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Biomedical subjects

M Adam

Publications and source records attributed to M Adam.

At least 55 records · Page 3Linked to original sources
PubMed1996

Identification of fatty acid methyl ester as naturally occurring transcriptional regulators of the members of the peroxisome proliferator-activated receptor family.

The nuclear hormone receptors NUC-1 (PPAR delta) and PPAR alpha are members of the peroxisome proliferator-activated receptor (PPAR) family. The members of this receptor family are activated by agents that stimulate peroxisome proliferation, free fatty acids, prostaglandin 12 metabolites, and agents considered for the therapy of insulin-independent diabetes mellitus. To identify putative physiological agents that activate NUC-1, we tested the ability of acetone extracts of various rat tissues to activate the transcription of an MMTV-luciferase reporter gene, via a GR/NUC-1 hybrid receptor. GR/NUC-1 contains the ligand binding region of the NUC-1 receptor and the DNA binding domain of the glucocorticoid receptor. Using this assay, we found stimulatory activity in the pancreas, which upon purification and characterization was identified as methyl-palmitate, known to be enriched in pancreatic lipids. In addition, we determined that ethyl esters of palmitic and oleic acids are also potent activators of this receptor. Thus, fatty acid ester formation may control the cellular concentrations of fatty acids, and acyl-ester formation may play a role in the control of metabolic pathways and the activation of the PPAR.

Animals↗
PubMed1996

Morphological features of bone healing under the effect of collagen-graft-glycosaminoglycan copolymer supplemented with the tripeptide Gly-His-Lys.

The authors prepared 7.5% and 12.5% collagen gel, and supplemented it with the tripeptide Gly-His-Lys (GHK), perfloxacine and hypersulphated glycosaminoglycan (HSGAG). By means of 125l marking, its absorption was followed from small polyurethane sponges placed under the skin of rats. The absorption of gel without HSGAG was found to be faster. Antibodies against collagen (type I, II and III) or collagen gel were generated either in rabbits or in minipigs, in which collagen gel was tested. Microbiological tests proved the sterility of the collagen gel. The collagen gel supplemented with GHK, pefloxacine and HSGAG was named Colladel, and was used in a model experiment in guinea-pigs for filling artificially created bone defects in diaphyses of femurs, and with cementless endoprostheses. The healing process was followed by means of RTG and NMR, and histologically. The slowest healing process was found in unfilled bone defects. Defects filled with Colladel without GHK healed substantially more quickly, and the most accelerated healing was connected with complete Colladel application. When Colladel was used with cementless endoprostheses, vivid osteogenic activity at the interface of trabecular bone and metal stem was detectable in the course of the experiment.

4-Quinolones↗
PubMed1996

[Myelofibrosis regressing under corticotherapy and intravenous immunoglobulins in an infant].

BACKGROUND: Primary myelofibrosis is rare in infants and children; its association with auto-immune markers has only been reported in adults. CASE REPORT: An 8 month-old girl was admitted because of severe anemia and neutropenia. The marrow aspirate showed dysgranulopoiesis and partial interruption of maturation after the myelocyte level. The bone marrow biopsy revealed reticulinic myelofibrosis. The condition worsened with development of agranulocytosis and thrombocytopenia. Investigations ruled out malignant hemopathy, metastatic infiltration of the marrow and osteopathy. A myelodysplastic syndrome was discussed, but presence of anti-granulocyte auto-antibodies and positive Coombs test led to consider an autoimmune etiology. A corticosteroid therapy was attempted, effective only on the platelet lineage. Addition of intravenous gammaglobulin therapy corrected the problem. After a 24 month-course of the disease, it was necessary to prolong therapy. CONCLUSION: The efficacy of gammaglobulins may be an additional argument for auto-immunity, although no other auto-immune pattern has been observed in our patient, contrary to reported cases in adults.

Combined Modality Therapy↗
PubMed1996

Simplified strategies for minimal residual disease detection in B-cell precursor acute lymphoblastic leukaemia.

We have developed a simplified fluorescent run-off (FluRO) based IgH PCR strategy in order to facilitate follow-up of large numbers of B-cell precursor (BCP) acute lymphoblastic leukaemias (ALL) in a routine molecular diagnostic laboratory. DNA samples from 26 BCP-ALL and one B-cell line were amplified using IgH FR1 and FR2 consensus primers and analysed in parallel either by ethidium bromide non-denaturing PAGE or, after rendering the PCR products fluorescent with an internal JH consensus primer, by high-resolution analysis on an automated fragment analyser. The latter led to a minimum of one log increase in sensitivity of detection in 62% of alleles from 19 samples (16/28 in FR1; 11/15 in FR2) tested in parallel on log DNA dilutions, and to at least a 10(-2) level of sensitivity of detection in 15/19. The improved resolution allowed an approximate 20% increase in the number of clonal alleles detected, and consequently doubled the incidence of oligoclonality (6/26; 23%). Using these strategies, 6/17 (35%) of children analysed prospectively showed residual IgH positivity in the post induction complete remission bone marrow sample. Both early deaths occurred within this subgroup of patients and of the three of four surviving patients tested, two remained positive 2-3 months later. Although this simplified strategy is, as expected, less sensitive than anti-V-D-J junction specific strategies, it enables detection of a category of 'slow-remitters' which may have prognostic significance at a stage where therapeutic decisions are taken.

Burkitt Lymphoma↗
PubMed1996

Detection of intracellular calcium elevations in Xenopus laevis oocytes: aequorin luminescence versus electrophysiology.

Detection of receptor expression in Xenopus oocytes often relies upon functional coupling to second messengers such as Ca2+ or cyclic adenosine monophosphate. To detect intracellular Ca2+, electrophysiological measurement of the endogenous Ca(2+)-activated chloride current (ICl(Ca)) is often used (Dascal, 1987). An alternative utilizes the Ca2+ sensing, bioluminescent protein aequorin (Parker and Miledi(1986) Proc. R. Soc. Lond. B, 228: 307-315; Giladi and Spindel (1991) BioTechniques, 10: 744-747). In the present study the sensitivities of aequorin and electrophysiology for detecting receptor-mediated Ca2+ transients were compared. Assays were performed on the same batches of oocytes using either animal serum or ligands of exogenous receptors to generate inositol 1,4,5-trisphosphate (InsP3) and ultimately elevate intracellular Ca2+. Signal amplitudes were controlled by titrating the concentration of animal serum, or titrating the amount of receptor mRNA injected. Both assays detected signals with high concentrations of animal serum, or with high receptor density. However, aequorin signals were not detected in experiments with average ICl(Ca) current amplitudes below 200 nA. To further evaluate the differences between these two techniques, membrane current and bioluminescence were measured simultaneously. Results of these studies suggest that the signals differ due to the spatial distribution of aequorin, the chloride channels, and the calcium release sites.

Animals↗
PubMed1996

Different target specificities of phospholipid-dependent antibodies.

Phospholipid dependent antibodies are usually measured with assays for antiphospholipid/anticardiolipin antibodies (aPLA) or for lupus anticoagulant (LA) activity. Most of them are targeted to complexes of beta 2-glycoprotein I (beta 2-GPI) and anionic phospholipids (PLP) or to prothrombin for some LA. New understandings allow a better standardisation and optimisation of assays' reactivity. Antigenic targets of phospholipid dependent antibodies were studied on plasmas from 38 patients with the antiphospholipid syndrome (APS) and presenting aPLA and/or LA. Using human beta 2-GPI-PLP complexes as solid phase antigen offers the highest sensitivity for measuring aPLA. Many aPLA, but not all, also react with beta 2-GPI coated on solid phase, however there is no evidence until now that this latter reactivity shows a closest association with the clinical context. Most of the patients with LA present an immunological reactivity to beta 2-GPI alone or to prothrombin, when these proteins are coated on solid phase. In two cases there was a reactivity to only beta 2-GPI-PLP complexes. For the various immunoassays, using NUNC type I plates offers a good binding capacity for coating antigens. They are then present at enough density on solid phase for insuring an efficient binding of autoantibodies. This is an important factor for assay sensitivity and reproducibility. Interestingly, in 1 case with LA, autoantibodies were reactive with coated beta 2-GPI alone but not with its PLP-complexes. In another case reactivity to beta 2-GPI was much higher than that to beta 2-GPI-PLP.

Antibodies, Antiphospholipid↗
PubMed1996

[The calcium tolerance test in thyrotoxicosis, Hashimoto's thyroiditis and after total thyroidectomy].

Basic indicators of calcium and bone metabolism and the calcium tolerance test were investigated in patients with untreated hyperthyroidism, Hashimoto's thyroiditis, after total thyroidectomy and in controls. In florid hyperthyroidism elevated values of indicators of bone new formation (osteocalcin and bone fraction of alkaline phosphatases) and of bone resorption (urinary pyridinoline and deoxypyridinoline) were found and a protracted return of the serum calcium level to baseline values (during the 120th minute after completed infusion containing calcium). The calcitonin serum levels were significantly reduced after completion of the infusion up to the 120th minute. These results suggest a reduced calcitonin activity in hyperthyroidism, caused probably by exhaustion of C cells during the prolonged tendency of hypercalcaemia associated with untreated hyperthyroidism. In patients with autoimmune Hashimoto's thyroiditis no significant deviations in basal indicators of the calcium and bone metabolism were found nor deviations of the serum calcium and calcitonin levels during the calcium tolerance test. After total thyroidectomy the basal values of calcitoninaemia are significantly reduced and during the calcium tolerance test the elevated serum calcium level persists from the end of the calcium containing infusion up to the 120th minute. The calcitonin values are at all time intervals significantly low or cannot be detected (during the 120th and 240th minute). Changes of the serum calcium and calcitonin level are due to the surgical removal of the calcitonin source, thyroid C cells. The investigation confirmed the impaired calcium and bone metabolism and impaired calcium tolerance test in hyperthyroidism and in particular after total thyroidectomy. Reduced calcitonin values in these patients can act as a factor promoting the development of osteoporosis.

Adult↗
PubMed1995

Detection of adenylate cyclase-coupled receptors in Xenopus oocytes by coexpression with cystic fibrosis transmembrane conductance regulator.

To detect heterologous expression of receptors coupled via G proteins to the stimulation of adenylate cyclase in Xenopus laevis oocytes, the receptor of interest is coexpressed with the cystic fibrosis transmembrane conductance regulator (CFTR)--a cAMP-dependent Cl- channel. The binding of an agonist to the expressed receptor stimulates adenylate cyclase resulting in intracellular cAMP elevation, which in turn activates the CFTR. The CFTR-mediated Cl- current response is then measured using the standard two-electrode voltage-clamp technique. This method has allowed us to detect functional expression in oocytes of the human EP2 and IP prostanoid receptors. This method should prove valuable for expression and identification of putative G protein-coupled receptors signaling through stimulation of adenylate cyclase, for structure/function studies, and for analysis of receptor antagonists and agonists.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Chromosomal localization of the human prostanoid receptor gene family.

Prostaglandins (PGD2, PGE2, PGF2 alpha, and PGI2) and thromboxane A2 (TXA2) are biologically active molecules derived from the metabolism of arachidonic acid by cyclooxygenases. They produce a wide variety of physiological and pathophysiological effects mediated through specific G protein-coupled cell surface receptors. In this study, we have mapped the chromosomal positions of the human genes that encode the PGE2 receptor subtypes (PTGER1, PTGER2, and PTGER3), the PGF2 alpha receptor (PTGFR), the PGI2 receptor (PTGIR), and the TXA2 receptor (TBXA2R) using in situ hybridization. The PTGER1, TBXA2R, and PTGIR genes mapped to chromosome 19 at positions 19p13.1, 19p13.3, and 19q13.3, respectively. The PTGFR and PTGER3 genes mapped to chromosome 1 at positions 1p31.1 and 1p31.2, respectively, and PTGER2 gene mapped to chromosome band 5p13.1.

Chromosome Mapping↗
PubMed1995

Direct determination of the sequence recognition requirements of the SH2 domains of SH-PTP2.

SH-PTP2 is a widely-expressed protein tyrosine phosphatase with two tandem SH2 (src homology 2) domains and a C-terminal catalytic domain. Glutathione S-transferase fusions of the SH2 domains alone and of a catalytically inactive full-length mutant were made, and binding assays were developed using the purified fusion proteins to directly determine what residues are involved in the recognition of binding targets by the SH2 domains. The binding kinetics of the SH2 domains to a phosphotyrosyl-containing peptide of the sequence surrounding Tyr1009 of the platelet-derived growth factor receptor (PDGFR) beta subunit [DTSSVL(pY)TAVQPN] were determined by surface plasmon resonance, confirming that this is a high-affinity binding ligand. Using various N- and C-terminal truncations of this peptide as competitors in the binding assays, the minimum peptide that served as a high-affinity binding ligand was found to be VL(pY)TAV. Systematic Ala substitutions of this peptide indicated that in addition to the phosphotyrosine (pY), the critical residues for recognition and binding are at pY + 1 and pY + 3 as previously reported, and notably at pY-2 as well. Binding competition results with these and other PDGFR, IRP, and IRS-1 peptides suggested some general rules for sequence recognition by the SH2 domains of SH-PTP2. Peptides that bind to the SH2 domains in the binding assays were also found to stimulate the phosphatase activity of SH-PTP2.

Amino Acid Sequence↗
PubMed1995

Protein-tyrosine phosphatase SH-PTP2 (PTPN11) is localized to 12q24.1-24.3.

A 2.1-kb cDNA probe encoding the human SH2-domain containing protein-tyrosine phosphatase SH-PTP2 (PTPN11) was hybridized to human metaphase chromosomes in three independent experiments. In each instance, hybridization was maximal to chromosome 12q24.1-q24.3. The presence of SH-PTP2 cDNA crosshybridizing sequences located on a number of other chromosomes suggested that SH-PTP2-related genes or pseudogenes are present in the human genome.

Chromosome Mapping↗
PubMed1995

Reactivity and the fate of some composite bioimplants based on collagen in connective tissue.

Three composite materials based on collagen gel prepared with ISC40 and supplemented with the tripeptide glycine-histidine-lysine (GHK) mixed with bioglass (BAS O), pure hydroxyapatite (grain: 0.5-1.0 microns and 13-23 microns) and shredded beef bone were tested in the laboratory rat and guinea-pig. In the course of 49 d, no signs of infectious inflammation or incompatibility were observed around subcutaneously installed bioimplants. Particles of glass and hydroxyapatite became subject to phagocytosis. Bone fragments were completely resorbed, and no signs of osteogenic activity were detected in the neighbourhood.

Amino Acid Sequence↗
PubMed1995

Effect of the tripeptide glycyl-L-histidyl-L-lysine on the proliferation and synthetic activity of chick embryo chondrocytes.

Under certain conditions chondrocytes form lattices with cartilage collagens, which may serve as cartilage implants. It is necessary to find the optimal conditions for culturing chondrocytes. Three different supports are compared: (a) plastic; (b) cartilage collagens; and (c) insoluble skin collagen solubilized under denaturing conditions (ISC-40). The effect of culture medium supplementation with the tripeptide (Gly-His-Lys)2.Cu.2H2O.2NaCl (GHK) on chondrocyte proliferation and synthetic activity is studied, with particular attention paid to collagen types I, II and III. The collagen supports stimulated chondrocyte proliferation, but on the ISC-40 support they started to dedifferentiate rather early. In the primary culture, chondrocytes on all three supports synthesized mainly collagen type II, and only small amounts of types I and III. In the first passage the synthesis of these two collagen types increased, relative to collagen type II, at least on the cartilage collagen support. Supplementation of culture medium with GHK stimulated chondrocyte proliferation in the primary structure mostly on the ISC-40 support. On the other two types of supports the stimulatory effect of GHK was expressed mostly in the first passages. The collagen synthetic rate was increased by GHK on both of the collagen supports; on the cartilage collagen support collagen type II was synthesized predominantly and on the ISC-40 support types I and III were mostly formed. It is suggested that supplementation of culture medium with GHK may be useful in the preparation of cartilage implants.

Amino Acid Sequence↗
PubMed1995

[Hemophagocytic syndrome in children infected by HIV. Apropos of 3 cases].

BACKGROUND: The hemophagocytic syndrome has previously been reported in different infectious diseases (EBV, CMV, tuberculosis...) but rarely in adults with AIDS and never in children suffering from AIDS. CASE REPORTS: A hemophagocytic syndrome was recognized during the follow-up of 3 children with AIDS. The first, a 9-year-old girl developed an acute EBV coinfection and was treated with shots of corticosteroids and vepesid but died shortly afterwards. The second patient, a 3-year-old girl was infected with Aspergillus fumigatus for which she was given amphotericin B with a rapid improvement. The third patient, an 8-year-old boy had multi-resistant Streptococcus pneumoniae otitis and pneumonitis; his condition improved rapidly with adapted antibiotherapy. DISCUSSION: The HIV-hemophagocytic syndrome is not exceptional in HIV infection because of the association of immunodeficiency and resulting superinfections. Its diagnosis and treatment should be etiologic. Severe cases without etiology could benefit from chemotherapy. CONCLUSION: Management and outcome of this potentially lethal syndrome might depend on the identification of a curable infectious cause.

AIDS-Related Opportunistic Infections↗
PubMed1995

Development and validation of two enzyme-linked immunosorbent assay (ELISA) methods for recombinant hirudin.

Recombinant hirudin is currently being developed as a potential prophylactic and therapeutic antithrombotic drug in various clinical indications such as angina and deep venous thrombosis. In this report, we have discussed the production of specific polyclonal antibodies to recombinant hirudin (rH) and the development of two ELISA methods to measure rH concentrations in biological fluids: a sandwich and a competitive ELISA method. Intra- and inter-assay variations in the two methods are extremely low (3-7%). The competitive ELISA method is rapid, simple and highly reproducible. Saturation binding curves, selection of appropriate incubation times, recovery of different hirudin variants and reactivity in the presence of thrombin are discussed. The methods can be easily adapted to monitor hirudin concentrations in the clinical laboratory for diagnostic purposes as well as for performing pharmacokinetic studies.

Antibody Specificity↗
PubMed1995

Replication-defective adenovirus type 5 as an in vitro and in vivo gene transfer vector in chickens.

The capacity of E1A gene-deleted and thus replication-defective adenovirus type 5 (Ad5) to transduce foreign genes in chicken embryo fibroblasts (CEF) as well as in chickens was investigated. The lacZ and luciferase genes were successfully transduced in CEF by replication-defective Ad5, demonstrating that these cells possess receptor(s) for binding and penetration of Ad5. A single intramuscular inoculation of Ad-gD, a replication-defective Ad5 harbouring the gD gene of pseudorabies virus, in adult and 1-day-old chickens led to the production of a very high titres of specific antibodies. These gD-specific antibodies persisted for at least 56 days. These results demonstrate that replication-defective Ad5, despite its mammalian origin and the deletion of the E1A gene, is a good candidate for developing non-spreading vaccines in poultry.

Adenovirus E1A Proteins↗
PubMed1995

Isogenic adenoviruses type 5 expressing or not expressing the E1A gene: efficiency as virus vectors in the vaccination of permissive and non-permissive species.

We have previously constructed a recombinant adenovirus type 5 (Ad5) vector expressing the gD gene of pseudorabies virus (Ad-gD). In this virus the E1A gene was deleted, so that Ad-gD was replication defective. When high doses of this virus were inoculated into target species, it was nevertheless able to elicit a strong and protective immune response. We have restored a functional E1A gene in Ad-gD, by rescuing the E1A transcription unit in an ectopic position, far from the enhancer sequences of E1A. Unlike Ad-gD, this new virus (Ad-gD-E1A) was able to replicate in non-transcomplementing Vero cells. The kinetics of replication of Ad-gD-E1A were similar to that of the wild-type Ad5 when cells were infected at medium or high m.o.i. Nevertheless, the level of virus replication was low when Ad-gD-E1A was plated at low m.o.i. Cotton rats and mice were vaccinated once with 10(9) to 10(5) TCID50 of each of the two viruses. In both species, Ad-gD and Ad-gD-E1A induced similar antibody responses for each dose tested. Both were able to give a high level of protection against the challenge. However, the protective dose of Ad-gD-E1A was at least 250-fold lower in cotton rats, an Ad5-permissive species, than that of Ad-gD. In mice, a species restricted for Ad5 replication, the protective dose of Ad-gD-E1A was still not significantly different from that of Ad-gD. These data indicate that the protective dose of such an adenovirus vector vaccine for an animal species is strictly linked to the capacity of the virus to replicate in this species, at least in the model used. So, in designing an adequate vector one needs to balance the use of low dose vaccines against risks in terms of biosafety. Nevertheless, for diseases in which the protective immune response is mainly based on the antibody response, defective and non-defective adenovirus vectors should demonstrate similar protective doses.

Adenoviridae Infections↗