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Biomedical subjects

R Roy

Publications and source records attributed to R Roy.

At least 163 records · Page 9Linked to original sources
PubMed1997

TGF beta 2 in rabbit blastocoelic fluid regulates CD4 membrane expression: possible role in the success of gestation.

PROBLEM: During pregnancy, major changes occur in the decidual cell population. One of these changes involves some phenotypical transformations of lymphocyte sub-populations. Since these variations might be due to the presence of the embryo, the current study was designed to investigate the implication of blastocoelic fluid (BF) in these changes and to determine the mechanism by which this phenomenon occurs. METHOD: Lymphocytes isolated from human peripheral blood (PBL) were cultured for 72 h in RPMI-FCS 10% and with or without BF day 12 (BF d-12) or Concanavalin A (ConA). After 72 h, T cells were labelled with anti-CD4 antibodies and Quantum Simply Cellular microbeads were used as a standard to evaluate the antibody binding capacity (ABC). RESULTS: Treatment of human PBL with BF d-12 decreases the percentage of CD4 and TCR positive cells, as compared to non-stimulated cells, but has no significant effect on CD2, CD3, and CD8 positive cells. It was also demonstrated, for the first time, that transforming growth factor beta-2 (TGF beta 2) in BF day 12 diminishes the percentage of CD4 positive cells by downregulating CD4 membrane expression on leucocytes. CONCLUSION: These findings suggest that the embryo plays a role in its own protection. Furthermore, it is predicted that any tissue producing TGF beta 2, such as certain types of tumor, downregulates the immune response, thus allowing tumor growth.

Animals↗
PubMed1997

Infrared fluorescent detection of D1S80 alleles from blood and body fluid collected on IsoCode devices.

A genetic locus D1S80 (pMCT 118) containing variable number of tandem repeats (VNTR) has been used extensively in forensic analysis and paternity testing. In the current research, DNA was isolated from blood saliva and nasal secretions collected on two types of IsoCode paper-based devices. The D1S80 locus was amplified using PCR technology, and the alleles were separated by gel electrophoresis and then detected using an infrared (IR) fluorescence automated DNA sequencer. IR-labeled amplification products were generated from human genomic DNA using oligonucleotide primers, which were covalently linked to an infrared fluorescent dye (IRD41) at the 5' end. This system combines IR fluorescence chemistry and laser technology, thus eliminating the need for post-electrophoretic gel handling for the detection of the alleles. Real-time detection after separation of the alleles is valuable for visualization of the data. The VNTR alleles are displayed as familiar autoradiogram-like images, which can also be analyzed by computer. Since DNA is eluted from the IsoCode devices only with sterile distilled water and without time-consuming methods of extraction, amplification can be performed from numerous samples within a short period of time.

Alleles↗
PubMed1997

Infrared fluorescent detection of PCR amplified gender identifying alleles.

An automated DNA sequencer utilizing high sensitivity infrared (IR) fluorescence technology together with Polymerase Chain Reaction (PCR) methodology was used to detect several sex differentiating loci on the X and Y chromosomes from various samples often encountered in forensic case work. Amplifications of the X-Y homologous amelogenin gene, the alpha-satellite (alphoid) repeat sequences and the X and Y chromosome zinc finger protein genes ZFX and ZFY (ZFX/ZFY) were performed. DNA extracted from various forensic specimens was amplified using either Taq, Tth or ThermoSequenase. Multiplexing using primers for all three loci in one reaction tube was achieved using Tth and ThermoSequenase. Two IR labeling strategies for detection of PCR products were utilized. In the first strategy, one of the PCR primers contained a 19-base extension at its 5' end identical to an IR-labeled universal M13 Forward (-29) primer which was included in the amplification reactions. During PCR the tailed primer generates sequence complementary to the M13 primer which subsequently primes the initial amplification products, thereby generating IR-labeled PCR products. In the second strategy, dATP labeled with an IR dye (IR-dATP) was included in the amplification reaction. During amplification IR-dATP was utilized by the polymerase and incorporated into the synthesized DNA, thus resulting in IR-labeled PCR products. X and Y specific bands were readily detected using both labeling methodologies. Amplified products were electrophoretically resolved using denaturing Long-Ranger gels and detected with an automated detection system using IR laser irradiation. A separation distance of 15 cm allowed run times of less than 2 h from sample loading to detection. Because the gels could be run more than once, at least 120 samples (2 loads x 60 samples/load) can be typed using a single gel.

Alleles↗
PubMed1997

MR imaging, MR angiography, and MR spectroscopy of the brain in eclampsia.

PURPOSE: To compare the MR imaging and MR angiographic changes with in vivo proton MR spectroscopic findings and to determine the spectral differences between edema and ischemia in patients with eclampsia. METHODS: Spin-echo MR imaging, MR angiography, and single-voxel proton MR spectroscopy were performed in 10 patients with eclampsia. MR studies were obtained within 3 to 5 days of diagnosis and repeated after 2 weeks with identical parameters. RESULTS: Multifocal subcortical/cortical hyperintensities were noted in all 10 patients on T2-weighted images; in two patients, hyperintensities were seen in both cerebral hemispheres. In nine patients, MR angiograms showed narrowing of the major vessels constituting the circle of Willis that resolved after 2 weeks. In one patient with subtle imaging changes, MR angiography showed mild bilateral narrowing of the proximal middle and posterior cerebral arteries that did not change after 2 weeks, whereas imaging abnormalities worsened. Findings at single-voxel MR spectroscopy of the reversible T2 hyperintense lesions were significantly different from findings in the control group for N-acetylaspartate (NAA)/creatine ratios. One patient with mild abnormalities at MR imaging and MR angiography had lactate and decreased creatine and NAA, and on a follow-up study had a further decrease of NAA and creatine as well as a decrease in lactate. CONCLUSION: In vivo proton MR spectroscopy may help to differentiate cerebral edema from ischemia in patients with eclampsia and thus may help to determine the prognosis for these patients.

Adolescent↗
PubMed1997

Seasonal dynamics of the tissue levels of total protein, free amino acid and ribonucleic acid in an Indian air-breathing teleost, Channa punctatus.

The tropical freshwater air-breathing teleost, Channa punctatus, reveals a seasonal metabolic reorganization with reference to its biochemical tissue compositions. The summer-adapted and winter-adapted C. punctatus seem to be two biochemically distinct populations. Besides, the different reproductive phases viz. prespawning, spawning, post-spawning, preparatory-I and preparatory-II are also biochemically distinct.

Acclimatization↗
PubMed1996

Immunosuppression with monoclonal antibodies and CTLA4-Ig after myoblast transplantation in mice.

Various combinations of monoclonal antibodies specific for lymphocyte cell surface antigens and a recombinant molecule (CTLA4-Ig) were used to immunosuppress mice after transplantation of MHC-incompatible myoblasts. To assess the effectiveness of the immunosuppression, the donor myoblasts were obtained from a transgenic mouse (TnI LacZ1/29) containing a beta-galactosidase (beta-gal) reporter gene under the control of a muscle-specific promoter. No muscle fibers expressing beta-gal were observed 1 month after the myoblast transplantation, when the animals were not immunosuppressed or were treated with CTLA4-Ig alone. Approximately 50% of the muscle fibers expressed the beta-gal reporter gene 1 month after transplantation in mice treated with CTLA4-Ig combined with an anti-CD4 monoclonal antibody and in mice treated with a combination of anti-CD4, anti-CD8, and anti-lymphocyte function-associated antigen-1. The percentage of beta-gal-labeled muscle fibers increased to 94% when this combination of the three monoclonal antibodies was administrated weekly for 3 weeks. Although excellent graft survival rates were obtained 1 month after transplantation, reflecting an effective immunosuppression by these three treatments, no beta-gal-positive fibers were found 2 months after the transplantation, indicating the inability of these immunosuppressive agents to maintain long-term graft survival and induce tolerance to the myoblasts and muscle fibers of donor origin.

Abatacept↗
PubMed1996

The domains of mammalian base excision repair enzyme N-methylpurine-DNA glycosylase. Interaction, conformational change, and role in DNA binding and damage recognition.

Repair of a variety of alkylated base adducts in DNA is initiated by their removal by N-methylpurine-DNA glycosylase. The 31-kDa mouse N-methylpurine-DNA glycosylase, derived by deletion of 48 amino acid residues from the 333-residue wild type protein without loss of activity, was analyzed for the presence of protease-resistant domains with specific roles in substrate binding and catalysis. Increasing proteolysis with trypsin generated first a 29-kDa polypeptide by removal of 42 amino-terminal residues, followed by production of 8-, 6-, and 13-kDa fragments with defined, nonoverlapping boundaries. The 8- and 13-kDa domains include the amino and carboxyl termini, respectively. Based on DNA-affinity chromatography and the protease protection assay, it appears that the 6- and 13-kDa domains are necessary for nontarget DNA binding and that the 8-kDa domain, in cooperation with the other two domains, participates in recognition of damaged bases. Furthermore, chemical cross-linking studies indicated that, in the presence of substrate DNA, the 8- and 6-kDa domains undergo conformational changes reflected by both protection from proteolysis and reduced availability of cysteine residues for the thiol-exchange reaction.

Amino Acid Sequence↗
PubMed1996

Leishmania donovani: metabolite mapping of promastigotes using proton nuclear magnetic resonance spectroscopy.

Proton nuclear magnetic resonance spectroscopy was used for studying the intracellular metabolite profile of promastigotes of Leishmania donovani. The major intracellular metabolites observed in the promastigotes were acetate, alanine, succinate, glycine, alpha-glycerophosphorylcholine, acetoacetate, arginine and ethanol. A comparative study of the intracellular metabolite profile of promastigotes of different strains of L. donovani showed that, all the major intracellular metabolites were present in promastigotes of different strains. A quantitative estimation of metabolites showed a strain specific (Finger print) metabolite profile which can be used for strain/species identification/differentiation.

Acetates↗
PubMed1996

Regulation of lysyl oxidase and cyclooxygenase expression in human lung fibroblasts: interactions among TGF-beta, IL-1 beta, and prostaglandin E.

Prostaglandin E2, transforming growth factor-beta, and interleukin-1 beta variably regulate the expression of cyclooxygenase 1, cyclooxygenase 2, and lysyl oxidase in IMR90, human embryo lung fibroblasts. Prostaglandin E2 at 100 nM upregulates cyclooxygenase 1 mRNA by approximately three-fold while it downregulates lysyl oxidase mRNA levels. Notably, prostaglandin E2 suppresses the enhancing effect of TGF-beta on basal levels of lysyl oxidase mRNA. These changes in steady state mRNA levels reflect transcriptional level control, at least in part. Corresponding changes are seen in the protein levels of lysyl oxidase, cyclooxygenase 1 and cyclooxygenase 2 and catalytic activities of these enzymes, including net prostaglandin E2 synthesis. Cyclooxygenase 2 mRNA(t1/2, 30 min) is considerably less stable than that of cyclooxygenase 1 (t1/2, 4 h) while lysyl oxidase mRNA is unusually stable (t1/2 > 14 h). Taken together with the differing kinetics with which these genes respond to perturbation by these cytokines, the present results suggest a coordinated, autocrine-like mechanism of regulation of cyclooxygenase 1 and cyclooxygenase 2 and further point to the potential of their metabolic product, prostaglandin E2, to suppress the expression of lysyl oxidase in the inflammatory response to injury.

Cyclooxygenase 1↗
PubMed1996

Increased interferon-gamma mRNA expression following alloincompatible myoblast transplantation is inhibited by FK506.

Myoblasts from C57BL/10SnJ+/+ were transplanted in major histocompatibility complex (MHC)-compatible mice (i.e., C57BL10SnJ+/+ and C57BL/10SnSc mdx/mdx) and in MHC-noncompatible (BALB/c+/+) mice. The recipients were killed 1-21 days after transplantation. C57BL10SnJ+/+ myoblasts were also transplanted in a few BALB/c+/+ mice treated with FK506 and killed 7 days thereafter. Our results showed that after MHC-noncompatible transplantation, interferon-gamma (IFN-gamma) mRNA expression is increased from day 5 to day 21, indicating the presence of a cellular immune reaction. Short-term immunosuppressive treatment with FK506 inhibited the transcription of IFN-gamma mRNA compared with that in untreated mice. Myoblast-specific antibodies were also detected 2 and 3 weeks after MHC-incompatible transplantation, indicating that the cellular immune reaction, revealed by the increase in IFN-gamma, was followed by a humoral reaction.

Animals↗
PubMed1996

Finger printing of Mycobacterium tuberculosis in patients with intracranial tuberculomas by using in vivo, ex vivo, and in vitro magnetic resonance spectroscopy.

In vivo, ex vivo, and in vitro proton magnetic resonance spectroscopy was performed in 12 patients with intracranial tuberculomas with an aim of detecting the biochemical constituents of Mycobacterium tuberculosis in a granuloma. One dimensional (1D) single pulse and spin-echo sequences and 2D correlative spectroscopy were used for the ex vivo study to confirm the resonances seen on in vivo study. Spectroscopic studies of the perchloric acid and lipid extract of granuloma and M. tuberculosis were performed to look for similarity of resonance. In vivo study showed the presence of lipids at 0.9, 1.3, 2.0, 2.8 ppm, and phosphoserine at 3.7 ppm. All these resonances were confirmed on ex vivo study. In addition, distinct resonances of serine and phenolic lipids were seen on ex vivo and in vitro study of tuberculous granuloma, which have not been observed in other intracranial tumors. Lipid extract of granuloma and M. tuberculosis showed phenolic lipids at 7.1 and 7.4 ppm, a constituent of the cell wall of the bacteria in a tuberculoma. It appears that it may be possible to finger print the biochemicals of the cell wall of M. tuberculosis in a tuberculous granuloma and thus may help in detection and diagnosis of such lesions.

Amino Acids↗
PubMed1996

Scrokoelziside A, a triterpene glycoside from Scrophularia koelzii.

Scrokoelziside A, isolated from the aerial parts of Scrophularia koelzii was shown to be 3-O-([alpha-L-rhamnopyranosyl-(1-->3), beta-D-glucopyranosyl-(1-->2)]-beta-D-fucopyranosyl-(1-->4)-beta-D- glucopyranosyl]-13 beta,28-epoxyolean-11-en-23-ol, on the basis one- and two-dimensional NMR homo- and hetero-nuclear spectroscopic evidence.

Carbohydrate Sequence↗
PubMed1996

Selecting valid in vitro biocompatibility tests that predict the in vivo healing response of synthetic vascular prostheses.

We have investigated the usefulness of six in vitro biocompatibility tests in predicting the healing performance of polyester vascular prostheses as observed in previous canine in vivo trials. Vascular grafts were evaluated by using (i) a direct contact (DC) assay, (ii) an extract dilution (ED) assay on murine fibroblast cells, (iii) a DC assay on endothelial cells, (iv) a complement activation study, (v) a leucocyte activation study of CD18 integrin subunit expression on human polymorphonuclear cells (PMNs) and (vi) interleukin-2 receptor expression on lymphocytes. Uncleaned polyester grafts had previously been associated with poor healing and gelatin-impregnated polyester grafts with delayed but satisfactory healing, whereas commercially cleaned polyester grafts had demonstrated excellent healing. Lightweight and heavyweight knitted and woven polyester grafts supplied specifically for this project were studied, each with a different surface condition, i.e. commercially available (CP), uncleaned (UP) and impregnated with gelatin (GP). The UP grafts induced fibroblast cytotoxicity according to the ED assay, poor migration and viability of endothelial cells, and an elevated expression of CD18 and interleukin-2 receptor on PMNs and lymphocytes, respectively. In contrast, the CP grafts promoted good endothelial cell growth, no evidence of cytotoxicity and a weaker cell activation, and the GP grafts were found to be non-cytotoxic, to exhibit a good cellular response and to moderate cell activation. The complement activation assay and the DC assay on fibroblasts were found to be less useful and less discriminating. From this, it is concluded that the two cell activation measurements, the DC assay on endothelial cells and ED assay on fibroblasts, are useful in predicting the in vivo healing response of arterial polyester substitutes.

Analysis of Variance↗
PubMed1996

An albumin-coated polyester arterial graft: in vivo assessment of biocompatibility and healing characteristics.

The albumin-coated vascular graft (ACG) and its uncoated polyester substrate, the Vascular II (V-II), were evaluated in terms of biocompatibility and biofunctionality using two in vivo animal studies. Biocompatibility and immunoreactivity were assessed by implanting intraperitoneally in the rat small segments of the ACG and the V-II graft and harvesting them with their surrounding tissue 3d, 1, 2 and 4 weeks later. Cytofluorometric determination of total T cells (CD3), the ratio of CD4/CD8 subsets and the percentage of IL-2 receptor-positive T cells in the peripheral blood has revealed that no significant difference in any of the T cell populations was found between the ACG and the V-II graft. The cellular reactivity of the ACG in terms of acid phosphatase activity at the implant side was significantly greater at 3 d but not at longer periods. Biofunctionality was evaluated by implanting both grafts as a thoracoabdominal vascular bypass in dogs for 11 different periods ranging from 4 h to 6 months. The rate of albumin resorption was such that traces were still present at 1 month, but no longer observable at 2 months. Tissue incorporation into the graft wall was earlier for the V-II (2 weeks) than for the ACG (4 weeks), which showed complete encapsulation, tissue incorporation and endothelialization after 2 months in vivo. Only small differences were observed between both grafts in terms of platelet and fibrin uptake on the luminal surface. The prostacyclin/thromboxane A2 ratio increased to a level higher that 1.0 aorta within 1 month for the V-II and 4 months for the ACG. In conclusion, the Bard ACG has demonstrated excellent biocompatibility in terms of blood T cell behaviour and acid phosphatase activity at the implant site. Finally, its healing response is equivalent to that of the uncoated Dacron prosthesis once the albumin coating has been resorbed.

Acid Phosphatase↗
PubMed1996

Successive injections in mdx mice of myoblasts grown with bFGF.

We studied the effects of single and repeated sets of injections in the same muscle of mdx mice of myoblasts grown with or without a high concentration 100 ng ml-1 of basic fibroblast growth factor (bFGF). The injected myoblasts were obtained from non-dystrophic transgenic mice expressing the beta-galactosidase gene under the control of a muscle-specific promoter. In these experiments, the host muscle was not irradiated to prevent muscle regeneration by host myoblasts. The host muscle was not damaged before myoblast transplantation with notexin, marcaïne or cold to trigger a regeneration-degeneration cycle. Without such pretreatments, the first set of injections of myoblasts grown without bFGF produced only 8% beta-galactosidase-positive and dystrophin-positive muscle fibers 1 month after transplantation. The percentage of muscle fibers containing the donor reporter gene increased, however, to 26% following a second set of injections in the same muscle. The percentage of muscle fibers expressing the donor reporter gene was significantly higher when the myoblasts were grown with a high dose of bFGF. Indeed the first set of injections produced 34% beta-gal-positive fibers while a second set of injections raised this percentage to 54%. In all cases, the percentage of dystrophin-positive fibers was similar to that of beta-gal-positive fibers. Therefore a high percentage of muscle fibers of donor origin can be obtained without preliminary damaging treatments of the mdx muscle when myoblasts grown with bFGF are injected several times. The effects of bFGF is not produced by increasing the percentage of myoblasts in a primary muscle culture since improvement of myoblast transplantation was obtained with a pure myoblast clone even with a lower concentration (10 ng ml-1) of bFGF.

Animals↗