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Biomedical subjects

A Albertini

Publications and source records attributed to A Albertini.

At least 91 records · Page 5Linked to original sources
PubMed1993

Progesterone induced expression of alkaline phosphatase is associated with a secretory phenotype in T47D breast cancer cells.

In our previous work we reported on stimulation of a tissue unspecific alkaline phosphatase (liver/bone/kidney, L/B/K) in the human breast cancer cell line T47D by progestins. Here we show that in these cells the synthetic progestin R5020 (Promegestone) induces transcription of a 2.7-kilobase ALP mRNA. In this cel line, maximal induction is reached after 24 hours, decreases to 50% after 72 hours and is sensitive to inhibitors of protein synthesis. The induction of ALP mRNA and enzyme activity is specific for R5020 and dexamethasone and is completely inhibited in the presence of 10(-7) M RU486. When treated with R5020 for 48 hours, T47D cells exhibit a substantially altered phenotype, lipid vacuoles accumulate all over the cytoplasm, conferring to the cells a secretory morphology. This phenotype is associated with increased ALP enzyme activity which is also maximal at 48 hours. This effect is progestin specific since other steroids do not lead to the same macroscopical changes.

Alkaline Phosphatase↗
PubMed1993

Extensive deletion of immunoglobulin heavy chain constant region genes in the absence of recurrent infections: when is IgG subclass deficiency clinically relevant?

This report describes two children with undetectable serum levels of IgA1, IgG2, IgG4, and IgE due to a homozygous deletion encompassing the A1-E genes. The father is a heterozygous carrier of the same deletion and the mother a heterozygous compound carrying the deletion on one chromosome and duplication on the other. In both children, serum IgG, IgG1, and IgG3 were higher than in normal children and IgG antibody response to tetanus toxoid and polysaccharide antigens was normal with increased IgG1 and IgG3 response compared to controls. The latter can be interpreted as a compensatory mechanism for the subclass deficit and may explain the lack of significant infections in both children. The importance of distinguishing IgG subclass deficiency due to gene deletion from that due to immunoregulatory dysfunction is discussed.

Adolescent↗
PubMed1993

Digital subtraction angiography--a new approach to brain death determination in the newborn.

The diagnosis of brain death in the newborn infants is elusive and often difficult. The lack of cerebral blood flow has become an identified criterion for loss of cerebral function. The diagnosis can be obtained by the technique of digital subtraction angiography, which is presented in two case reports demonstrating the utility of this technique.

Angiography, Digital Subtraction↗
PubMed1993

Real-time kinetic analysis applied to the production of bispecific monoclonal antibodies for radioimmunodetection of cancer.

An automated biosensor system designed for measuring molecular interactions in real-time and without labelling of the reactants has been used to evaluate the association/dissociation rate and affinity constants of bivalent monoclonal antibodies and a monovalent bispecific monoclonal antibody. Observed differences in affinity between parental and bispecific antibody produced were related to the association rate constants, since the dissociation rate constants were in the same range. Values were also closely related to radioimmunochemical data. These results indicate that the biosensor system, besides presenting several advantages for characterizing antigen-antibody interaction, is valuable for selecting monoclonal antibodies with properties which might be useful in the development of bispecific monoclonal antibodies.

Antibodies, Bispecific↗
PubMed1993

Plasma levels of atrial natriuretic factor (ANF) and urinary excretion of ANF, arginine vasopressin and catecholamines in children with congenital heart disease: effect of cardiac surgery.

We studied the changes in the plasma concentration of atrial natriuretic factor (ANF) and the urinary excretion of ANF, arginine vasopressin (AVP) and catecholamines in 22 children with congenital heart disease, divided into two groups. Group 1 included 11 children with congestive heart failure (CHF), treated with digitalis and diuretics. Group 2 included 11 children without CHF and without medical treatment. Each group was compared with a control group of 15 healthy, age-matched children. The plasma concentration of ANF was raised in both groups, but it was significantly higher in group 1 (235.5 +/- 82.9 pg/ml), compared to group 2 (48.4 +/- 29.4 pg/ml, P < 0.002). Urinary excretion of ANF was measurable in both groups and higher in group 1 (185.9 +/- 116.2 pg/kg per h) than in group 2 (48.5 +/- 30.7 pg/kg per h), but not significantly so. Urinary excretion of AVP and catecholamines was not different in children with congenital heart disease and healthy children. Twenty-four hours after surgery, plasma ANF diminished in group 1 (from 235.5 +/- 82.9 to 93.4 +/- 53.8 pg/ml, P < 0.003), but did not change in group 2. The urinary excretion of ANF was unchanged in both groups. In contrast, urinary excretion of AVP and catecholamines rose significantly in both groups. These data show that plasma ANF is increased in children with congenital heart disease, even in the absence of CHF. The measurement of urinary ANF is less reliable than a plasma assay. The postoperative increases in AVP and catecholamine urinary excretions could be responsible for the vasoconstriction and water retention typical of the postoperative period.

Adolescent↗
PubMed1993

Kinetic analysis of TNF-alpha oligomer-monomer transition by surface plasmon resonance and immunochemical methods.

In this work we have studied the kinetic parameters of oligomeric tumour necrosis factor alpha (TNF-alpha) dissociation using biospecific interaction analysis (BIA), based on surface plasmon resonance (SPR) of TNF-alpha immobilized on a sensor chip, and by an ELISA technique able to detect TNF-alpha oligomers in solution. Validation studies, carried out with sensor chips bearing TNF-alpha oligomers or bovine albumin monomers, verified that: (a) TNF-alpha can be immobilized in the oligomeric form onto sensor chips; (b) the covalent linkage between TNF-alpha and sensor chips is stable under the experimental conditions; (c) TNF-alpha monomers are present on the sensor chips after dissociation; (d) immobilization and dissociation rate constant (kdiss) measurements are reproducible. The kdiss of recombinant TNF-alpha, measured under non denaturing conditions at pH 7.4 by BIA and ELISA were in good agreement, being 0.92 x 10(-5)/s and 1.1 x 10(-5)/s respectively (corresponding to a half life of about 20.9 h and 17.5 h, respectively). The dissociation rate was found to be significantly affected by the presence of detergents and by the pH of the solution, suggesting that TNF-alpha, at low concentrations, exists in solution with different molecular forms depending on the time of storage and buffer composition. Real-time BIA is rapid and does not require particular antibodies or reagents. Thus, the stability of the quaternary structure of natural or recombinant TNF-alpha from human or animal species as well as that of other oligomeric cytokines can probably be studied using this method.

Animals↗
PubMed1993

Application of molecular analysis to genetic counseling in the Wiskott-Aldrich syndrome (WAS).

The Wiskott-Aldrich syndrome (WAS) is a severe X-linked, recessive disorder, with a high mortality rate at early age due to hemorrhages, infections, and lymphoid malignancies. The molecular pathogenesis of the disease is unknown. Carrier females of WAS are clinically and immunologically normal, thus precluding carrier detection by simple laboratory tests. Major advances in molecular genetics have allowed mapping of the WAS gene to the pericentromeric short arm of the X chromosome, and have made carrier detection and prenatal diagnosis feasible by segregation analysis with closely linked polymorphic DNA markers. Furthermore, the observation that carriers of WAS exhibit a unilateral inactivation of the X chromosome in hematopoietic cells has provided a new tool for carrier detection. However, critical interpretation of molecular analysis data is essential to provide accurate genetic counseling to WAS families.

Dosage Compensation, Genetic↗
PubMed1993

Gene expression of cardiac beta 1-adrenergic receptors during the development of hypertension in spontaneously hypertensive rats.

OBJECTIVE: To study adrenergic receptors in the heart tissues of genetically hypertensive rats by evaluating the gene expression and the membrane protein density of beta 1-adrenergic receptors using steady-state messenger RNA (mRNA) levels and a radioligand binding assay, respectively. DESIGN: We compared prehypertensive (5-week-old) and early-hypertensive (13-week-old) spontaneously hypertensive rats (SHR) with age-matched Wistar-Kyoto (WKY) normotensive control rats. METHODS: Polyadenylated RNA was extracted from individual hearts and analysed by the slot-blot technique using a beta 1-adrenergic receptor complementary DNA probe. beta-Adrenergic receptors in myocardial membranes were studied by radioligand binding assay using [125I]-cyanopindolol and the beta 1- and beta 2-selective antagonists CGP 207.12A and ICI 118.551, respectively. RESULTS: beta 1-Adrenergic receptor mRNA levels were slightly higher, and membrane protein density was similar in prehypertensive SHR and age-matched WKY rats. However, both beta 1-adrenergic receptor mRNA levels and beta 1-adrenergic receptor density were lower in the hypertensive SHR than in the control rats. beta 1-Adrenergic receptor mRNA was significantly reduced in older rats of both strains, and this reduction was most evident in the SHR. CONCLUSIONS: The absence of downregulation of beta 1-adrenergic receptors in young SHR, despite published data indicating a higher cardiac noradrenaline turnover than in WKY rats, may suggest that the cardiac hyperadrenergic activity observed in prehypertensive SHR is maintained, at least in part, by the participation of peripheral, postsynaptic component(s) involving beta 1-adrenergic receptor dysregulation. In addition, the present data suggest that the previously reported evidence of an age-related decrease in cardiac beta 1-adrenergic receptors in rats may be determined at the transcriptional level.

Animals↗
PubMed1993

Detection of BCR/ABL transcripts in chronic myeloid leukaemia by polymerase chain reaction and DNA enzyme immunoassay: a DNA probe assay without DNA labelling.

The detection of the t(9;22) translocation, typical of chronic myeloid leukaemia (CML), can be accomplished by cytogenetical detection of Philadelphia (Ph1) chromosome or by molecular analysis of the bcr/abl fusion gene with nucleic acid probes after amplification by polymerase chain reaction (PCR). PCR-based approaches are now widely used for follow up of CML patients during therapy or after bone marrow transplantation (BMT). We describe here a microtitre, colorimetric assay (DNA Enzyme Immunoassay, DEIA) for analysis of t(9;22) translocation after enzymatical amplification of RNA from CML patients. This assay is based on the use of a monoclonal antibody specifically reacting with double stranded DNA, i.e. with hybridized DNA. The assay represents a nonisotopic alternative to other current hybridization assays and requires no modifications of primers, probe or target DNA.

Adult↗
PubMed1993

The effect of the concentration ratio of avidin and biotin on a single step sandwich enzyme immunoassay.

The interaction between biotin and avidin, used in a single-step enzyme-immunoassay for ferritin determination, has been studied. The antigen is simultaneously bound by an antibody coated to a polystyrene bead and by an antibody coupled to biotin which reacts with avidin conjugated to peroxidase. We have assessed the optimal ratio between avidin, conjugated to peroxidase, and biotin, coupled to antibodies, to give rise to the best signal for a quantitative enzyme-immunoassay. We have found that a careful balance between biotinylated antibody and conjugated avidin is necessary for our purpose and a biotinylated antibody excess should be avoided since it causes a signal decrease. This ratio is uninfluenced by both the presence and the absence of the antigen. Thus, an avidin-biotin single-step methodology, which has proved to be reliable for routine use, was developed.

Antigen-Antibody Complex↗
PubMed1992

New heteromyeloma cell lines for the production of human monoclonal antibodies.

The aim of this study was to establish hybridomas capable of long-term production of human monoclonal antibodies (mAbs). Heterohybridization was performed between the mouse myeloma cell line P3X63Ag8.653 and activated human peripheral blood lymphocytes (PBL). In order to achieve better retention of human chromosomes, as well as to improve the stability of the heterohybrids, one HAT-sensitive immunoglobulin (Ig)-non-secreting human x mouse (h x m) heteromyeloma was fused for a second time with activated human PBL. In this way, a panel of HAT-sensitive Ig-non-secreting h x h x m heteromyelomas was obtained and tested for its ability to generate stable human Ig-secreting heterohybrids with activated human PBL. Six lines were selected on the basis of their enhanced characteristics of fusion efficiency and genetic stability. When fused with in vitro immunized human PBL, they generated several h x h x h x m hybridomas stably secreting high yields (10-23 micrograms/ml/24 h) of human mAbs reactive with recombinant HBV core antigen (rHBcAg). Moreover, a continuous production of human Ig was observed when two h x h x m heteromyelomas, previously made ouabaine-resistant, were hybridized with EBV-transformed lymphoblastoid cell lines. These h x h x m heteromyelomas are ideal fusion partners for the production of human mAbs.

Animals↗
PubMed1992

Evidence of H- and L-chains have co-operative roles in the iron-uptake mechanism of human ferritin.

The ability to incorporate iron in vitro was studied in homopolymers of human ferritin L-chain, human ferritin H-chain and its variants and in homopolymer mixtures. The H-chain variants carried amino acid substitutions in the ferroxidase centre and/or in carboxy residues on the cavity surface. Iron incorporation was examined by gel electrophoresis of the reaction products by staining for iron and protein. It was found that inactivation of the ferroxidase centre combined with the substitution of four carboxy groups on the cavity abolished the ability of H-chain ferritin to incorporate iron. Competition experiments with limited amounts of iron showed that, at neutral pH, L-chain ferritin is more efficient in forming iron cores than the H-chain variants altered at the ferroxidase activity or in the cavity. Competition experiments at pH 5.5 demonstrated that L-chain apoferritin is able to incorporate iron only when in the presence of H-chain variants with ferroxidase activity. The results indicate that L-chain apoferritin has a higher capacity than the H-chain apoferritin to induce iron-core nucleation, whereas H-chain ferritin is superior in promoting Fe(II) oxidation. The finding of cooperative roles of the H- and L-chains in ferritin iron uptake provides a clue to understanding the biological function of isoferritins.

Apoferritins↗
PubMed1992

Characterization of a Ca (II)-independent monoclonal antibody that lacks reactivity with Gla-domainless human factor VII.

Eight murine monoclonal antibodies to human factor VII have been produced and characterized. As tested by direct ELISA, all antibodies bound to immobilized factor VII and this interaction did not require Ca(II). On immunoblotting three antibodies reacted only with native factor VII and activated factor VII (rFVIIa), two with native factor VII, activated factor VII and with the light chain of reduced activated factor VII and three with reduced activated factor VIIa (light chain) only. When coupled to Sepharose five antibodies were capable of immunodepleting factor VII from normal plasma and inhibited coagulant activity with varied potencies. Among these antibodies, one (10C12.2) had peculiar characteristics, lacking reactivity with Gladomainless FVII and with the Ca(II)-dependent conformation. This antibody has been used to develop a two-site ELISA which appears to measure native factor VII but not poorly carboxylated, inactive forms of the molecule.

Antibodies, Monoclonal↗
PubMed1992

Enzyme-linked immunosorbent assay of human factor VII based upon a monoclonal antibody that recognizes the native conformation of the protein.

An enzyme-linked immunoassay (ELISA) was developed for measuring human factor VII antigen using two monoclonal antibodies, one of them reacting only with fully carboxylated factor VII. This assay permits to measure factor VII antigen in concentrations ranging from 0.78 to 100 ng/ml, with within- and between-assay coefficients of variation of less than 7%. In 53 normal subjects, 32 patients with liver cirrhosis, 21 pregnant women and 53 patients on oral anticoagulant therapy the plasma levels of FVII antigen were very similar to those of factor VII coagulant activity measured with a bioassay. The ELISA also gave very similar values of factor VII antigen in plasma and in serum, and in plasma before and after exposure to cold, indicating that the assay is not affected by factor VII activation. In five of 8 patients with severe congenital deficiency of factor VII values of factor VII antigen were higher than those of factor VII activity. The close concordance of factor VII values obtained by ELISA and bioassay in the majority of plasmas, including those from patients on oral anticoagulant therapy, indicates that the assay measures native factor VII and can perhaps replace the bioassay systems in clinical conditions associated with normal or high levels of factor VII.

Adult↗