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Biomedical subjects

M Pascual

Publications and source records attributed to M Pascual.

At least 127 records · Page 7Linked to original sources
PubMed1994

Proteolytic cleavage of CR1 on human erythrocytes in vivo: evidence for enhanced cleavage in AIDS.

The number of complement receptor type 1 (CR1; CD35) on human erythrocytes (E) decreases during normal in vivo aging. Patients with acquired immunodeficiency syndrome (AIDS) have an acquired deficiency of CR1 on E. The possible mechanisms responsible for the loss of CR1 from E include the release of small vesicles from the E membrane and proteolytic cleavage of CR1. When compared to E of normal donors and of asymptomatic human immunodeficiency virus HIV+ subjects, E of patients with AIDS had fewer CR1/E (p < 0.001), but had the same number of two glycosylphosphatidylinositol-anchored proteins, decay-accelerating-factor (DAF) and CD59. When compared to young E, old E separated by density gradients on Percoll had fewer CR1 [six normal subjects, mean loss: 50.4 +/- 4.9 (SEM) %], DAF (34.4 +/- 1.2%) and CD59 (34.5 +/- 2.7%). The loss of CR1 was significantly higher than the loss of DAF and CD59 (p < 0.02). In vitro, ATP depletion of E is responsible for the release of vesicles from the E surface, a reaction that has been called in vitro aging. CR1, DAF and CD59 were lost on ATP-depleted E; however, the loss of CR1 and DAF were identical (six experiments, mean loss of CR1: 28.7 +/- 2.7%, DAF: 26.3 +/- 4.6% and CD59: 20.5 +/- 4%). Thus, the release of vesicles from E cannot explain the specific loss of CR1 in patients with AIDS and would explain only incompletely the loss of CR1 during in vivo aging. In vitro experiments indicated that CR1 was more sensitive to trypsin and papain cleavage than DAF and CD59. Enhanced chemiluminescence Western blotting, using a monoclonal antibody (E11) recognizing fragments of CR1 down to 43 kDa on E exposed to trypsin or papain, indicated that normal E bear fragments of CR1, which are not found on polymorphonuclear leukocytes or on CR1-bearing vesicles in urine. The relative amount of these fragments was increased in patients with AIDS. Taken together these data suggest that the specific loss of CR1 on E in AIDS is due to proteolytic cleavage. The loss of CR1 during in vivo aging also involves proteolytic cleavage, although part of the loss might be explained by other mechanisms including the release of vesicles by E.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Soluble complement receptor type 1 (CD35) is released from leukocytes by surface cleavage.

The soluble form of complement receptor type 1 in human plasma (sCR1) might correspond to the shedding of the receptor by proteolytic cleavage at the cell surface. A new enzyme-linked immunosorbent assay (ELISA) was established to specifically measure membrane-bound CR1 using a rabbit polyclonal antibody against a 19-amino acid peptide corresponding to the C-terminal sequence of the intracellular domain of CR1 (mCR1-ELISA). This ELISA measured CR1 from solubilized erythrocyte membranes, polymorphonuclear leukocytes (PMN), a B lymphocyte cell line and renal podocyte-derived urinary vesicles in a dose-dependent manner. In contrast, and similarly to recombinant soluble CR1 which lacks the intracellular domain of CR1, plasmatic sCR1 was not recognized, suggesting that sCR1 corresponds to an extracellular fragment of whole CR1. In vitro, PMN were shown to release a soluble form of CR1 which was also not recognized in the mCR1-ELISA, and whose size was smaller (5 kDa) than the CR1 of PMN cell membranes. The release of soluble CR1 was highest for PMN and HL60 cells, followed by U937 cells and three different B lymphocyte cell lines, whereas T lymphocyte cell lines did not release soluble CR1. The levels of CR1 gene expression were also higher in PMN compared to remaining blood leukocytes and the different cell lines tested above. Incubation of PMN with formyl-methionyl-leucyl-phenylalanine, tumor necrosis factor-alpha or lipopolysaccharide accelerated the release of soluble CR1, and incubation with granulocyte/macrophage colony-stimulating factor resulted in sustained CR1 gene expression and higher total soluble CR1 release. Our results suggest that soluble CR1 is produced by cleavage of cell surface CR1, and that a large fraction of human plasma sCR1 is cleaved from PMN. The release of sCR1 by leukocytes may play a role in the control of complement activation at sites of inflammation.

Amino Acid Sequence↗
PubMed1993

Circulating soluble CR1 (CD35). Serum levels in diseases and evidence for its release by human leukocytes.

C receptor type 1 (CR1, CD35) is present in a soluble form in plasma (sCR1). Soluble CR1 was measured with a specific ELISA assay in normal individuals and in patients with different diseases. The mean serum concentration of sCR1 in 31 normal donors was 31.4 +/- 7.8 ng/ml, and was identical in plasma. An increase in sCR1 was observed in 36 patients with end-stage renal failure on dialysis (54.8 +/- 11.7 ng/ml, p < 0.0001), and in 22 patients with liver cirrhosis (158.3 +/- 49.9 ng/ml, p < 0.0001). The mean sCR1 levels dropped from 181 +/- 62.7 to 52.1 +/- 24.0 ng/ml (p < 0.001) in nine patients who underwent liver transplantation, and was 33.5 +/- 7.3 in 10 patients with functioning renal grafts, indicating that the increase in sCR1 was reversible. Soluble CR1 was elevated in some hematologic malignancies (> 47 ng/ml), which included B cell lymphoma (12/19 patients), Hodgkin's lymphoma (4/4), and chronic myeloproliferative syndromes (4/5). By contrast, no increase was observed in acute myeloid or lymphoblastic leukemia (10) or myeloma (5). In two patients with chronic myeloproliferative syndromes, sCR1 decreased rapidly after chemotherapy. The mean concentration of sCR1 was not significantly modified in 181 HIV-infected patients at various stages of the disease (34.8 +/- 14.4 ng/ml), and in 13 patients with active SLE (38.3 +/- 19.6 ng/ml), although in both groups the number of CR1 was diminished on E. There was a weak but significant correlation between sCR1 and CR1 per E in HIV infection and SLE (r = 0.39, p < 0.0001, and r = 0.60, p < 0.03 respectively). In vitro, monocytes, lymphocytes, and neutrophils were found to release sCR1 into culture supernatants. In vivo, sCR1 was detected in the serum of SCID mice populated with human peripheral blood leukocytes. The sCR1 levels correlated with those of human IgG (r = 0.97, p < 0.0001), suggesting synthesis of sCR1 by the transferred lymphocytes. The mechanisms underlining the increased levels of sCR1 and its biologic consequences remain to be defined.

Animals↗
PubMed1993

Release of vesicles enriched in complement receptor 1 from human erythrocytes.

Vesicles released from human E by Ca(2+)-loading, ATP-depletion, or storage are enriched in several glycosylphosphatidylinositol-anchored proteins such as acetylcholinesterase (AchE) and decay-accelerating factor (DAF). As a result of this, the remaining E are depleted of these proteins. We analyzed whether vesiculation induced by ATP-depletion in vitro was also responsible for a loss of C receptor 1 (CR1), which is a transmembrane protein arranged predominantly in small clusters. ATP-depleted E had lost 15.4% to 33.9% of their CR1. This loss was similar to that of AchE and DAF. The released vesicles contained CR1. The number of CR1 per band 3 protein was 1.7 to 2.7 that in the original E, indicating an enrichment of CR1 in vesicles. This enrichment was similar to that observed for AchE and DAF (1.83- and 2.6-fold, respectively). The capacity of the vesicles and the ATP-depleted E to bind C3b-coated immune complexes correlated with the CR1 number, suggesting that there was no preferential loss of CR1 clusters. Vesicles released from human E during C attack also contained CR1. In conclusion, in vitro aging induced by ATP-depletion is responsible not only for a loss of glycosylphosphatidylinositol-anchored proteins, but also of CR1. Whether vesiculation explains the loss of CR1 from aging E in vivo and from E of patients with SLE or AIDS remains to be studied.

Adenosine Triphosphate↗
PubMed1993

[Erythrocyte CR1 receptor: binding and transport of immune complexes in the blood circulation].

Immune complexes (IC), after reacting with the complement system, bear C3b fragments (opsonized IC) and bind to the CR1 receptor that is present on human erythrocytes (CR1 = complement receptor type 1). This efficient binding reaction prevents random vascular IC deposition, and allows IC to be transported through the circulation to the fixed macrophage system of the liver and spleen, where they are safely eliminated. The structure of CR1, with multiple C3b binding sites, and the clustered distribution of CR1 on the erythrocyte surface favor the multivalent binding of opsonized IC to erythrocytes. CR1 on erythrocytes serves as a cofactor for the inactivation of C3b by factor I, thus allowing the release of IC from the erythrocyte surface and their transfer to fixed macrophages. Under normal circumstances, the erythrocyte plays a major role in the processing of IC in humans.

Antigen-Antibody Complex↗
PubMed1993

Inhibition of complement alternative pathway in mice with Fab antibody to recombinant adipsin/factor D.

Mouse adipsin is a serine protease secreted mainly by adipocytes. Similarly to factor D of human complement, it cleaves factor B. That adipsin is the equivalent of human factor D in the mouse is further suggested by their structural homology. Specific antisera against recombinant mouse adipsin (r-adipsin) were produced in rabbits. Anti-r-adipsin IgG was shown to bind to radiolabeled r-adipsin and to inhibit its hemolytic activity. In vitro, these antibodies Ab and Fab fragments thereof inhibited the adipsin/factor D hemolytic activity of mouse serum. They also blocked C3 activation induced by cobra venom factor (CVF), but did not interfere with classical pathway function. After intravenous injection of anti-r-adipsin Fab into BALB/c mice, the adipsin/factor D hemolytic activity of serum was abolished during a 4-h period. The C3 depleting effect of CVF injected intravenously was significantly delayed in BALB/c mice which had been pretreated with anti-r-adipsin Fab. These experiments demonstrate that mouse adipsin is the only form of mouse factor D and that anti-r-adipsin antibody can be used to produce a specific inhibition of the alternative pathway in vivo.

Animals↗
PubMed1993

Specific interactions of polystyrene biomaterials with factor D of human complement.

The contact of blood with some biomaterials results in complement activation, primarily by the alternative pathway (AP). Insoluble polystyrene derivatives bearing isolated sulphonate groups (PSSO3) deplete complement, whereas identical surfaces substituted with both sulphonate and hydroxymethyl groups (PSCH2OH-SO3) are non-activators. Polystyrene sulphonate derivatives possess high adsorptive properties, particularly for serine proteases of the coagulation cascade. Thus, we studied the interactions between polystyrene derivatives and factor D, an enzyme essential for AP activation. C3 was activated when normal human serum (NHS) was incubated with PSSO3, whereas PSCH2OH-SO3 did not induce any specific C3 activation. Both polymers adsorbed factor D from serum, as shown by the loss of haemolytic factor D from NHS incubated with the polymers and by the specific adsorption of radiolabelled factor D. When bound to the polymers, factor D was not functional. The disappearance of factor D was in contradiction to the observed complement activation induced by PSSO3. When other AP components were studied, it was evident that PSSO3 adsorbed factor H even more rapidly and efficiently than factor D. Thus, the net effect was an immediate deregulation of the AP resulting in C3 activation, followed by inhibition of the AP when factor D was finally depleted. Pre-exposure of PSSO3 to NHS prevented any complement activation because the polymer was saturated with factor H, but still adsorbed factor D. Such properties could be beneficial during haemodialysis with membranes for uremic patients who have increased levels of factor D in their serum.

Biocompatible Materials↗
PubMed1993

Tissue protein turnover in animals treated with the mixed beta-agonist metaproterenol: influence of dose, route and pattern of administration.

beta-Adrenergic agonists have been shown to increase protein deposition as a result of changes in the balance between protein synthesis and degradation rates. The aim of this study is to investigate the effect of the treatment with the non-selective beta-adrenergic agonist, metaproterenol, on protein metabolism in rats as well as the influence of the route and pattern of administration. A short- and long-term experimental trial were carried out. After the short-term treatment with the beta-agonist (1 mg/kg), neither protein nor nucleic acids were affected in liver or gastrocnemious muscle, while cathepsin A activity, an index of protein degradation, significantly increased in muscle. However, cathepsin A activity was reduced in muscle by the oral administration during 21 days of metaproterenol (2 ppm/day), but not by the subcutaneous injections (0.1 mg/kg/day). On the other hand, RNA/DNA, an index of protein synthesis capacity, and protein/DNA, an indicator of cell size, significantly diminished in muscle after the subcutaneous long-term treatment but did not change in the liver of treated rats. Our study has demonstrated a different outcome of a mixed beta-adrenergic agonist on protein metabolism depending on the duration of the treatment and the route of administration.

Administration, Oral↗
PubMed1993

Adsorption of complement factor D by polyacrylonitrile dialysis membranes.

Factor D, an essential enzyme of the alternative pathway (AP) of complement, accumulates in end-stage renal failure (ESRF). Polyacrylonitrile (PAN) membrane activates complement poorly and has been shown to adsorb C3a, the main anaphylatoxin released during complement activation. In the present work we investigated whether factor D might be adsorbed on PAN. In vitro there was a loss of hemolytic factor D when normal human serum (NHS) was incubated with PAN dialysis fibers, whereas no loss was observed with cuprophan (Cu) fibers. There was a dose and time dependent binding of purified radiolabeled factor D to PAN but not to Cu. The hemolytic function of factor D released from PAN by 2 M NaCl was normal. By contrast, factor D was inactive while adsorbed to PAN fibers. When 50 ml of NHS or 100 ml of whole blood were passed through a new hollow fiber PAN dialyzer 95% of factor D was adsorbed. The eluate from a PAN filter which had been used for dialysis in a patient with ESRF contained 38.4 mg of hemolytic factor D, representing 34% of the proteins eluted. By immunoblotting, antigenic factor D from the PAN eluate was identical to purified human factor D. In six patients there was a 81.4% decrease in hemolytic factor D in blood after dialysis with PAN, contrasting with a 9.6% decrease in those dialyzed with cellulose acetate. No factor D was found in the dialysis fluid of PAN dialyzers, indicating that PAN removed factor D mainly by adsorption. In conclusion, PAN has a significant capacity to adsorb factor D, a reaction that might contribute to the diminished capacity of PAN membrane to activate the AP of complement. Whether the efficient removal of large amounts of factor D might be beneficial in uremic patients remains to be defined.

Acrylic Resins↗
PubMed1992

[Intervention against tobacco dependence in primary care. A study of 4 modalities of action].

OBJECTIVE: To study the feasibility and evaluate the outcome over a years of four methods of combatting tobacco addiction at the primary care level: one directed towards the team's staff and three aimed at patients (counselling of smokers and specific treatment with nicotine chewing-gum and group or individual support). To compare the results of group and individual support. DESIGN: Uncontrolled intervention study. SITE. Primary care. PATIENTS AND OTHER PARTICIPANTS: 19 members of the Team. 121 smoking patients who attended for consultation. 62 people who requested treatment. INTERVENTIONS: Consensus on the attitude taken to tobacco within the team. Systematic counselling to smokers. Treatment with nicotine chewing-gum and group or individual support. MAIN MEASUREMENTS AND RESULTS: Numbers of people who gave up after a year: among the 12 smokers in the team, 4 (33%); 8 out of the 103 people who received counselling (7.7%) (Cl 2.60-12.94%); zero out of the 20 people who never attended for treatment or left the course after the first interview; zero out of the 20 treated in a group; and 8 out of the 20 treated individually (40%) (Cl 18.53-61.47%, p less than 0.001 regarding the treatment in group). CONCLUSIONS: The results support the effectiveness of three of the methods of intervention at the Primary Care level: intervention within the team itself, systematic counselling to smokers and treatment using nicotine chewing-gum and individual support. Group support must be reserved for staff with a specific training on Tobacco Addiction and the dynamics of group treatment.

Chewing Gum↗
PubMed1992

Serum TNF in patients with severe malaria treated by exchange transfusion.

The authors report on three cases of severe P. falciparum malaria successfully treated by iv quinine and exchange transfusion. Serum concentrations of Tumor Necrosis Factor (TNF) were determined before and during treatment. After an initial decrease, serum levels of TNF remained markedly elevated during the first 48 hours despite exchange transfusion. Though exchange transfusion accelerates the elimination of parasites from the blood, it seems to have no immediate effects on reducing serum levels of cytokines such as TNF.

Adult↗
PubMed1991

A prospective evaluation of the immediate reproducibility of the signal-averaged ECG.

The purpose of this investigation was to prospectively evaluate the immediate reproducibility of the signal-averaged electrocardiogram (SAECG). A total of 114 patients undergoing evaluation for ventricular arrhythmias were enrolled in this protocol. Two consecutive SAECG's (40 Hz bidirectional high-pass filtering with a computer-automated system) were performed 10 minutes apart. Abnormal SAECG parameters were defined as (1) vector QRS duration more than 120 msec, (2) terminal root mean square (RMS) voltage less than 20 microV, and (3) low-amplitude signal (LAS) duration more than 40 msec. An SAECG was defined as abnormal if at least one vector parameter was abnormal. There was close correlation between vector parameters during the two SAECG observations: QRS duration had the highest reproducibility (r2 = 0.97, p less than 0.001) followed by terminal RMS voltage (r2 = 0.92, p less than 0.001), and LAS duration (r2 = 0.90, p less than 0.001). The mean (+/- SD) percentage of change between the two recordings was 2% +/- 2% of the QRS duration, 13% +/- 22% for terminal RMS voltage, and 7% +/- 11% for LAS duration. The reproducibility of an initially normal SAECG was 92% and of an initially abnormal SAECG, 96%. Seventeen patients (15%) had a change in one of the three vector parameters between the two recordings. There were no clinically significant differences between the 17 patients in whom the SAECG was nonreproducible and the 97 patients in whom the SAECG was reproducible. However, reproducibility was significantly higher in patients with an initially normal versus an initially abnormal SAECG (92% vs 76%, p = 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)

Arrhythmias, Cardiac↗
PubMed1991

A truncated P element is inserted in the transcribed region of the Cu,Zn SOD gene of an SOD "null" strain of Drosophila melanogaster.

The decreased Cu,Zn SOD activity (less than 5%) in a "null" SODCA1 Drosophila melanogaster strain isolated in our laboratory is due to the insertion of a truncated P element into the transcribed region of the Cu,Zn SOD gene. Using a cDNA Cu,Zn SOD probe from a wild type D. melanogaster (F allele) we isolated an EcoRI Cu,Zn SOD clone from an EMBL3 genomic library of the SODCA1 strain, subcloned it, restriction-mapped and partially sequenced it. The 2.5 kb clone consists of a wild-type 1.84 kb EcoRI fragment containing the Cu,Zn SOD gene previously isolated in our laboratory, with an insertion of 0.68 kb derived (by an internal deletion) from an autonomous, 2.9 kb P element. The insertion starts 21 bp upstream from the coding sequence and causes an 8 bp target site duplication characteristic of P elements. A point mutation in the second exon results in a replacement of Asn by Lys at position 96, confirming that the mature protein encoded by the SOCCA1 is the same one encoded by the S allele, commonly found in natural populations. The diminished expression of SODCA1 allele is most possibly due to a reduction of the rate of transcription attributable to the insertion of the P element.

Alleles↗
PubMed1991

[Factor D of complement: Uremic toxin?].

Factor D is an essential enzyme of the alternative pathway of complement. Its catabolism is mainly renal. The concentration of factor D increases with renal failure, and is approximately 10-fold higher in patients with end-stage renal disease. The accumulation of factor D is responsible for an enhancement of alternative pathway activation. Whether this excess of factor D has pathophysiological consequences remains to be determined, however, complement activation might participate in adverse effects during hemodialysis and in the progression of renal injury.

Complement Factor D↗