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Biomedical subjects

P Riccio

Publications and source records attributed to P Riccio.

At least 19 recordsLinked to original sources
PubMed1995

Release of myelin basic protein-degrading proteolytic activity from microglia and macrophages after infection with Theiler's murine encephalomyelitis virus: comparison between susceptible and resistant mice.

Theiler's murine encephalomyelitis virus (TMEV) produces a chronic inflammatory demyelinating disease in its natural host, the mouse. A delayed-type hypersensitivity (DTH) response to viral antigens generally correlates with susceptibility to the disease and is thought to play an important role in the pathogenesis of demyelination in this model of human multiple sclerosis (MS). The hallmark of DTH responses is the recruitment by activated Th-1 cells of lymphoid cells and especially macrophages in infected areas. It is believed that soluble factors released by these cells would produce tissue damage, particularly myelin breakdown. In the present study, we compared TMEV-infected macrophages and microglia, isolated from both susceptible SJL/J and resistant C57BL/6 mice, for their ability to secrete proteolytic enzymes capable of degrading myelin basic protein. In addition, we studied whether supernatants from infected microglia/macrophages were also capable of killing oligodendrocytes in the same in vitro system. As detected by SDS-PAGE, MBP-degrading proteolytic activity was found only in supernatants from infected SJL/J microglia and macrophages, but not in supernatants collected from infected C57BL/6 microglia and macrophages, or in supernatants from mock-infected SJL/J and C57BL/6 cells. Similarly, incubation of E20.1 cells, an immortalized line of oligodendrocytes, with infected SJL/J, but not C57BL/6 supernatants, resulted in cytotoxic activity. When cells from resistant C57BL/6 mice were treated with LPS, they became susceptible to infection and also secreted proteolytic enzymes. The proteolytic activity released from infected microglia and macrophages was found to be dose-dependent, was inactivated by heat, and was inhibited by phenylmethylsulphonyl fluoride (PMSF). These results indicate that a serine protease is released from infected microglia and macrophages and suggest a role for proteases in TMEV-induced myelin injury.

Animals↗
PubMed1994

Myelin degrading activity in the CSF of HIV-1-infected patients with neurological diseases.

Degradation of purified myelin basic protein (MBP) was studied by SDS gel electrophoresis after addition of CSF samples obtained from HIV-1-infected patients. An increase in MBP degradation was detected in patients with neurological complications, such as AIDS dementia complex (ADC) or progressive multifocal leukoencephalopathy (PML), when compared with patients with no neurological symptoms (NA) or with other neurological opportunistic infections (OI). In the ADC and PML patients, in addition to CSF proteolytic activity, an increase in CSF-MBP levels and presence of white matter lesions were also observed by neuroimaging (MRI). In other opportunistic infections of the brain, MBP levels but not anti-MBP proteolytic activity increased. Results suggest the involvement of proteases in the virus-induced demyelination.

AIDS Dementia Complex↗
PubMed1994

A new detergent to purify CNS myelin basic protein isoforms in lipid-bound form.

We have previously shown that CNS myelin basic protein (MBP) can be purified in the lipid-bound, native-like form by using a procedure based on myelin solubilization with detergents at pH above 7, and on the filter-like use of hydroxyapatite to separate non-adsorbed MBP from other myelin proteins. Here, we report on the isolation of MBP in the zwitterionic detergent 3-((3-cholamidopropyl)dimethylammonio)-1-propane sulfonate (CHAPS), which does not interfere at 280 nm and can be removed by dialysis. This detergent appears to improve MBP purification and to be suitable for fluorescence and reconstitution studies that can be useful to understand both structure and function of MBP in its natural environment.

Animals↗
PubMed1994

Fluorochromes with long-lived fluorescence as potential labels for pulsed laser immunocytofluorometry: photophysical characterization of pyrene derivatives.

An apparatus for laser-induced time-resolved fluorescence measurements, in conjunction with pyrene derivatives endowed with long-lived excited singlet states, was employed for immunocytofluorometric measurements. N-(1-pyrene)maleimide, 1-pyrenesulfonyl chloride and 1-pyreneisothiocyanate were conjugated with immunoglobulin, antimouse-IgG, and the fluorescence decays of both free and conjugated forms were investigated. Bi-exponential decays were obtained in all cases with time constants of the short-lived component in the range 3-4.7 ns and the long-lived one in the range 20-55 ns. Only the spectral distribution of the two components is essentially affected upon conjugation. The persistence of the long-lived component, well above the lifetime of autofluorescence, and of the antibody specificity, as shown by immunodiffusion tests, upon conjugation indicates that this technique could be advantageously adopted in immunocytofluorometry.

Animals↗
PubMed1994

Fluorometric determination of the kinetics of anthracyclines uptake by cells.

Fluorometric measurements on extracellular medium are shown to allow kinetic parameters of in vitro anthracycline uptake by cells to be calculated. The method provides influx and efflux rates, as well as the time dependence of both influx and efflux. It is applied to a normal thyroid epithelial cell line (FRTL-5) and a cell line (MPTK-6) derived from the lung metastases of a thyroid carcinoma exposed to daunorubicin at concentrations within the range of 250 to 1000 ng/ml. The results show that the number of cells influences the dependence of the kinetics upon the extracellular drug concentration and that the MPTK-6 cells are endowed with very efficient efflux mechanisms.

Animals↗
PubMed1994

Myelin basic protein interaction with zinc and phosphate: fluorescence studies on the water-soluble form of the protein.

The interaction of myelin basic protein (MBP) with zinc and phosphate ions has been studied by using the emission properties of the single tryptophan residue of the protein (Trp-115). The studies have been carried out by means of both static and time-resolved fluorescence techniques. The addition of either zinc to MBP in the presence of phosphate or phosphate to MBP in the presence of zinc resulted in an increase of fluorescence intensity and a blue shift of the emission maximum wavelength. Furthermore, a concomitant increase in the scattering was also detected. Anisotropy decay experiments demonstrated that these effects are due to the formation of MBP molecules into large aggregates. A possible physiological role for such interaction is discussed.

Animals↗
PubMed1994

Laser photosensitization of cells by hypericin.

Administering a light dose of 90 J/cm2 at 599 nm during incubation with hypericin to a highly differentiated normal epithelial cell line (FRTL-5), derived from Fisher rat thyroid, and to a neoplastic cell line (MPTK-6), derived from the lung metastases of a thyroid carcinoma induced in Fisher rats, produces cell kill at drug doses 1000 times lower than those necessary to cause the same mortality in the dark. The photocytocidal activity of this polycyclic quinone drug on neoplastic cells is superior to that of antitumor anthraquinone drugs, such as daunomycin and mitoxanthrone, and to the photosensitized antiviral activity previously reported for hypericin.

Animals↗
PubMed1993

Induction of experimental autoimmune encephalomyelitis in rats and immune response to myelin basic protein in lipid-bound form.

Encephalitogenic activity of myelin basic protein (MBP) isolated in a form retaining binding to all myelin lipids was tested in Lewis rats. Immunization with this new stable lipid-bound and native-like preparation (LB-MBP), induced experimental autoimmune encephalomyelitis (EAE) as intensively as the classical lipid free MBP (LF-MBP). During the course of the disease, high affinity specific response to LB-MBP and high frequency of LB-MBP specific precursors was observed in peripheral lymphoid organs, indicating that the disease occurred in presence of anti LB-MBP specific T-cell responsivity. Short term lines, generated from lymphocytes collected at the onset of the disease from LB-MBP immunized rats, showed a strong dose-dependent response to LB-MBP, but not to LF-MBP. The present data indicate that in rat, LB-MBP maintains encephalitogenic activity and induces expansion of a specific T-cell population. These data suggest also that LB-MBP is a new autoantigen that may be relevant in human diseases.

Animals↗
PubMed1993

Antibodies specific for the lipid-bound form of myelin basic protein during experimental autoimmune encephalomyelitis.

On the hypothesis that myelin basic protein isolated with surrounding lipids may constitute an autoantigen in demyelinating diseases, we studied the antibody response to the lipid-free and lipid-bound form of myelin basic protein during the course of experimental autoimmune encephalomyelitis induced in rats with either form of protein. Immunization with the lipid-bound form of myelin basic protein induced high titres of antibodies directed to the protein, accompanied by no antibodies to cerebroside 30 days after immunization. Antibodies specifically directed to the lipid-bound form of myelin basic protein were revealed after removal of antibodies recognizing the delipidated myelin basic protein. Anti lipid-bound myelin basic protein antibodies could already be detected at day 10 post-immunization, reaching a maximum at day 20 post-immunization. Demonstrations of antibodies entirely specific for the lipid-bound form of myelin basic protein suggests that this molecule may present epitopes not to be found in its already extensively studied primary structure, possibly the result of conformational changes following lipid binding.

Animals↗
PubMed1992

Identification of water-soluble proteases in myelin preparations.

Sodium chloride extracts obtained from purified bovine brain myelin were found to contain proteolytic activity capable of degrading isolated myelin basic protein as assessed by SDS gel electrophoresis. Using gels copolymerized with gelatin as substrate, two bands at about 54 and 117-125 KDa, respectively, were detected. Activity corresponding to the 54 KDa band was inhibited by zinc. Data presented in this article suggest that proteolytic activity can be released from the myelin sheath in water-soluble form and recognize MBP as substrate.

Animals↗
PubMed1992

Cerebrospinal fluid myelin basic protein as predictive marker of demyelination in AIDS dementia complex.

Myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF) of patients with acquired immunodeficiency syndrome (AIDS) dementia complex (ADC) in order to investigate the degree of white matter destruction. Results show that increased CSF levels of MBP were detected in all patients with severe ADC (10/10) and, less often, in subjects with mild (2/7) or moderate dementia (7/16). No evidence of MBP-elevated concentration was observed in 14 human immunodeficiency virus (HIV)-seropositive subjects without neurological disorders and in nine HIV-seronegative controls. Our findings suggest that the measurement of CSF MBP concentration may represent a predictive marker of myelin injury and neurologic damage during the course of ADC.

AIDS Dementia Complex↗
PubMed1991

On the spontaneous adherence of myelin basic protein to T lymphocytes.

We have applied a double tagging system in order to study whether purified myelin basic protein is able to adhere to normal human peripheral T lymphocytes without the need to purify cells. Evaluation of myelin basic protein adherence to peripheral blood mononuclear cells was determined with biotinylated myelin basic protein and fluoresceinated avidin, and lymphocyte population was identified by the corresponding phycoerythrinated monoclonal antibody. The observed adherence of myelin basic protein to T lymphocytes was found to depend on protein conformation.

Animals↗
PubMed1991

Detection of cerebrospinal fluid antibodies against myelin basic protein in patients with AIDS dementia complex.

We evaluated cerebrospinal fluid (CSF) antibody levels against a lipid-free, denatured form of myelin basic protein (LF-MBP) in 11 patients with AIDS dementia complex (ADC) by using an enzyme-linked immunosorbent assay (ELISA). In 9 out of 11 patients, anti-LF-MBP antibody levels were significantly higher than those observed both in 15 human immunodeficiency virus (HIV)-infected patients without neurological disorders and in 9 anti-HIV-negative subjects affected by other neurological diseases. Furthermore, we followed up anti-MBP levels in 5 out of the 11 ADC patients and detected a strict relationship with the encephalopathy progression. At the same time, with the aim to detect early demyelinating events we investigated CSF antibody levels against a lipid-bound, native-like form of MBP (LB-MBP). Results did not show any significant difference between LF-MBP and LB-MBP in terms of antibody reactivity. The detection of anti-MBP antibodies in CSF may provide the opportunity to assess a diagnostic tool for discovering demyelinating lesions in ADC patients.

AIDS Dementia Complex↗