In vitro studies on the effect of delaminated a-C:H film fragments on bone marrow cell cultures

Amorphous hydrogenated carbon (a-C:H) films have many outstanding properties required for a protective coating material on load bearing medical implants. Recently, titanium doped a-C:H films have been evaluated regarding their effects on bone marrow cell cultures. But many materials that are well-tolerated in bulk form are able to induce toxic reaction if present particulate form. In order to further assess biocompatibility aspects of these two coatings, film delamination has been mimicked in exposure to fluids. In the present study, particles from a-C:H, a-C:H/Ti and a-C:H-a-C:H/Ti bilayer films were added to bone marrow cell cultures in vitro. The results showed that plain a-C:H and to a certain extent a-CH/Ti particles were inert. Both kinds of particles did not significantly stimulate the osteoclast-related enzyme tartrate resistant acid phosphatase (TRAP). A slight increase in cell proliferation and total culture TRAP was found in cultures treated by a-C:H-a-C:H/Ti bilayer films. Latter effect can probably be traced back by the relative high percentage of small particles of a size of around 2 microm. However, if corrected by the cell number also no differences between particle-treated and untreated control cultures could be found, indicating the absence of a toxic effect from delaminated a-C:H coatings.     

Determination of Red Blood Cell Velocity by Video Shuttering and Image Analysis

A novel modification of conventional video imaging techniques has been developed to determine the velocity of red blood cells (RBCs), which offers compatibility with existing video-based methods for determining blood oxygenation and hemoglobin concentration. Traditional frame-by-frame analysis of video recordings limits the maximum velocity that can be measured for individual cells in vivo to about 2 mm/s. We have extended this range to about 20 mm/s, by electronic shuttering of an intensified charge-coupled device camera to produce multiple images of a single RBC in the same video frame. RBCs were labeled with fluorescein isothiocyanate and the labeled cells (FRBCs) were used as probes to determine RBC velocities in microvessels of the hamster retractor muscle. Velocity was computed as the product of the distance between centroids of two consecutive image positions of a FRBC and the shuttering frequency of the camera intensifier. In vitro calibrations of the system using FRBC and Sephadex beads coated onto a rotating disk yielded an average coefficient of variation of about 6%. Flow conservation studies at bifurcations indicated that the maximum diameter of microvessels below which all the FRBCs in the lumen could be detected was 50 m. The technique was used to estimate mean-FRBC velocity distributions in vessels with diameters ranging from 8 to 50 m. The mean-FRBC velocity profiles were found to be blunter than would be expected for Poiseuille flow. Single FRBCs tracked along an unbranched arteriole exhibited significant temporal variations in velocity. © 1999 Biomedical Engineering Society. PAC99: 8719Tt, 8717Jj, 4279Pw, 8780Tq, 8719Ff, 4230Va, 0705Pj     

Inhibition of HIV-1 by modification of a host membrane protease

While it is clear that CD4 Is the receptor for the gp120 envelopeprotein of HIV-1, substantial evidence suggests that other hostcell proteins are required for successful membrane fusion. Studieswere initiated to examine the potential for a protein receptorwhich has an elastase-like character to participate in fusionof HIV-1 with permissive host cells. A synthetic elastase inhibitorwas shown to significantly reduce HIV-1 infectivity when presentduring, but not after, the initial contact between virus andcells. A human T cell elastase-like membrane component was purifiedand shown to be lipid-associated. By competitive Inhibition,the purified protein was shown to bind gp160 within the HIV-1fusion domain. The binding parameters of whole T cell membraneextract, with a hydrophobic pentapeptide representative of thefusion domain, suggested an elastase-like protein is the single,secondary T cell receptor for HIV-1 (K = 1x10³ M^–1). Thepentapeptide interacted with porcine and human (epithelial andpolymorphonuclear leukocyte), but not murine, elastase isoforms,suggesting its participation In the permissiveness of host cellsto infection.     

Cytogenetic and comparative genomic hybridization findings in four cases of breast cancer after neoadjuvant chemotherapy

To assess a potential common pattern of genetic alterations in chemotherapy-resistant tumors we analyzed four tumors from breast cancer patients (patients 1-4) after neoadjuvant chemotherapy, by comparative genome hybridization (CGH) and conventional chromosome banding analysis. All patients showed structural aberrations involving chromosomes 1, 5, 11, 16, and 17. In CGH analysis, the patients showed typical imbalances for ductal breast cancer: gains of 1q (3 patients), 5q (2 patients), 8q (3 patients), and X (4 patients) and losses of 1p33 approximately p36 (3 patients), 16q (3 patients), 17p (3 patients), 19 (4 patients), and 22q (4 patients). Other recurrent imbalances of atypical pattern for ductal breast cancer were gain of 4q21 approximately q32 (2 patients), 20q21 approximately q22 (2 patients), and 21 (2 patients) and loss of 20p (3 patients). Three patients showed involvement of several regions bearing genes of drug resistance (MDR1 [HUGO symbol: ABCB1], BCRP [HUGO symbol: ABCG2], MRP1 [HUGO symbol: ABCC1], RFC1); the fourth patient displayed an amplification in the region of MYC (alias c-myc), thus providing--at the level of the light microscope--an explanatory background for the ability of their tumors to survive anthracycline-, taxane- and cyclophosphamide-based chemotherapy. Conventional cytogenetic analysis and CGH displayed highly coincidental findings in the tumors of four patients after neoadjuvant chemotherapy for breast cancer.     

Mechanisms of respiration and phosphorylation in Ascaris muscle mitochondria

In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electron transport this process appears to be coupled to energy conservation at phosphorylation site I. The branchpoint from which electrons are taken from the main respiratory chain to either the alternative oxidase or fumarate reductase is likely to be on the oxygen side of the NADH dehydrogenase segment.Malate and succinate are the only substrates which appreciably support respiration in the mitochondrion of the nematode. Regardless of the presence or absence of oxygen malate is utilized by an oxidation-reduction reaction resulting in the formation of pyruvate, acetate, succinate, propionate and CO₂. In addition, aerobically, hydrogen peroxide is formed as the product of oxygen reduction. Succinate accumulation was found to be significantly higher in the anaerobic as compared to the aerobic incubation mixtures. This effect was accompanied by an increase in anaerobic malate consumption. ATP generation and the formation of pyruvate, acetate and propionate were found to be similar in the presence and absence of oxygen.In malate-supported respiration of intact Ascaris mitochondria reducing equivalents (NADH) are produced exclusively through pyruvate and acetate formation. These enzymatic reactions are functionally coupled to the electron transport-linked reductions of fumarate to succinate and oxygen to hydrogen peroxide, respectively. In accordance with the position of the redox potentials of the fumarate/succinate and O₂/H₂O₂ couples, anaerobic and aerobic respiration was found to be associated with relatively low energy conservation efficiencies. Thus one molecule of ATP was conserved per 2e^? transferred to fumarate or oxygen, respectively. No evidence could be obtained for a significant activity of energy conservation sites II and III and electron transfer through the alternative oxidase pathway was shown not to be coupled to phosphorylation.     

Prolonged Replication of a Type 1 Vaccine-Derived Poliovirus in an Immunodeficient Patient

VP1 sequences were determined for poliovirus type 1 isolates obtained over a 189-day period from a poliomyelitis patient with common variable immunodeficiency syndrome (a defect in antibody formation). The isolate from the first sample, taken 11 days after onset of paralysis, contained two poliovirus populations, differing from the Sabin 1 vaccine strain by ~10%, differing from diverse type 1 wild polioviruses by 19 to 24%, and differing from each other by 5.5% of nucleotides. Specimens taken after day 11 appeared to contain only one major poliovirus population. Evolution of VP1 sequences at synonymous third-codon positions occurred at an overall rate of ~3.4% per year over the 189-day period. Assuming this rate to be constant throughout the period of infection, the infection was calculated to have started ~9.3 years earlier. This estimate is about the time (6.9 years earlier) the patient received his last oral poliovirus vaccine dose, approximately 2 years before the diagnosis of immunodeficiency. These findings may have important implications for the strategy to eliminate poliovirus immunization after global polio eradication.     

Effects of interleukin 4 on CD25+CD4+ regulatory T cell function

CD25+CD4+ regulatory T cells (Tregs) contribute to the maintenance of peripheral tolerance against self and non-self. The modulatory effects of cytokines, such as interleukin 4 (IL-4) on the function of Tregs have not been explored in detail. We here report that IL-4 prevents spontaneous apoptosis and the decline of foxp3 mRNA which were found to occur during culture of isolated Tregs. Tregs exposed to IL-4 were more potent in suppressing the proliferation of na?ve CD4+ T cells and they better inhibited IFN-gamma production by CD4+ T cells as compared to Tregs cultured in medium. IL-4 also enhanced membrane IL-2Ralpha (CD25) expression on Tregs above the levels observed on freshly isolated cells. IL-4-mediated effects on Treg function persisted in Tregs from Stat6-/- mice, pointing to a Stat6-independent intracellular transduction pathway. In conclusion, our data suggest that the anti-inflammatory function of IL-4 could partly be mediated by effects on Tregs function.