop a stably resistant phenotype or increased catalase activity, suggesting that chronic exposure is required for the development of this phenotype. This study investigates the mechanism underlying increased catalase activity in the H2O2- and O2-resistant cell lines. In H2O2- and O2-resistant cells, catalase activity was found to be 20-30-fold higher than that in the parental HA1 cells and correlated with increased immunoreactive catalase protein and steady-state catalase mRNA levels. Resistant cell lines also demonstrated a 4-6-fold increase in catalase gene copy number by Southern blot analysis, which is indicative of gene amplification. Chromosome banding and in situ hybridization studies identified a single amplified catalase gene site located on a rearranged chromosome with banding similarities to Z-4 in the hamster fibroblast karyotype. Simultaneous in situ hybridization with a Z-4-specific adenine phosphoribosyltransferase (APRT) gene revealed that the amplified catalase genes were located proximate to APRT on the same chromosome in all resistant cells. In contrast, HA1 cells contained only single copies of the catalase gene that were not located on APRT-containing chromosomes, indicating that amplification is associated with a chromosomal rearrangement possibly involving Z-4. The fact that chronic exposure of HA1 cells to either HO2 or 95% O2 resulted in gene amplification suggests that gene amplification represents a generalized response to oxidative stress, contributing to the development of resistant phenotypes. These results support the hypothesis that chronic exposure to endogenous metabolic or exogenous environmental oxidative stress represents an important factor contributing to gene amplification and genomic instability.     

Poly(ethylene oxide) Grafted to Silicon Surfaces: Grafting Density and Protein Adsorption

Poly(ethylene oxide) (PEO) polymer, in linear and star form, was covalently grafted to silicon surfaces, and the surfaces were tested for their ability to adsorb proteins. Linear PEG of molecular weight 3400, 10 000, and 20 000 g/mol and star PEO molecules were coupled via their terminal hydroxyl groups activated by tresyl chloride to aminosilane-treated silicon wafers. The amount of PEO coupled to the surface was varied by changing the concentration of the tresyl-PEO solution. The dry PEO thickness on the surface was measured using X-ray photoelectron spectroscopy (XPS) and ellipsometry, from which the grafting density was calculated. The PEO surfaces were exposed to solutions of each of three proteins: cytochrome-c, albumin, and fibronectin. The degree of adsorption of each protein was determined by XPS and ellipsometry and recorded as a function of PEO grafting density. All three proteins were found to reach zero adsorption at the highest grafting densities on all three PEG surfaces, which for all three PEG surfaces was a PEO content of 100 +/- 10 ng/cm2. On both star PEO surfaces, albumin and fibronectin decreased to zero adsorption at intermediate values of grafting density, whereas cytochrome-c continued to adsorb at all grafting densities, although with a decreasing trend. A physical model of the surface helped explain these protein adsorption results in terms of the spacing and degree of overlap of grafted PEO chains.     

Influence of gravity and light on the developmental polarity of Ceratopteris richardii fern spores

Starburst dendrimer, a structurally defined, spherical macromolecule composed of repeating polyamidoamino subunits, was investigated to augment plasmid-mediated gene transfer efficiency in a murine cardiac transplantation model. The grafts were directly injected with naked pCH110, a plasmid encoding beta-galactosidase (beta-Gal), or pCH110-dendrimer complex, and reporter gene expression determined by X-Gal staining. The grafts injected with pCH110-dendrimer demonstrated widespread and extended beta-Gal expression in both myocytes and the graft infiltrating cells from 7 to 28 days, compared to the grafts injected with naked pCH110 that expressed beta-Gal only in myocytes for less than 14 days. p alphaMHC-vIL-10, as plasmid encoding viral interleukin-10 (vIL-10) under the control of alpha-myosin heavy chain promoter, was able to prolong allograft survival from 13.9 +/- 0.9 days to 21.4 +/- 2.3 days (p < 0.005). When dendrimer G5EDA was used with p alphaMHC-vIL-10, 60-fold less DNA resulted in significant prolongation of graft survival to 38.6 +/- 4.7 days (p < 0.0005). The dose of DNA, the charge ratio of DNA to dendrimer, and the size generation of the dendrimers were all determined to be critical variables for prolongation of allograft survival in this model system. Thus, the use of the Starburst dendrimer dramatically increased the efficiency of plasmid-mediated gene transfer and expression. Production of immunosuppressive cytokines at higher amounts for longer periods of time in a greater expanse of tissue enhanced the immunosuppressive effect and prolonged graft survival further.     

Chemical sulfonation and anticoagulant activity of acharan sulfate

Acharan sulfate is a glycosaminoglycan prepared from the giant African snail, Achatina fulica. This polysaccharide has a repeating disaccharide structure of -->4)-2-deoxy-2-acetamido-alpha-D-glucopyranose (1-->4)-2-sulfo-alpha-L-idopyranosyluronic acid (1-->). Its structure is related to heparin and heparan sulfate but is distinctly different from all known members of these classes of glycosaminoglycans. Because of its structural similarities to heparin, chemically modified acharan sulfate was studied to understand the chemical structure effected its anticoagulant activity. After de-N-acetylation, acharan sulfate was N-sulfonated using either chlorosulfonic acid-pyridine or sulfur trioxide-trimethylamine complex. The sulfate level in these products ranged from 22 to 24%(w/w), significantly less than that of heparin at 36%. The molecular weight of both N-sulfoacharan sulfates were comparable with that of heparin. In vitro anticoagulant activity assays showed that N-sulfoacharan sulfate derivatives were moderately active for the inhibition of thrombin and neither product showed any measurable anti-factor Xa activity. The differences in the activities of N-sulfoacharan sulfates produced by these two methods are probably ascribable to a small level of concomitant O-sulfonation obtained when using chlorosulfonic acid-pyridine.     

Interaction of the baculovirus anti-apoptotic protein p35 with caspases. Specificity, kinetics, and characterization of the caspase/p35 complex

The anti-apoptotic protein p35 from baculovirus is thought to prevent the suicidal response of infected insect cells by inhibiting caspases. Ectopic expression of p35 in a number of transgenic animals or cell lines is also anti-apoptotic, giving rise to the hypothesis that the protein is a general inhibitor of caspases. We have verified this hypothesis by demonstrating that purified recombinant p35 inhibits human caspase-1, -3, -6, -7, -8, and -10 with kass values from 1.2 x 10(3) to 7 x 10(5) (M-1 s-1), and with upper limits of Ki values from 0.1 to 9 nM. Inhibition of 12 unrelated serine or cysteine proteases was insignificant, implying that p35 is a potent caspase-specific inhibitor. Mutation of the putative inhibitory loop to favor caspase-1 resulted in a substantial decline in caspase-3 inhibition, but minimal changes in caspase-1 inhibition. The interaction p35 with caspase-3, as a model of the inhibitory mechanism, revealed classic slow-binding inhibition, with both active-sites of the caspase-3 dimer acting equally and independently. Inhibition resulted from complex formation between the enzyme and inhibitor, which could be visualized under nondenaturing conditions, but was dissociated by SDS to give p35 cleaved at Asp87, the P1 residue of the inhibitor. Complex formation requires the substrate-binding cleft to be unoccupied. Taken together, these data revealed that p35 is an active-site-directed inhibitor highly adapted to inhibiting caspases.     

Guanine nucleotide binding properties of Rac2 mutant proteins and analysis of the responsiveness to guanine nucleotide dissociation stimulator

A case-control study of the associations of retinoids and specific carotenoids with breast cancer using concentrations of these nutrients in breast adipose tissue was conducted among women attending a breast clinic in the Boston area in 1989-1992. Breast adipose tissue was collected during breast biopsy. Cases (n = 46) were women whose biopsies revealed invasive or in situ breast cancer; control subjects (n = 63) were women whose biopsies revealed benign disease. We observed inverse associations between breast adipose concentrations of retinoids and carotenoids and risk of breast cancer, although not all were statistically significant. The multivariate-adjusted odds ratio comparing women above the median value of the control group for retinol with those below or equal to the median was 0.71 (95% CI: 0.26, 1.93; NS); corresponding odds ratios were 0.61 (95% CI: 0.23, 1.64; NS) for retinyl palmitate, 0.30 (95% CI: 0.11, 0.85) for beta-carotene, 0.32 (95% CI: 0.11, 0.94) for lycopene, and 0.68 (95% CI: 0.27, 1.73; NS) for lutein/zeaxanthin. There was a nonsignificant positive correlation (r = 0.23, P = 0.15) between breast adipose tissue concentrations of retinol and dietary intake of preformed vitamin A, including supplements measured by using a food-frequency questionnaire. No correlation was found between breast adipose concentrations of carotenoids and intake of dietary carotenoids. These data suggest that higher breast adipose concentrations of retinoids and some carotenoids may be associated with decreased risk of breast cancer and that further examination of these relations is warranted.     

Pharmacokinetics of isosorbide dinitrate in healthy volunteers after 24-hour intravenous infusion

No studies have examined the pharmacokinetics of isosorbide dinitrate (ISDN) after infusion of long duration, even though such infusions are used in patients. We therefore measured ISDN and its active metabolites, isosorbide-5-mononitrate (IS5MN) and isosorbide-2-mononitrate (IS2MN), in plasma of 9 healthy volunteers who received a continuous intravenous infusion of ISDN for 24 hours at a dose rate that lowered diastolic blood pressure by 10% during the first 30 minutes of infusion. All subjects tolerated the infusion except one who experienced intolerable headache. Five subjects received 1 microgram.min-1.kg-1, one 2 micrograms.min-1.kg-1, and two 4 micrograms.min-1.kg-1 ISDN, whereas the full rate of 6 micrograms.min-1.kg-1 was used continuously in one subject. At all infusion rates the plasma concentrations of ISDN were higher at 24 hours than at earlier times, suggesting that a steady-state condition had not been reached at that time. The same was true for the mononitrate metabolites, which reached higher plasma concentrations and were cleared more slowly than the parent compound after the end of the infusion. Apparent elimination half-lives of ISDN, IS2MN, and IS5MN were 67 +/- 10 minutes, 115 +/- 13 minutes, and 272 +/- 38 minutes, respectively. Comparison of low-rate infusions (1 and 2 micrograms.min-1.kg-1) with high-rate infusions (4 and 6 micrograms.min-1.kg-1) showed that the plasma concentration ratios at 24 hours of mononitrate metabolites to parent drug and apparent plasma clearance of ISDN were almost halved at the higher infusion rates.     

Low incidence of myelodysplastic syndrome following transplantation using autologous non-cryopreserved bone marrow

Between May 1984 and October 1995 we performed 114 autologous stem cell transplants for lymphoma in our centre; 77/114 (68%) were transplanted after primary therapy. The conditioning regimen varied according to diagnosis; 26 patients were conditioned with melphalan and total body irradiation, 66 received melphalan and etoposide and the remainder (50) were conditioned with melphalan alone. The median follow-up is 62 months. Only two new haematological malignancies have occurred, both in patients with Hodgkin's disease. One patient developed Ph+ chronic myeloid leukaemia 18 months post-transplant. In this case, because of the timing of the haematological disorder, we considered the malignancy to be concurrent with or to have preceded the transplant. A second patient developed acute myeloid leukaemia 20 months post-transplant. She had been treated for Hodgkin's disease for 10 years and was transplanted in third complete remission. Cytogenetic analysis in this case showed trisomy 11. We believe this to have been an unequivocal second malignancy. Our finding of a 1.1% incidence of secondary haematological malignancy (95% CI 0.02-4.96) from a census population adds weight to the hypothesis that haematological problems post-transplant reflects prior chemotherapy rather than toxicity from the transplant procedure itself.