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Biomedical subjects

F Marcucci

Publications and source records attributed to F Marcucci.

At least 37 records · Page 2Linked to original sources
PubMed1994

Changes in nasal specific IgE to mites after periods of allergen exposure-avoidance: a comparison with serum levels.

Variations of serum and nasal specific IgE to Dermatophagoides pteronyssinus (Der p) during alternate periods of antigen avoidance-exposure have been evaluated with an open design in a group of allergic children with asthma and rhinitis at the residential house Istituto Pio XII (Misurina, BL, Italy), at 1756 m, in the Italian Dolomites. A method based on direct incubation of allergen coupled substrate on the nasal mucosa has been employed to evaluate the levels of nasal IgE. Serum specific IgE decreased respectively from (median) 117-89.3 kU/l (P < 0.001) during an initial period of 3 months of allergen avoidance and from 88.2 to 78.4 kU/l (P < 0.0002) during a subsequent period of allergen avoidance. No significant increase in serum specific IgE was, in contrast, observed during two periods, 22 and 9 days, of antigen exposure, changing respectively from 89.3 to 88.2 and from 78.4 to 89.1 kU/l. In contrast, nasal IgE has been significantly influenced by the alternate periods of antigen exposure-avoidance, showing a decrease from 19.75 to 4.01 kU/l (P < 0.0001) after the initial period of avoidance, followed by an increase to 9.95 kU/l (P < 0.0001) after 22 days of exposure. A significant decrease to a value of 2.37 kU/l (P < 0.0001) was also observed during the subsequent period of avoidance, followed again by an increase to 7.87 kU/l (P < 0.002) after 9 days of exposure. The evaluation of the kinetics of changes in nasal specific IgE revealed a significant decrease (P < 0.01) as soon as antigen avoidance was implemented for 3 days.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Suramin induces deoligomerization of human tumor necrosis factor alpha.

Suramin inhibits the biological activity of human tumor necrosis factor alpha (TNF) through a direct action on the ligand rather than on its receptors (Grazioli, L., Alzani, R., Ciomei, M., Mariani, M., Restivo, A., Cozzi, E., and Marcucci, F. (1992) Int. J. Immunopharmacol. 14, 637-642). In order to clarify the mechanism whereby suramin leads to inhibition of TNF, we investigated the possibility that suramin might modify the quaternary structure of TNF which is biologically active as a trimer. For this purpose we used a new assay (double streptavidin sandwich assay) designed for the rapid detection of oligomer-monomer conversion of proteins. Taking advantage of this assay we observed, upon incubation with suramin, dissociation of TNF. Suramin-induced dissociation of TNF was confirmed by gel filtration chromatography. Under conditions of partial dissociation, two molecular species were separated. One of higher molecular weight, corresponding to trimeric TNF, was biologically active, whereas the other, corresponding to monomeric TNF, was inactive. These results are at variance with others recently reported, where suramin has been shown to induce microaggregation of several polypeptides (Middaugh, C. R., Mach, H., Burke, C. J., Volkin, D. B., Dabora, J. M., Tsai, P. K., Bruner, M. W., Ryan, J. A., and Marfia, K. E. (1992) Biochemistry 31, 9016-9024). This suggests that suramin inhibits the bioactivity of different protein molecules through opposite effects on their quaternary structure. The present results are, to our knowledge, the first demonstration of a drug inhibiting a target molecule through dissociation of its quaternary structure.

Animals↗
PubMed1993

Evidences that syngeneic alpha-type anti-idiotypic antibodies may non-competitively inhibit idiotype/oligomeric antigen interactions by affecting idiotype avidity.

The effects of syngeneic anti-Id antibodies on the multivalent interaction between human TNF-alpha, a homotrimeric Ag, and an anti-TNF mAb (mAb(1)78) have been studied. Eight anti-mAb(1)78 Ig secreting hybridoma, able to inhibit TNF binding in a competitive or non-competitive mode, have been generated. Two representative clones (mAb(2)1G3 and mAb(2)9F1) were selected for studying the inhibition mechanism of TNF-mAb(1)78 interaction. Idiotype-paratope topography studies indicated that mAb(2)1G3 (IgG2a) and mAb(2)9F1 (IgG1) bind two sterically distinct idiotopes on mAb(1)78 (IgG1) V regions. In particular, mAb(2)1G3 was found to bind an idiotope located within (or spatially close to) the Ag combining site suggesting that competitive inhibition of TNF binding to mAb(1)78 by mAb(2)1G3 occurs through paratope blockade. On the other hand, mAb(2)9F1 recognizes an idiotope located outside the paratope, being able to bind mAb(1)78 even in the presence of saturating amounts of TNF. mAb(1)78-TNF molar ratio in complexes, at stoichiometric equivalence, was unchanged in the presence of a large excess of mAb(2)9F1, suggesting that the functional bivalency of mAb(1)78 was not impaired by this anti-Id antibody. However, bivalent mAb(2)9F1 was able to partially inhibit the binding of bivalent mAb(1)78 to oligomeric TNF in liquid-phase as well as in solid-phase assays, whereas no inhibition was observed with monovalent mAb(2)9F1-F(ab) or mAb(1)78-F(ab). This suggests that inhibition is based on a decrease of the avidity of bivalent mAb(1)78 and not on allosteric effects on antigen binding sites. The effect of mAb(2)9F1 on mAb(1)78 arm flexibility and paratope orientation is discussed. In conclusion, the results indicate that anti-Id antibodies may inhibit Ag-antibody multivalent interactions by paratope blockade or by affecting the antibody avidity.

Animals↗
PubMed1993

Expression, in Escherichia coli, of a soluble human tumor necrosis factor receptor.

We have expressed in Escherichia coli a soluble, truncated form of the human 55 kDa Tumor Necrosis Factor (TNF) receptor. For this purpose a plasmid was constructed which contains the extracellular domain of the 55 kDa TNF receptor fused to the coding sequence of the IgG binding domains of protein A from Staphylococcus aureus. The fusion product (TNFR-PA) obtained in E. coli is a soluble protein which bound human TNF alpha (huTNF alpha) with high affinity. In ligand-blotting experiments huTNF alpha bound to a single 52 kDa protein, a molecular mass corresponding to that expected for the monomeric fusion product. In gel filtration experiments binding activity was recovered from fractions that eluted at a volume corresponding to 140-150 kDa. TNFR-PA neutralized huTNF alpha in an in vitro cytotoxicity assay.

Chromatography, Gel↗
PubMed1993

Mode of interaction between tumor necrosis factor alpha and a monoclonal antibody expressing a recurrent idiotype.

Tumor Necrosis Factor alpha (TNF alpha) is an inflammatory cytokine which exists mainly as a 51kD complex built up of 3 identical, noncovalently-linked polypeptide subunits. We have raised monoclonal antibodies (mAb) against human TNF alpha (huTNF alpha). One of these mAb (mAb78, mouse IgG1k) was studied in detail. mAb78 expresses a recurrent idiotype typical of the BALB/c anti-huTNF alpha antibody response. HuTNF alpha bound to mAb78 with an affinity constant (Kobs) of 3.2 x 10(10)M-1. The number of huTNF alpha-binding sites per mAb78 molecule was approximately 0.7. At concentrations higher than the Kobs mAb78 neutralized huTNF alpha at a approximately 1.3:1 molar ratio. mAb78 precipitated huTNF alpha in a double immunodiffusion assay in agar. Gel-filtration experiments of mAb78-huTNF alpha mixtures, that had been set up in large antigen excess, detected complexes of 570 kD as the smallest ones formed under these conditions. We propose that these results are accommodated best by a model according to which cyclic complexes built up of 3 mAb78 and 2 huTNF alpha molecules are the smallest units formed upon interaction of the reagents. In view of this model we discuss how huTNF alpha and mAb78 can undergo a precipitin reaction.

Animals↗
PubMed1993

Double blind, placebo controlled study of nedocromil sodium in asthma.

After a two week baseline, 209 asthmatic children (mean age 10 years, range 6-17) were randomly allocated to receive 4 mg nedocromil sodium (n = 110) or placebo (n = 99) four times daily for 12 weeks in addition to their current treatment. The children completed daily diary cards and visited the clinic at four week intervals. Statistically significant differences in favour of nedocromil sodium were seen for clinician assessment of asthma severity and diary card symptom scores, pulmonary function and inhaled beta 2 bronchodilator use. Total symptom score decreased by 50% from baseline in the nedocromil sodium group and by 9% in the placebo group during the final four weeks. Nedocromil sodium was considered very or moderately effective by 78% of children/parents (placebo 59%) and 73% of clinicians (placebo 50%). Nausea, headache and sleepiness, and dyspnoea led to withdrawal of one child from nedocromil sodium and placebo treatments, respectively. Reports of sore throat and headache were marginally greater with the nedocromil sodium treatment. It is concluded that nedocromil sodium was both effective and safe in the treatment of asthma in children.

Adolescent↗
PubMed1992

Detection of mycoplasma contamination through modulation (stimulation or inhibition) of thymidine incorporation by unstimulated mouse spleen cells.

In the present report we describe a rapid and sensitive assay for mycoplasma detection in cell cultures. The assay is based on the ability of contaminated culture supernatants to modulate [3H]TdR incorporation by unstimulated mouse splenocytes. Several mycoplasma species (Mycoplasma orale, culturable and non-culturable strains of Mycoplasma hyorhinis) inhibited [3H]TdR incorporation and permitted the detection of some contaminated cell cultures that would otherwise have escaped detection in assays measuring [3H]TdR incorporation by mitogen-stimulated splenocytes. On the other hand, several other mycoplasma species (Mycoplasma arginini, Mycoplasma hominis) strongly enhanced [3H]TdR incorporation by unstimulated splenocytes. This enhancement was optimally detectable on day 2 after initiation of the cultures. The sensitivity of the assay was determined for a mycoplasma species (culturable M. hyorhinis) that inhibited as well as for one (M. arginini) that enhanced [3H]TdR incorporation. In both cases, the sensitivity was such that 1-3 x 10(2) mycoplasma colony-forming units (CFU) could be detected.

Animals↗
PubMed1992

Oligomeric tumour necrosis factor alpha slowly converts into inactive forms at bioactive levels.

The stability of oligomeric human tumour necrasis factor alpha (TNF) at bioactive levels has been studied by two immunoenzymatic assays: one able to specifically detect oligomeric and not monomeric TNF (O-e.l.i.s.a.) and the other able to detect both forms (OM-e.l.i.s.a.). The selectivity of O-e.l.i.s.a. and OM-e.l.i.s.a. for oligomeric and monomeric TNF was demonstrated with isolated forms prepared by partial dissociation of recombinant TNF with 10% (v/v) dimethyl sulphoxide and gel-filtration h.p.l.c. Evidence for instability of oligomeric TNF were obtained in physiological buffers, as well as in serum and cell-culture supernatants, as a function of TNF concentration. In particular, only a half of the TNF antigen was recovered in the oligomeric form after 72 h incubation (37 degrees C) at 0.12 nM, whereas no apparent dissociation was detected at 4 nM. The structural changes observed at picomolar concentrations were rapidly reversed by raising the concentration of TNF to about 2 nM by ultrafiltration, suggesting that subunit dissociation and reassociation reactions occur in the picomolar and nanomolar range respectively. The cytolytic activity of L-M cells correlates with oligomeric-TNF levels after incubation at picomolar concentrations. Moreover, isolated oligomeric TNF was cytotoxic towards L-M cells, whereas monomeric TNF was virtually inactive. In conclusion, the results suggest that bioactive oligomeric TNF is unstable at picomolar levels and slowly converts into inactive monomers, supporting the hypothesis that quaternary-structure changes in TNF may contribute to the fine regulation of TNF cytotoxicity.

Animals↗
PubMed1992

Antigenic regions of tumor necrosis factor alpha and their topographic relationships with structural/functional domains.

The immunogenic regions of human Tumor Necrosis Factor alpha (huTNF) have been mapped by studying the interaction between various mouse anti-huTNF sera and synthetic huTNF fragments, spanning the entire sequence of huTNF. Three main immunogenic regions were identified within residues 1-23, 95-116 and 137-157 of huTNF and two other less immunogenic regions within residues 117-136 and 37-55. The same huTNF regions were found to contain antigenic sites by binding studies with cognate anti-peptide sera. Competitive binding experiments with shorter synthetic subfragments provided evidence for the location of strong antigenic sites within residues 1-10, 17-23, 104-112 and 137-143. In particular the immunodominant site was found to be located within residues 104-112. huTNF regions corresponding to residues 24-36, 56-75, 76-94, and 147-157 resulted to be not or poorly antigenic. However, treatment of huTNF with Triton X-100 under conditions that partially dissociate the oligomeric quaternary structure resulted in the exposition of sites recognized by sera against peptides huTNF [56-75] and huTNF [76-94], suggesting that antigenic sites not accessible in the oligomeric huTNF are exposed in the dissociated form. The principal antigenic sites in the oligomeric molecule are localized in the flexible N-terminal part and in hydrophilic segments located in the "middle/top" region of the molecule, opposite to the C-terminus. Protein segments of the "bottom" region, close to the C-terminus, were poorly immunoreactive. Neutralization assays of TNF cytolytic activity on L-M cells showed that binding of antibodies to epitopes located in the "middle/top" regions of huTNF does not affect its cytolytic activity, supporting the hypothesis of a receptor binding site location at the "bottom" of TNF trimer.

Animals↗
PubMed1992

Inhibitory effect of suramin on receptor binding and cytotoxic activity of tumor necrosis factor alpha.

The effect of suramin on the binding of human Tumor Necrosis Factor alpha (huTNF alpha) to specific cell-surface receptors as well as on its cytotoxic activity in vitro was investigated. Suramin inhibited both activities in a dose-dependent manner. Experiments designed to discriminate if suramin exerted its inhibitory activity on the ligand or on the receptor showed that the ligand (huTNF alpha) was the most likely target for suramin in this system. These results may explain, in part, the immunosuppressive activities of suramin that have been observed in vivo and suggest that suramin could be useful in those disease states in which hyperproduction of huTNF alpha has been shown to play a pathogenic role.

Cell Survival↗
PubMed1989

In vivo effects in mice of an anti-T cell immunotoxin.

We have evaluated both the proliferative response as well as the Thy-1 Ag expression of lymphocytes from mice treated in vivo with an anti-Thy-1 immunotoxin (IT). The IT was a rat IgG2c mAb recognizing the Thy-1 Ag, disulfide-linked to a ribosome-inactivating protein isolated from the seeds of the plant Saponaria officinalis (soapwort). Toxicity studies showed that a single i.v. injection of doses up to 20 micrograms IT/mouse was well tolerated and allowed indefinite survival. The Con A-induced proliferative response of spleen cells from mice killed 1 day after treatment with sublethal doses of IT was inhibited in a dose-dependent manner, with complete inhibition observed at doses of greater than or equal to 5 micrograms IT/mouse. Control experiments showed that the inhibition was due to the IT and not to its single components. Moreover, the IT effect was abolished by a large (100-fold) excess of anti-Thy-1 mAb alone given concurrently, but not by an unrelated, isotype-matched rat mAb. At all IT doses, the proliferative response to a B cell mitogen (LPS) was normal. Kinetic studies showed a time- and dose-dependent reconstitution of Con A responsiveness. In limiting dilution cultures of spleen cells from mice treated with 5 micrograms IT 1 or 4 days before death, a 97% depletion of T lymphocytes capable of proliferation was observed. Limiting dilution cultures showed that also the thymus of IT-treated mice was depleted by more than 90% of growth-competent T lymphocytes. Cytofluorographic studies of Thy-1+ cells from the spleens of IT-treated mice gave results which did not correlate with those obtained in functional assays. Thus, a dose-dependent reduction, followed by a time-dependent reconstitution of Thy-1+ cells was observed in this case too, but the depletion occurred at later time points and was less complete than that observed in functional assays. Moreover, the mean fluorescence intensity of the residual Thy-1+ cells decreased below normal levels.

Animals↗
PubMed1989

A new method for IgE detection in nasal mucosa.

A new method for determining IgE antibodies on mucosal surfaces has been developed with the purpose of overcoming the main problems of nasal secretion sampling and standardization. The method is based on the principle of performing the incubation of the solid-phase coupled allergen with IgE antibody directly on the mucosal surface by means of a proper applicator. About two times higher values of specific IgE have been obtained with 5 min incubation on septal mucosa, behind the internal ostium, than with 3 hr in-vitro incubation with native secretion. In a study of 53 children with allergic rhinitis and asthma and 10 healthy non-atopic controls the sensitivity and specificity of this new method were evident. Because of its reliability and easy execution the new method could be widely used in diagnosis of allergic disease. Further, it seems to offer a good opportunity to study the IgE-mediated reaction in the target organ more extensively.

Child↗
PubMed1986

An interferon analogue, [Ala 30,32,33]HuIFN-alpha 2, acting as a HuIFN-alpha 2 antagonist on bovine cells.

We have studied the biological and receptor binding properties of a human alpha 2-interferon (HuIFN-alpha 2) analogue, [Ala30,32,33] HuIFN-alpha 2, which is shown in the accompanying paper (1) to be biologically inactive on homologous cells. Here we demonstrate that this analogue is also devoid of biological activity on bovine MDBK cells. However, whereas the analogue did not inhibit the binding of radiolabeled HuIFN-alpha 2 to WISH cells, it did compete for binding to receptors on the bovine cells. This behavior suggested that [Ala30,32,33] HuIFN-alpha 2 could act as an antagonist of HuIFN-alpha 2 on bovine cells and indeed coaddition of the analogue and native HuIFN-alpha 2 to MDBK cells competitively inhibited both the antiviral and antiproliferative activity of HuIFN-alpha 2.

Animals↗
PubMed1986

Production of interferon-beta by murine T-cell lines induced by 10-carboxymethyl-9-acridanone.

Besides the established T-cell property of producing gamma interferon (IFN-gamma), murine T cells additionally possess the ability to produce IFN-alpha and IFN-beta when appropriate inducers such as 10-carboxymethyl-9-acridanone (CMA) or Newcastle disease virus (NDV) are used. Interleukin 2 (IL-2)-dependent murine T-cell lines, but not purified resting splenic T cells, responded to CMA and NDV with production of IFN-alpha, beta. The IFN production by these T cells was not restricted to a special subset, since T cells expressing the Lyt 1+2- and the Lyt 1-2+ phenotype responded to these inducers with IFN production. After prolonged passaging of the T-cell lines in IL-2-containing medium, the ability to respond to CMA with production of antiviral activity was sustained longer than the ability for concanavalin A-induced IFN-gamma production. Whereas the NDV-induced T-cell supernates contained both IFN-alpha and IFN-beta, the induction with CMA resulted exclusively in the synthesis of IFN-beta by the T-cell lines.

Acridines↗
PubMed1985

Effect of swimming on protein degradation: 3-methylhistidine and creatinine excretion.

The effect of 5-km noncompetitive swimming (moderate exercise) and 2-km competitive speed swimming (intensive exercise) on protein breakdown was studied in a group of young male volunteers (16-20 years old) who followed a 3-MH-free diet throughout the study. Urinary 3-MH and creatinine were determined over a period of 24 and 48 hr as an index of protein degradation. Basal 3-MH levels in the two groups of swimmers were 2.85 and 3.07 mumole X kg-1 X day-1. Mean rates of 3-MH excretion were, respectively, 1.54 and 1.94 mumole X kg-1 X day-1 for the 48 hr after moderate exercise and the 24 hr after intensive exercise. The decrease in 3-MH urinary excretion was still evident when calculated as the urinary 3-MH-to-creatinine ratio.

Adolescent↗
PubMed1985

Specific IgE to food and inhalant allergens in intestinal washings of children affected by atopic eczema.

Serum and rectal total and specific IgE were measured in eleven children with atopic dermatitis and eight with atopic dermatitis and associated wheezing. Specific IgE to food and inhalant allergens in rectal washings were found in fourteen patients. Of the seventy-six allergens which gave positive results, twenty were positive in both serum and intestine, thirty in serum alone and twenty-six in intestines alone. Specific intestinal IgE were confirmed by food challenge in three out of four patients whose skin-prick test and serum RAST were both negative. Local production of these antibodies was demonstrated by the 'double ratio' of Deuschl and Johansson, and the 'specific activity ratio' of Platts-Mills. Positive ratios (greater than 1) were obtained with both formulas for twelve of fourteen allergens tested. These data suggest that gut-associated lymphoid tissue may play a role in the pathogenesis of atopic disease.

Allergens↗