Biomedical subjects

M A Moscarello

Publications and source records attributed to M A Moscarello.

At least 55 records · Page 3Linked to original sources

PubMed1994

ADP-ribosylation of human myelin basic protein.

When isolated myelin membranes were ADP-ribosylated by [32P]NAD+ either in the absence of toxin (by the membrane ADP-ribosyltransferase) or in the presence of cholera toxin, the same proteins were ADP-ribosylated in both cases and myelin basic protein (MBP) was the major radioactive product. Therefore, cholera toxin was considered a good model for ADP-ribosylation of myelin proteins. Although purified human MBP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular mass of 20 kDa, the microheterogeneity that is masked under these conditions can be clearly demonstrated on alkaline-urea gels at pH 10.6. At this pH, MBP is resolved into several components that differ one from the other by a single charge (charge isomers). These charge isomers can be resolved on CM52 columns at pH 10.6, and several can be ADP-ribosylated. Component 1 (C-1), the most cationic charge isomer, incorporated 1.79 mol of ADP-ribose/mol of protein. C-2 and C-3 (which differ from C-1 by the loss of one and two positive charges, respectively) incorporated slightly less at 1.67 and 1.63 mol of ADP-ribose/mol of protein, respectively, whereas C-8, the least cationic, incorporated less than 0.11 mol/mol of protein. In the presence of neutral hydroxylamine, the ADP-ribosyl bond was shown to have a half-life of about 80 min, suggesting an N-glycosidic linkage between ADP-ribose and an arginyl residue of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗

PubMed1994

Myelin in multiple sclerosis is developmentally immature.

The etiology of multiple sclerosis (MS) is considered to involve genetic, environmental, infective, and immunological factors which affect the integrity of a normally assembled myelin sheath, either directly or indirectly resulting in demyelination. In a correlative study involving protein chemical, mass spectrometric, and electron microscopic techniques we have determined that myelin obtained from victims of MS is arrested at the level of the first growth spurt (within the first 6 yr of life) and is therefore developmentally immature. The data supporting this conclusion include (a) the pattern of microheterogeneity of myelin basic protein (MBP); (b) the NH2-terminal acylation of the least cationic component of MBP ("C-8"); (c) the phase transition temperature (Tc) of myelin isolated from victims of MS correlated with the increased proportion of the least cationic component of MBP; and (d) immunogold electron microscopy using an antibody specific for "C-8" showed that the distribution of gold particles in a 2-yr-old infant was similar to the distribution found in a victim of MS. We postulate that this developmentally immature myelin is more susceptible to degradation by one or a combination of factors mentioned above, providing the initial antigenic material to the immune system.

Acylation↗

PubMed1993

Demyelination in a transgenic mouse: a model for multiple sclerosis.

A transgenic mouse containing 70 copies (ND4) of the transgene encoding DM20, a myelin proteolipid protein, appeared clinically normal up to 3 months of age. By 8-10 months, it showed tremors, unsteady gait, and died shortly thereafter. We concluded that the clinical symptoms correlated with demyelination based on the following criteria: 1) at 10 months of age only 17% of the amount of myelin obtained from normal mice was isolated from the ND4 mice; 2) astrogliosis, a prominent feature of demyelinating disease was minimal at 3 months of age but prominent by 10 months; 3) at the electron microscopic level disrupted myelin was seen at 8 months of age in the ND4 mice and ingested myelin debris was found in astrocytes; 4) lymphocytic infiltration in association with endothelial cells was observed routinely in the ND4 mice; 5) sections through optic nerves showed denuded and thinly myelinated axons in the 8 month old ND4 mice. Although the mechanism by which demyelination takes place is not fully understood, measurements of the amounts of PLP suggest it is down-regulated by the large amount of DM20. Since DM20 is a major proteolipid in the young but a minor one in the adult, the persistence of high levels in the adult results in improperly assembled myelin which is prone to disruption. Therefore demyelination in the ND4 mouse appears to result from the persistence of immature myelin into the adult.

Animals↗

PubMed1993

Localization of basic proteins in human myelin.

The myelin basic protein (MBPs) represent a family of proteins (charge isomers) which account for 35% of the total myelin protein. Localization studies have been inconclusive because MBP is not a single protein. Antibodies obtained by injection of MBP into animals recognized all members of the MBP family. In the studies reported here, we have fractionated the MBPs into specific components or charge isomers. One of these which contains citrulline accounts for about 20% of the total MBP. We report the localization of this single MBP to the intraperiod line of myelin by immunoelectron microscopy. For these studies several specific antibodies were used including antibodies raised against total MBP, specific MBP peptides, and against a tetracitrulline peptide. This latter antibody was specific for component 8 (C-8) of MBP. Since C-8 is the only MBP which contains citrulline it was used to localize this particular form of MBP principally to the intraperiod line by immunogold electron microscopy, while antibody against total MBP (consisting of all charge isomers C-1-->C-8) labelled both the major dense line and the intraperiod line. When the anti-citrulline antibody was used with a 3 nm gold conjugated Fab fragments prepared from the secondary antibody, 66.5% of the gold particles were localized to the intraperiod line, while 11.2% of gold particles were localized to the major dense line. On the other hand, with the monoclonal anti-MBP antibodies reactive with residues 69-74, 59.4% of the gold particles were localized to the major dense line and 23.6% of gold particles at the intraperiod line. Other supporting evidence includes increased labelling of myelin by 125I labelled anti-citrulline IgG when isolated myelin was swollen, a process known to take place at the intraperiod line. Gold particles were demonstrated at the intraperiod line in swollen and recompacted myelin. C-8 was shown to associate preferentially with lipids asymmetrically localized to the intraperiod line.

Amino Acid Sequence↗

PubMed1993

Carbon-13 "magic-angle" sample-spinning nuclear magnetic resonance studies of human myelin, and model membrane systems.

We have obtained high-field (11.7 Tesla), high-resolution carbon-13 solid-state "magic-angle" sample-spinning nuclear magnetic resonance (NMR) spectra of a variety of phospholipids, sphingolipids, myelin and white matter samples, resolving and assigning over 40 resonances in the spectra of human and bovine myelin. The NMR results indicated no large spectral changes due to sample preparation, sample freezing, or brain location, and also no changes in myelin structure detectable via light microscopy, electron microscopy, thin layer chromatography, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, attributable to the sometimes lengthy NMR data acquisition process. Human myelin and white matter chemical shift assignments were made based on 13C "magic angle" sample spinning (MAS) NMR spectra of individual model lipids, as well as on spectra of lipid mixtures. In all myelin samples there were essentially no features attributable to membrane proteins, with the exception of one small feature due to C zeta of Arg residues, primarily in the myelin basic proteins. The general similarity between the model lipid and intact myelin spectra suggested no major effects of protein on lipid mobility. We have also investigated human myelin samples as a function of developmental age (4, 15, 48 months and adult), and our results showed only small changes in overall lipid composition, although there were significant decreases in lipid hydrocarbon chain unsaturation with age, as determined by computer line-shape simulations of myelin and model compounds. The spectrum of an infant leukoencephalopathy myelin showed marked decreases in galactocerebrosides. Overall, the ability to resolve and assign over 40 resonances in the 13C MAS NMR spectra of myelin, and to detect changes as a function of development and disease, should provide a useful starting point for further more detailed studies of myelin membrane molecular motions, and function.

Adult↗

PubMed1993

Stimulation of bovine brain phospholipase C activity by myelin basic protein requires arginyl residues in peptide linkage.

We reported previously a highly purified phosphatidylinositol-specific phospholipase C (PI-PLC) from bovine brain and from human myelin which was stimulated by myelin basic protein. In this paper we report that the stimulation of the PI-PLC activity by myelin basic protein (MBP) requires arginine residues in peptide linkage. MBP and poly-L-arginine were able to stimulate the PI-PLC activity by 250% while other basic poly amino acids were unable to stimulate the PI-PLC activity. Neither free arginine nor benzoyl-arginine ethylester was able to stimulate the activity of the enzyme. These results suggested a requirement for the guanidino group of arginine and arginine in peptidyl linkage. The arginyl residues of MBP were modified chemically with 1,2-cyclohexanedione, or enzymatically by cholera toxin which ADP-ribosylated arginyl groups, or by peptidylarginine deiminase which converted the guanidino group of arginine to the ureido group of citrulline. ADP-ribosylation did not affect the stimulation while the 1,2-cyclohexanedione modified MBP and the peptidylarginine deiminase-treated MBP showed a reduced ability to stimulate the PI-PLC activity which correlated with the number of arginyl residues modified. Sequence analysis of the peptidylarginine deiminase-treated MBP established that specific arginyl residues had been converted to citrulline to a greater extent than others. When 70% of Arg 25 and Arg31 were converted to citrulline little stimulatory activity remained, whereas the conversion of 100% of Arg 170 did not affect the ability of C1 to stimulate the enzyme. A role for "active" arginine in this MBP peptide is suggested by our data.

Animals↗

PubMed1993

Immunological analysis of the amino terminal and the C8 isomer of human myelin basic protein.

The citrullination and N-terminus acylation of myelin basic protein (MBP) increases the heterogeneity among the MBP isoforms. The present study was undertaken to further characterize the immune response to the citrullinated form (C8) of MBP as well as to the variably acylated N-terminus of MBP. Six well-characterized murine monoclonal antibodies (mAbs) to human MBP-C8 or MBP peptides (four mAbs to MBP acetyl 1-9, one mAb to MBP 10-19 and one mAb to MBP 80-89), one murine T cell line (PL11) to human MBP peptide acetyl 1-9 and one Lewis rat T cell line (RT-1) to guinea pig (GP) MBP peptide 68-88 were used to assess reactivity with MBP-C1, MBP-C8, and MBP peptides including a series of MBP peptide 1-21 containing 0, 2, 4, 6, 8 or 10 carbon fatty acids. Enzyme-linked immunosorbent assay (ELISA) results revealed that all of the mAbs reacted with human MBP-C1 and MBP-C8 except anti-MBP 10-19 and anti-MBP-C8. The former reacted only with MBP-C1 and the latter only with MBP-C8. The presence and length of acylation of MBP peptide 1-21 modified reactivity. Three mAbs to MBP acetyl 1-9 reacted only with acetyl 1-21, and one mAb anti-MBP actyl 1-9 reacted with all of MBP 1-21 preparations whether acylated or not. mAb anti-MBP-C8 generally reacted better with acylated MBP 1-21 having longer fatty acids. The PL11 T cell line strongly proliferated to human MBP-C1, MBP-C8 and MBP acetyl 1-9, responded, but less well, to MBP 1-21 with longer fatty acids and failed to respond to nonacylated MBP peptide 1-21. The RT-1 cell line responded strongly to GP MBP peptide 68-88, marginally to MBP-C8 and failed to respond to MBP-C1 or any of the other MBP peptides. Specific immune responses to different MBP charge isomers and different N-terminal acylating groups of MBP may play a role in immune-mediated demyelination.

Acylation↗

PubMed1993

Deimination of human myelin basic protein by a peptidylarginine deiminase from bovine brain.

A peptidylarginine deiminase (PAD; EC 3.5.3.15) has been isolated from bovine brain and some of its characteristics have been studied. The enzyme showed an absolute requirement for Ca2+, a temperature optimum at approximately 50 degrees C, and two Km values when benzoylarginine ethyl ester was used as substrate, 0.78 mM and 11.2 mM. The higher Km has not been reported previously. Protein substrates for the enzyme included polyarginine and myelin basic protein but not histones. Because one of the components of MBP contains six citrullinyl residues per mole, enzymic deimination appeared to be a likely mechanism. When the most cationic component (C-1) was subjected to PAD in solution, 17 of the 19 arginyl residues were modified. From sequence analyses we concluded that the nature of the amino acid residues adjacent to the deiminated arginine were not modifiers of the reaction as arginyl residues in a variety of environments were deiminated. This deimination was reflected in a large increase in random structure, as measured by [theta]200. At 5 degrees C, the [theta]200 of the deiminated protein was -70 x 10(3) compared with -30 x 10(3) deg.cm2/dmol for the native protein. When the temperature was increased to 70 degrees C, the [theta]200 was -44 x 10(3) for the deiminated protein and -20 x 10(7) deg.cm2/dmol for the native C-1. When plotted as a function of temperature, [theta]200 decreased linearly from 5 degrees C to 50 degrees C for both proteins and did not change from 50 degrees C to 70 degrees C. PAD provides a mechanism for deimination of arginyl residues of myelin basic protein. The selective deimination of the six arginyl residues that are consistently found deiminated in C-8 may be determined by the orientation of the protein in the membrane and/or the more complex lipid composition of myelin may affect the selectivity of the deimination.

Amino Acid Sequence↗

PubMed1993

Characterization of basic proteins from goldfish myelin.

Myelin basic protein (MBP) from common goldfish (Carassius auratus) myelin was extracted with dilute mineral acid. Immunological cross-reactivity of the goldfish MBP, with polyclonal antisera raised against bovine MBP, suggested that the goldfish protein has epitopes for these antibodies. It also reacted with a monoclonal antibody specific for a seven amino acid epitope (130-137) conserved in the MBP of most mammalian species. To characterize the charge heterogeneity of this protein, we iodinated the protein with 125I and chromatographed it on a carboxymethyl cellulose-52 column together with a nonlabeled acid soluble fraction prepared from human white matter as a carrier protein. All of the goldfish protein was recovered in the unbound fraction, demonstrating that it was less cationic than the carrier protein (human MBP). We have also examined the urea alkaline gel profile of the goldfish MBP together with the human C-1, C-2, C-3, C-4, and C-8 components. The results from these experiments indicated that this MBP extracted from goldfish brain myelin lacked the microheterogeneity that is associated with MBPs from higher vertebrates. The MBPs from goldfish myelin were separated into their isoforms by reversed-phase HPLC. Amino acid compositions were determined for both the 17- and 14-kDa goldfish proteins. Amino acid analysis revealed similarities with the compositions of other MBPs; however, the serine content in both the 17- and 14-kDa proteins was higher than that of the human C-1, the mouse C-1 protein, and the shark proteins. The HPLC-purified 14-kDa goldfish protein was chemically cleaved with CNBr for partial sequence analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

A glycoprotein expressed by human fibrous astrocytes is a hyaluronate-binding protein and a member of the CD44 family.

We have isolated and characterized an antigen from normal human brain called p80, so called because it migrated with an M(r) of 80 kDa on SDS PAGE. The M(r) of 80 kDa consists of a protein of about 55-60 kDa and carbohydrate (20-25 kDa). The carbohydrate is almost entirely of the N-linked type, although a small amount of O-linked carbohydrate was detected. Cross-reactivity with monoclonal antibodies A3D8 and A1G3 showed that p80 could therefore be considered an isoform of the CD44 adhesion molecules. In addition, specific binding to hyaluronate which was not competed for by proteoglycan demonstrated that it involved different sites than the proteoglycan binding sites. We also observed that fucoidan and dextran sulphate increased the binding by 200-250% while chondroitin sulphate C also increased the binding but to a lesser extent. Heparin, heparan sulphate and chondroitin sulphates A and B did not have such an effect. The binding of p80 to hyaluronate was pH dependent with a maximum at pH 6.4. We concluded that p80 was an astrocyte specific adhesion molecule.

Astrocytes↗

PubMed1992

The N terminus of human myelin basic protein consists of C2, C4, C6, and C8 alkyl carboxylic acids.

Peptide 1-21, generated by cyanogen bromide cleavage of each of two highly purified components of human myelin basic protein, components 1 and 8, gave a series of peaks in the fast atom bombardment mass spectra with m/z 2299, 2327, 2355, 2383, and 2411, indicating additions of 42, 70, 98, 126, and 154 atomic mass units respectively with m/z 2327 and 2355 as the dominant species. The pentafluorobenzyl esters prepared from an acid hydrolysate analyzed by negative ion chemical ionization gas chromatography mass spectrometry confirmed that C6, C8, and C10 fatty acids were present. These data demonstrated (i) that the N terminus of a myelin basic protein is not simply acetylated but contains C2, C4, C6, C8, and C10 fatty acids with C4 and C6 as the dominant species, (ii) the two components studied (C-1 and C-8) showed different relative amounts of C2 and C8 in particular, and (iii) human myelin basic protein is the first protein to be reported with a complex N terminus consisting of several alkyl carboxylic acid species.

Amino Acid Sequence↗

PubMed1992

Reactivity of two anti-galactosyl ceramide antibodies towards myelin basic protein.

Two anti-galactosyl ceramide antibodies (polyclonal Ab142 and a monoclonal antibody) were characterized in terms of their reactivity towards purified lipids and myelin basic protein. Polyclonal Ab142 is a rabbit anti-mouse galactosyl ceramide (Gal C) IgG. Antigenic recognition is dependent on both galactose and ceramide since neither could inhibit galactosyl ceramide binding by more than 10%. MAb-Gal C is a monoclonal antibody raised in mice against galactosyl ceramide. Binding of MAb-Gal C to Gal-C was equally inhibited by ceramide and galactose to approximately 50%, indicating that both groups are important for antibody recognition. MAb-Gal C was also shown to be reactive with the structurally related lipids, sphingomyelin and sulfatide. Polyclonal Ab142, although raised against Gal C, was shown to be 3-fold more reactive with component 8 (C-8) of myelin basic protein than Gal C. On the other hand, the MAb-Gal C which also reacted with C-8 was 2-fold less reactive with C-8 than with Gal C. Neither of these antibodies were reactive with component 1 (C-1) of myelin basic protein. An anti-MBP IgG was shown to be reactive with C-1 and C-8 but unreactive with Gal C. In competitive inhibition ELISA, C-8 was able to compete out 44% and 41% of Gal C binding to polyclonal Ab142 and MAb, respectively. The reverse competition demonstrated that Gal C could inhibit 75% of C-8 binding to both antibodies. D-galactose was unable to inhibit C-8 binding to either antibody, whereas ceramide was as efficient as Gal C.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

An immunochemical comparison of human myelin basic protein and its modified, citrullinated form, C8.

An immunochemical analysis was conducted to compare the C1 isomer of human myelin basic protein (MBP) with the newly described and less cationic, citrullinated isomer of MBP referred to as C8. Ten polyclonal antisera directed at multiple epitopes or restricted regions of MBP were used in radioimmunoassays to examine MBP-C1 and MBP-C8. Antisera reactive with MBP peptide 1-14 clearly distinguished MBP-C1 from MBP-C8. Antisera to human MBP peptides 10-19 and 90-170, but not to MBP peptide 69-89, showed modest differences between MBP-C1 and MBP-C8. The MBP-C8s from multiple sclerosis (MS) and non-MS brain reacted essentially the same. With murine monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA), differences between MBP-C8 and other isomers were shown for anti-MBP 10-19 but not for anti-MBP 1-9 or anti-MBP 80-89. These findings imply differences in sequence or conformation in the structure of MBP-C7 compared to MBP-C1, most notably near the amino terminus.

Adult↗

PubMed1992

An antibody specific for component 8 of myelin basic protein from normal brain reacts strongly with component 8 from multiple sclerosis brain.

Myelin basic protein (MBP) consists of several components or charge isomers (C-1 through C-8) generated by one or a combination of posttranslational modifications. One of these, C-8, has been shown to contain citrulline (Cit) at defined sites formed by deimination of six arginyl residues. This unusual modification has allowed us to raise antibodies specific for this charge isomer only. To do this, a synthetic peptide, Gly-Cit-Cit-Cit-Cit, was coupled to keyhole limpet hemocyanin and injected into rabbits. The antibodies so generated reacted only with C-8 and not with any of the other charge isomers. A second antibody fraction was raised against the synthetic peptide ACitHGFLPCitHR naturally occurring between residues 24 and 33 of C-8 (all other charge isomers contain R instead of Cit at positions 25 and 31). These antibodies preferred C-8 but reacted with the other charge isomers, to the extent of approximately 25-30% of the reactivity shown with C-8. In studies with C-8 from multiple sclerosis (MS) MBP, much greater reactivity was obtained with these antibodies when compared with their reactivity with C-8 from normal MBP. Because the total number of Cit residues in C-8 from MS and normal MBP is the same, the difference in reactivity may be related to structural factors. The antibodies raised with the tetra-Cit peptide were reacted with three pairs of synthetic peptides: 24ARHGFLPRHR33 and ACitHGFLPCitHR; 120GQRPGFGYGGRAS132 and GQCitPGFGYGGCitAS; and 157GGRDSRSGSPMARR170 and GGCitDSRSGSPMACitR. They reacted only with the Cit-containing peptides in the order 157-170 greater than 120-130 greater than 24-33.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer Disease↗

PubMed1991

A 57-kDa phosphatidylinositol-specific phospholipase C from bovine brain.

A phosphatidylinositol-specific phospholipase C (PI-PLC) has been isolated from bovine brain (purification factor of 5.6 x 10(4)). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it had a Mr of 57,000. Neither amino nor neutral sugars were detected in the purified enzyme. The pH optimum was 7.0-7.5, and the activity decreased only slightly at pH 8.0. When phosphatidylinositol was used as a substrate, the optimum Ca2+ requirement was 4 mM, and Km was 260 microM. When phosphatidylinositol 4,5-bisphosphate was used, the optimum Ca2+ requirement was 10(-7) M, and the Km was reduced to 90 microM. Lipid specificity studies showed that equal amounts of inositol phosphate and diacylglycerol were released from phosphatidylinositol but 4 times as much inositol 1,4,5-trisphosphate was released from phosphatidylinositol 4,5-bisphosphate. Other lipids, phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin, were not substrates. Failure to detect phosphatidic acid confirmed the absence of a phospholipase D activity in the purified enzyme. Myelin basic protein (MBP) stimulated the PI-PLC activity between 2- and 3-fold. Histone had a small effect only, whereas bovine serum albumin and cytochrome C had no effect. Phosphorylation of MBP reduced the stimulatory effect. Protein-protein interactions between MBP and PI-PLC have been demonstrated both immunologically and by sucrose density gradients. A stoichiometry of 1:1 has been suggested by the latter method. A number of peptides have been prepared by chemical, enzymatic, and synthetic methods. Peptides containing the MBP sequences consisting of residues 24-33 and 114-122 stimulated the PI-PLC but were less effective than the intact protein.

Amino Acid Sequence↗

PubMed1991

Characterization of myelin basic protein charge isomers from adult mouse brain.

ADULT mouse MBP charge isomers (C1 or component 1, C2 or component 2, etc.) were purified from an acid soluble brain protein fraction by cation exchange chromatography. They were characterized by their elution profiles, their migration rates in alkaline-urea gels and by SDS-PAGE. Mouse C1 and C2 were both 14 kD in size, while C3, C4 and C5 consisted of the 18.5, 17 and 14 kD isoforms. Comparison of mouse and human MBP charge isomers showed that they were similar in that they both showed extensive charge heterogeneity, but different in that mouse MBP charge isomers were more cationic than their human counterparts. Finally, a possible explanation for the presence of the 14 kD MBP isoform in mouse myelin was suggested.

Animals↗

PubMed1991

Localization of CD44 (P80) on the external surface of a human astrocytoma cell.

A brain antigen, originally identified by a MAb 44D10, has been shown to be a glycoprotein with an Mr of 80 kDa. Cellular localization studies of sections of brain showed that the antigen was associated with the membrane of astrocytes. In the present study we demonstrate its localization to the membrane of an astrocytoma cell line by fluorescence and electron microscopic immunogold methods. Heavy labelling with immunogold was found along cellular processes. Labelling of the smooth surfaces of cells and the microvilli was also observed.

Antibodies, Monoclonal↗

PubMed1991

Localization of the CD44 glycoprotein to fibrous astrocytes in normal white matter and to reactive astrocytes in active lesions in multiple sclerosis.

The CD44 antigen is a proteoglycan recently implicated in several adhesion events including that of lymphocytes to endothelium. The CD44 antigen, reactive with monoclonal antibody (MAb) 44D10, has been shown previously to be expressed in normal human white matter homogenates and to be found at higher concentrations in brain homogenates of victims of multiple sclerosis (MS). The cellular localization of CD44 in human brain of normal individuals and in those afflicted with MS has now been determined. Monoclonal antibody 44D10 reacted with astrocyte-like cells in 40 microns thick paraformaldehyde-fixed sections but not in thin (6 microns) fixed sections. A double labeling experiment performed on a frozen brain section with MAb 44D10 and rabbit anti-glial fibrillary acidic protein (GFAP), a cytoplasmic marker of astrocytes, confirmed the co-localization of these two antigens. The reactivity with brain tissue sections of a rabbit antiserum produced against lymphocyte-CD44 could be absorbed by a preparation of the CD44 glycoprotein, purified 2,100-fold from a white matter homogenate. The antiserum was shown by Western blot analysis to be specific for p80 glycoprotein in brain extracts derived from a normal and MS patients. This antibody reacted with fibrous astrocytes predominantly in white matter; staining was also noted in subependymal and subpial regions. Inhibition studies using a cellular radioimmunoassay indicated that the highest concentrations of CD44 in three MS victims were found in plaques, followed by periplaques and non-involved areas of white matter which were higher than normal white matter. Reactive astrocytes, identified in active lesions, expressed high levels of CD44 on their surfaces. Thus, CD44 is associated with astrocytes in human brain and the increased expression observed in MS brain may reflect activation and/or proliferation of astrocytes implicated in the pathogenesis of this disease.

Astrocytes↗