Glia maturation factor: effect on chemical differentiation of glioblasts in culture.

A protein factor from the adult brain increases the concentrations of adenosine 3',5'-monophosphate and S-100 protein in glioblasts in culture. Such changes are correlated with the outgrowth of cell processes.

Tumor-associated immunoglobulins in pulmonary carcinoma.

In view of the uncertainty of location and significance of immunoglobulin in tumors found by elution or rosette formation (as reported in the literature), the presence of IgG, IgM, and IgA in human carcinoma of the lung was studied by means of the peroxidase-antiperoxidase method. Surgically obtained specimens from patients with known survival times were used in this study. Membranous as well as cytoplasmic location of IgG was demonstrated more frequently than was that of IgA or IgM. The number of tumor cells carrying immunoglobulin varied greatly, even within a given case. Albumin could be demonstrated in tumor cells in 10 of 20 specimens, but there was poor correlation with immunoglobuin. In some instances, only the necrotic part of the tumor or the stroma was immunoreactive. The results are discussed and suggest that Fc receptors are not involved in the binding of immunoglobin by pulmonary carcinoma cells.

The protein composition of bovine myelin-free axons.

The proteins of axons prepared from myelinated axons and isolated as myelin-free entities were separated by sodium dodecyl sulfate polyacrylamide electrophoresis and found to consist of more than 10 different molecular weight species. The molecular weights range from 13 000 to over 200 000 with a prominent protein of molecular weight 47 000. The amino acid composition of the seven major proteins showed that the protein with a molecular weigt of 47 000 is distinct from all the other proteins analyzed. A group of three low molecular weight proteins have amino acid compositions which are similar to each other as do a group of three high molecular weight proteins although the two groups are distinctly different from each other and the major axonal protein. Histones, DNA, myelin basic protein and glycoprotein were absent from the proteins but neurotubule protein was present as indicated by cochicine binding activity in the axonal preparations. The cellular origin of these proteins and their relationship to other central nervous system proteins are discussed.

Alpha tocopherol decreases lipid peroxidation, neuronal necrosis, and reactive gliosis in reaggregate cultures of fetal rat brain.

This study explored the effects of the lipid-soluble free radical scavenger, alpha tocopherol (vitamin E), on neuronal injury and glial protein accumulation in a well-characterized, three-dimensional, mixed neuronal and glial culture system derived from fetal rat prosencephalon. As these reaggregated spheroidal cultures grew and enlarged, they developed small central areas of necrosis (presumably due to nutritional compromise) which we used as a model for central nervous system injury. Treatment with vitamin E (delivered in egg phosphatidylcholine liposomes beginning on day 8 in vitro) did not alter the appearance of the central necrotic areas, but it strongly suppressed the reaction of adjacent astroglia and microglia. Histological examination showed that by day 35 in vitro control cultures which received liposomes without tocopherol were nearly devoid of neurons and contained many glial and microglial cells. In contrast, tocopherol-treated cultures contained many viable-appearing neurons and did not exhibit an overgrowth of glia. Both glial fibrillary acidic protein (as measured by immunoassay) and lipid peroxidation (as estimated by malondialdehyde levels) were markedly reduced in the tocopherol-treated cultures. We speculate that the vitamin exerts its protective effect on injured nervous tissues by scavenging free radicals, stabilizing cellular membranes, and quenching the cascade of biochemical events that follows necrosis in brain. This work suggests that the signal(s) to initiate gliosis are mediated, at least indirectly, by free radical formation.

Glial fibrillary acidic protein synthesized in vitro using messenger RNA from a human glioma cell line.

Messenger RNA (mRNA) extracted from a continuous human glioma cell line grown in culture or as a solid tumor was translated in an mRNA-dependent reticulocyte lysate system. Translation products labeled with [35S]methionine were immunoprecipitated with antiserum specific for glial fibrillary acidic (GFA) protein, separated by one- and two-dimensional polyacrylamide gel electrophoresis and analyzed fluorographically. Immunoprecipitates from both cell culture and tumor mRNA translations had a molecular weight of 49,000 daltons, consistent with GFA protein extracted from human tissue. In two dimensions, the 49,000-dalton band resolved into two to three spots at pH 5.7-5.9, the isoelectric point of GFA protein. Minor lower molecular weight products were detected in fluorographs of heavily overloaded gels or in film exposed for extended periods of time. These data indicate that the GFA protein produced by this glioma cell line is chemically and immunologically similar to normal human GFA protein, which suggests that the primary phenotypic expression of GFA protein in this tumor cell line is not altered by the neoplastic process.

Immunocytochemical studies of substance P and Met-enkephalin in the basal ganglia and substantia nigra in Huntington's, Parkinson's and Alzheimer's diseases.

Immunocytochemical studies of the distribution and intensity of Substance P and Met-enkephalin staining in the basal ganglia and substantia nigra were carried out in five cases each of brains from patients with Huntington's disease, Parkinson's disease, Alzheimer's disease, and normal controls. The usefulness of the peroxidase-antiperoxidase method for human autopsy material was confirmed. Substance P and Met-enkephalin fibers were distributed in essentially the same pattern as described in experimental animals and in human brains. In Huntington's disease brains decreased Substance P staining was found in the internal globus pallidus and the substantia nigra, in agreement with radioimmunoassay studies by others. Met-enkephalin staining in the external globus pallidus was of normal intensity, although present within a shrunken area. In Parkinson's and Alzheimer's diseases there was intense immunoreactivity for Substance P in the globus pallidus and substantia nigra, and for Met-enkephalin in the globus pallidus, at variance with reported decreases in Parkinson's disease by radioimmunoassay, but in essential agreement with other immunocytochemical studies. Immunocytochemical methods complement radioimmunoassays of human brain and may help in mapping neuropeptidergic pathways and in pinpointing abnormalities in these pathways in basal ganglia disorders.

The immunohistochemical application of three anti-GFAP monoclonal antibodies to formalin-fixed, paraffin-embedded, normal and neoplastic brain tissues.

The intermediate filament, glial fibrillary acidic protein (GFAP) has proven to be an important glial marker in diagnostic neuropathology. We report the histochemical application of three monoclonal antibodies (Mab) produced in this laboratory, 1B4, 2E1, and 4A11, which are monospecific to GFAP by radioimmunoassay, immunoblot electrophoresis, and immunoperoxidase histochemistry. The goal of this study was to compare the specificity and sensitivity of these Mab to GFAP on surgical brain biopsy specimens which had been routinely processed for diagnostic neuropathology with that of a high titer, highly specific, reference polyvalent anti-GFAP antiserum. The Mab stained astrocytes specifically in normal brain. When combined in a "cocktail" preparation, the quality of the immunoperoxidase detection of GFAP by these Mab closely approached that of the reference serum in 71 intracranial and intraspinal neoplasms. As these three Mab represent a continuous supply of a well defined, monospecific reagent, the monoclonal "cocktail" represents a standard reagent for large multi-institutional studies and for studies extending over a period of time.

Glial fibrillary acidic protein in hepatic encephalopathy. An immunohistochemical study.

Basal ganglia, thalamus, cerebral cortex, and subcortical white matter were studied in ten cases of hepatic encephalopathy (HE), including three cases of acquired hepatocerebral degeneration (HCD), and in thirteen age-matched controls using the peroxidase-antiperoxidase immunohistochemical staining technique for glial fibrillary acidic (GFA) protein. HE cases all had pronounced Alzheimer type II astrocytosis. The perikarya and processes of Alzheimer type II glia did not stain for GFA protein. Staining of perivascular endfeet was evaluated by first selecting blood vessels throughout the gray and white matter in hematoxylin and eosin-stained slides to eliminate bias. The vessels were then identified in sections stained for GFA protein and graded as to complete circumferential, partial circumferential, or absence of staining. Both the degree and frequency of staining in the basal ganglia, thalamus, and cerebral cortex were significantly decreased in cases of HE; no statistically significant differences were found for the white matter. There were no significant differences in staining between HCD and other HE cases. These findings show that the Alzheimer II change is associated with a loss of immunohistochemically detectable GFA protein in cerebral gray matter.

Heterogeneity of Genotypic and phenotypic characteristics of fifteen permanent cell lines derived from human gliomas.

Six new permanent cell lines were established from human gliomas and compared to nine other cell lines from human gliomas. All fifteen lines had individually distinct HLA phenotypes and all but two, which were from a black patient, had type B glucose-6-phosphate-de;hydrogenase isoenzymes. Morphologically, the lines could be classified into four patterns descriptively designated as fibroblastic, fascicular, epithelial, or glial. Four of the lines grew progressively and could be serially transplanted when injected into athymic mice; two others grew initially and then regressed. From none to 100% of cells developed elongated tapering processes and showed reduction in nuclear-cytoplasmic ratio in the presence of 1 mM cyclic AMP and theophylline. Levels of 2'-3' cyclic nucleotide 3'-phosphohydrolase activity ranged from nondetectable to 12.78 +/- 1.49 micromoles 2' AMP formed per hr mgm total protein. None of the lines had detectable S-100 protein, but two had readily demonstrable glial fibrillary acidic protein in indirect immunofluorescence. Fibronectin levels in spent culture supernatants ranged from undetectable levels to 21.4 micrograms/ml/10(5) cells. All but one line shared surface antigens with normal human adult or fetal brain, as detected in absorption analyses with nonhuman primate antiserum raised against glioblastoma multiforme tissue or cell line U-251 MG. Although there were many common properties of the lines, each line had a unique profile of the parameters evaluated. This heterogeneity most likely reflects the individuality of the tumors of origin and individual genotypes and capacity for a range of phenotypic expression of cells.

A glial fibrillary acidic protein-expressing and tumorigenic cell line derived from an avian sarcoma virus-induced rat astrocytoma.

A permanent cell line, S635c15, was derived from an anaplastic astrocytoma induced by the Schmidt-Ruppin strain of avian sarcoma virus (ASV) in a female F-344 rat. Persistent expression of the astrocytic differentiation protein, glial fibrillary acidic protein (GFAP), was detected both in cultured cells after 100 passages in vitro and in transplanted tumors. Subcutaneous and intracerebral transplantation of S635c15 cells in syngeneic rats resulted in a 100% tumor incidence and a reproducible mortality distribution. S635c15 cells formed discrete masses after subcutaneous injection but grew intracranially as infiltrative lesions. Tumor blood flow and blood-to-tissue transport studies yield comparable values to other rat glioma models; S635c15 intracranial tumors proved to be a homogeneous model with little variation within and between tumors with respect to morphology, GFAP expression, blood flow, and permeability. This cell line provides a GFAP-expressing brain tumor model that extends the use of autochthonous ASV-induced astrocytomas by allowing in vitro and in vivo studies. It may be useful for further studies in neurobiology and brain tumor biology, diagnosis, and therapy.

Structural and immunohistochemical characterization of insulin-like growth factor I and II receptors in the murine central nervous system.

The description of the cellular localization of insulin-like growth factor (IGF) receptors in the central nervous system (CNS) remains incomplete, as do the descriptions of changes in their characteristics with respect to different developmental stages. We, therefore, performed affinity labeling studies in microsomal membrane preparations of adult and fetal rat brain and liver tissues with [125I]IGF-I and [125I]IGF-II. These studies demonstrated tissue- and developmental stage-specific structural variants of type I receptor alpha-subunits as well as type II receptors. The adult rat brain type I alpha-subunit had an apparent mol wt (Mr) of 127,000, whereas those of adult and fetal rat liver measured 140,000. Fetal rat brain microsomes, however, had two types of type I receptor alpha-subunits measuring 130,000 and 120,000 Mr. The larger subunit from fetal brain consistently migrated at an apparent Mr of 3,000, greater than subunits from adult brain. Both type I and II receptors were more abundant in fetal liver and brain than in adult tissues. Affinity labeling was also performed directly to monolayers of cultured fetal brain neurons and newborn astrocytes. These studies detected both type I and II receptors on the surfaces of both types of cells. However, only the high Mr (140,000) form of the type I alpha-subunit was detected in cultured CNS cells, suggesting that expression of low Mr variant receptors is altered in vitro. Type II receptors were demonstrated by immunohistochemistry in adult rat hypothalamic neurons. However, the majority of neurons did not react with type II receptor antibody. This finding implies that only a minority of hypothalamic neurons are capable of responding to IGF-II via type II receptors. On the other hand, all astrocytes had striking type II receptor immunoreactivity. This signifies a more general biological role for this receptor in astrocytes compared with neurons. These results suggest that different tissue-, developmental stage-, and cell-specific processes are mediated by IGF receptors and suggests new directions in which to explore potential biological actions for these receptor-ligand systems in the CNS.

GFAP and astrogliosis.

One of the most remarkable characteristics of astrocytes is their vigorous response to diverse neurologic insults, a feature that is well conserved across a variety of different species. The astroglial response occurs rapidly and can be detected within one hour of a focal mechanical trauma (Mucke et al., 1991). Prominent reactive astrogliosis is seen; in AIDS dementia; a variety of other viral infections; prion associated spongiform encephalopathies; inflammatory demyelinating diseases; acute traumatic brain injury; neurodegenerative diseases such as Alzheimer's disease. The prominence of astroglial reactions in various diseases, the rapidity of the astroglial response and the evolutionary conservation of reactive astrogliosis indicate that reactive astrocytes fulfill important functions of the central nervous system (CNS). Yet, the exact role reactive astrocytes play in the injured CNS has so far remained elusive. This chapter summaries the various experimental models and diseases that exhibit astrogliosis and increase in glial fibrillary acidic protein (GFAP). Recent in vitro studies to inhibit GFAP synthesis are also presented.

Glial fibrillary acidic protein in the retina of the developing albino rat: an immunoperoxidase study of paraffin-embedded tissue.

The peroxidase-anti-peroxidase method was used on paraffin-embedded material to demonstrate the distribution of glial fibrillary acidic (GFA) protein, an astrocyte-specific protein, in the developing retina of the albino rat. At birth activity was scant and was confined to scattered, poorly differentiated cells in the inner retinal layers near the optic disc. At 3 days primitive astrocytes which displayed GFA protein activity were confined to the stratum opticum near the optic disc. With increasing age these cells were found at greater distances from the optic disc and began to assume the appearance of typical fibrous astrocytes. By 30 days the perikarya of these cells were confined almost exclusively to the region between the nerve fiber layer and the inner limiting membrane. The processes of these cells terminated either in suckerlike end-feet upon blood vessels or, to a lesser extent, ended in relation to axon fascicles of the nerve fiber layer. A second population of GFA protein-active cells existed as perivascular glia which were found upon vessels in the inner portion of the stratum opticum in young animals. In the mature retina perivascular glia were found on vessels throughout the stratum opticum and in the inner portion of the inner plexiform layer. Unequivocal staining of Müller cells or their processes was not obtained. The best staining was obtained with fixatives containing minimal concentrations of aldehydes, especially in tissue from younger animals. The fixative which gave the best preservation of cellular structure along with preservation of GFA protein antigenicity was Perfix (Fischer Scientific Company).

Glial fibrillary acidic protein in the optic nerve of the developing albino rat: an immunoperoxidase study of paraffin-embedded tissue.

The unlabeled peroxidase-anti-peroxidase method was used to stain glial fibrillary acidic (GFA) protein in the optic nerve of the developing albino rat. Optic nerves from animals ranging in age from the day of birth to adulthood were embedded in paraffin following fixation with various agents for times ranging from 30 minutes to 48 hours. GFA protein activity was demonstrable at birth in large astrocytic processes following fixation with alcohols or with Perfix for short intervals, but not with 4% or 2% buffered paraformaldehyde solutions. With increasing age, GFA protein could be demonstrated using higher aldehyde concentrations, longer fixation times, and longer paraffin embedding schedules. At all ages GFA protein activity was greater following treatment with nonaldehyde fixatives rather than those containing formaldehyde or glutaraldehyde. At birth the majority of GFA protein-containing processes were confined to planes which were perpendicular to the axons of the optic nerve. With increasing age, tangential and longitudinal processes became more numerous until, in the mature optic nerve, astrocytic processes were best characterized as being multidirectional.

The topographical distribution of S-100 and GFA proteins in the adult rat brain: an immunohistochemical study using horseradish peroxidase-labelled antibodies.

The cytological and topographical distribution of S-100 and glial fibrillary acidic (GFA) proteins in the adult rat brain has been compared using the horseradish peroxidase-labelled antibody technique. Both proteins are present in astrocytes and structures composed of astrocytic processes, namely the glial limitans and the perivascular membranes, but the cytological localization varies between the two proteins. S-100 is found in the nucleus and cytoplasm whereas GFA protein is confined to the cytoplasm. Neither is found in neurons, but S-100 is present in some oligodendroglia, suggesting a general regulatory role in glia. Although GFA protein in present in both protoplasmic and fibrous astrocytes, it is more prominent in the latter, confirming its association with astrocytic filaments.

Glial fibrillary acidic protein (GFAP): the major protein of glial intermediate filaments in differentiated astrocytes.

The glial fibrillary acidic protein (GFA protein or GFAP) is the major protein constituent of glial intermediate filaments in differentiated fibrous and protoplasmic astrocytes of the central nervous system. Proteins having similar molecular weights, isoelectric points, and immunoreactivity with GFAP have been found in cells of neural crest and ectodermal origin. A putative function ascribed to glial filaments is its role as a component of the cytoskeleton in defining and maintaining the shape of the astrocyte. Since 1980, over 350 reports have utilized antisera to GFAP for immunochemical and immunocytochemical studies.

[3H]thymidine labeling of astrocytes in experimental allergic encephalomyelitis.

In acute experimental allergic encephalomyelitis (EAE), astrocytes in spinal cord tissue hypertrophy and stain intensely with antibody to the glial fibrillary acidic protein (GFAP). We attempted to determine if this activation is a result solely of hypertrophy of existing astrocytes or if astrocyte division might also occur. Lewis rats in various stages of acute EAE were injected with [3H]thymidine, the spinal cord sections were prepared, immunostained for GFAP and processed for radioautography. In spinal cords from rats administered thymidine on days 11-15 after sensitization a large number of mononuclear cells showed radioactive label. Many of these labeled cells, most likely monocytes and lymphocytes, were associated with inflammatory lesions, but others were located in the CNS parenchyma at great distances from the lesions. Most cells staining for the GFAP were hypertrophied with greatly extended cell processes, and the nuclei of some of these cells identified as astrocytes were overlaid with silver grains, indicating uptake of [3H]thymidine. In addition a few ependymal cells appeared to be labeled. No GFAP-stained cells from the Freund's adjuvant controls contained radioactive label. Similar studies using SJL/J mice with chronic relapsing EAE yielded very few labeled inflammatory cells or astrocytes. This study indicates that division takes place in some astrocytes in acute EAE, but occurs much less frequently in chronic EAE. Probably most of the increase in GFAP-stained material is a result of hypertrophy of astrocytes rather than of massive cell division.

An ultrastructural and immunocytochemical study of astrocytic differentiation in vitro: changes in the composition and distribution of the cellular cytoskeleton.

Astroglia in cultures of dissociated neonatal rat optic nerves were studied by light microscopy, immunocytochemistry and electron microscopy to determine whether intermediate filaments play a role in defining the multipolar morphology of the mature astrocyte. Immature, polygonal astroblasts contained few glial filaments, in spite of exhibiting positive staining with antiserum against glial fibrillary acidic (GFA) protein. Microtubules were the most prominent cytoskeletal component at early stages of cytodifferentiation, but these were progressively reduced in number at later intervals and were gradually replaced by intermediate filaments. These observations suggest that microtubules are involved in the initial establishment of cytoplasmic asymmetry and process development. Subsequently, glial filaments may play a role in maintaining and stabilizing the overall geometry of the mature astrocyte.

Post-embedding immunoperoxidase staining of glial fibrillary acidic protein for light and electron microscopy.

Post-embedding peroxidase-antiperoxidase methods to stain glial fibrillary acidic (GFA) protein in Araldite-embedded sections for light and electron microscopy were developed. The Jimpy mouse spinal cord was used because it is gliotic and contains abundant glial filaments and GFA protein. For light microscopy, specific staining was obtained in thick and in ultrathin sections mounted on glass following removal of the plastic with sodium ethoxide. Consistent specific staining for GFA protein in ultrathin sections mounted on nickel grids required partial removal of the plastic with 1% sodium ethoxide and further treatment with 2% sodium dodecyl sulfate (SDS).

Processing techniques for the demonstration of myelin basic protein in paraffin-embedded optic nerve: an immunoperoxidase study of the developing albino rat.

A comparison was made of the effects of various fixation and processing conditions upon the antigenicity of myelin basic protein (MBP) in sections of paraffin-embedded optic nerve from the developing albino rat as judged by the unlabeled peroxidase-antiperoxidase technique. The fixatives used were: Perfix, 4% and 2% buffered paraformaldehyde (pH 7.4), 10% buffered formalin (pH 7.4); Bouin's, Clark's, and Carnoy's fixatives, and 20% formalin in a solution of HgCl2 that had been saturated at 1 degrees C. Perfix appeared to be the best fixative for the preservation of morphology and MBP antigenicity during the early stages of myelinogenesis but was not satisfactory during the later stages. The buffered aldehydes were slightly more destructive of MBP antigenicity than was Perfix, but they produced satisfactory results following the first postnatal week. Bouin's fixative was similar in effect to the buffered aldehydes, but nonspecific background staining was higher. HgCl2/formalin, Clark's and Carnoy's fixatives were unsuitable. No differences were noted in staining between material processed for embedding using 5, 30, or 60 min schedules.