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The antibody to the endothelial barrier antigen (anti-EBA) is localized to the luminal plasma membrane of endothelia that have a blood-brain barrier (BBB) but not to other vessels, for instance those in the circumventricular organs, which lack barrier function. We have examined EBA expression in the rat in certain tissues and in brain microvessels in models of brain angiogenesis such as development, wound healing and neural transplantation. All brain microvessels including pial ones stained for anti-EBA whereas those of the dura, median eminence and choroid plexus did not. Vessels of the iris which are characterized by tight junctions and barrier function expressed EBA strongly. Embryonic day 18 brain did not stain at all for anti-EBA although vessels were readily localized with anti-laminin. Following stab wounds to mature brain, directly injured and adjacent microvessels lacked EBA expression for a period of approximately 2 weeks which is a similar time frame of BBB breakdown. Following this period, EBA expression gradually returned to a normal pattern by 3-4 weeks. Likewise, in intraparenchymal transplants of fetal neocortex EBA expression was not observed for 2 weeks and while at later times transplant vessels expressed EBA whereas some interface vessels associated with inflammatory cells did not. Permeable choroid plexus vessels vascularizing intraventricular transplants did not stain for anti-EBA at any time period and neither did vessels in adrenal medulla transplants. The present study shows that while EBA expression is a postnatal event unlike the development of a barrier to serum protein, its expression may be lost or delayed in injured vessels or ones associated with inflammatory cells or reactive astrocytes.

Upon plotting of areas against optical densities in immunocytochemically stained tissue sections, hyperbolic curves were obtained which could be reduced to two straight lines, one representing variations in stained structures, and the other variations in background. The slopes of the stained structure lines reflected staining intensity independently of total area of stained structure in a section. The ratio of slopes of the stained structure and background lines reflected immunocytochemical sensitivity. A comparison of the peroxidase-antiperoxidase (PAP) method with the avidin-biotin complex (ABC) method showed that at usual antibody dilutions the PAP method was much more sensitive than the ABC method, while at impractically high antibody dilutions it was moderately more sensitive. Once sufficient dilutions of antibodies were reached, staining intensities dropped sharply with the PAP method. On the other hand, the dilution curves were flat with the ABC method. The ABC method consequently appeared unsuitable for estimating variations in concentration of antigen or for distinguishing high or low concentrations of antigen. The ABC method provided a stain for myelin even in the absence of any antibodies.

Active plaques from 4 patients with multiple sclerosis were examined for myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) using the peroxidase-antiperoxidase (PAP) immunocytochemical procedure applied to paraffin sections. MBP loss was intimately related to the presence of infiltrating macrophages which appeared to remove MBP-positive fragments directly off intact myelin sheaths. Phagocytosis of MAG-positive myelin sheaths was also observed. These findings support previous morphological studies that suggest that phagocytosis by macrophages of myelin attached to axons is an important mechanism of demyelination in multiple sclerosis.

Cerebrovascular amyloid is the main constituent of the perivascular and neuritic plaques typical of Alzheimer disease, whereas neurofilaments and microtubule-associated tau protein have been considered primary contributors to the formation of the characteristic Alzheimer tangles. Plaques and tangles and their constituents have at times been ascribed a role in pathogenesis of the disease. Normally, neurofilaments become phosphorylated only upon axonal entry. In many neurologic disorders, neurofilament phosphorylation, as detected by any of the available monoclonal antibodies (mAbs) to neurofilament phosphorylated epitopes is shifted from an axonal to a cell-body location. An exception is provided by Alzheimer disease, where tangles (which are neuronal cell-body-derived structures) exhibit only one phosphorylated epitope. However, the very presence of neurofilaments in tangles and plaques has been questioned because of a reported cross-reaction of mAbs to phosphorylated neurofilaments with tau protein. On reinvestigating this cross-reactivity we found that four of five mAbs to phosphorylated neurofilaments and four of five mAbs to nonphosphorylated neurofilaments failed to react with tau protein. A fifth mAb (07-5) to phosphorylated neurofilament cross-reacted with partially denatured tau protein at an affinity 1/1700th of that for denatured neurofilaments; nondenatured tau protein in tissue sections did not cross-react. A fifth mAb (02-40) to nonphosphorylated neurofilament also cross-reacted weakly. In Alzheimer disease normal-appearing axons were revealed with all the mAbs to phosphorylated neurofilaments, but tangles were revealed with only one of them (mAb 07-5). mAb to tau protein did not stain or did so indistinctly. Four of five mAbs to nonphosphorylated neurofilaments failed to reveal axons. Upon dephosphorylation of tissue, staining by mAbs to phosphorylated neurofilaments disappeared, and axons were revealed with the mAb to tau protein and all mAbs to the nonphosphorylated neurofilaments. Tangles became stained with tau mAb and one mAb to the nonphosphorylated neurofilaments (mAb 10-1). Quantitative evaluation of immunocytochemical staining intensities and immunoblot cross-reactivity showed that neurofilaments are, indeed, constituents of tangles--apparently exceeding the concentration of tau protein 17-fold. Contribution of both conformation and primary structure to IgG specificity may explain the lack of any cross-reaction of mAbs to neurofilaments with tau protein in intact tissue and the appearance of cross-reaction in immunoblots where conformation specificity may be largely lost. The present data extend earlier findings of abnormal processing of neurofilaments and tau protein in Alzheimer disease and, together with reported abnormal processing of cerebrovascular amyloid beta-protein, suggest that inhibition of the processing of multiple proteins is basic to the pathogenesis of Alzheimer disease, whereas formation of plaques and tangles could be merely the most striking histologic result.

Neurofilaments purified from adult rat brainstem by two methods were electrophoresed on NaDodSO4/polyacrylamide gels to separate the triplet proteins (approximate Mrs of 200,000, 155,000, and 68,000) which, in turn, were electroblotted onto nitrocellulose paper. On Coomassie blue-stained gels that were not electroblotted, the same banding pattern was seen with both methods of preparation. Immunocytochemical staining of the electroblots with each of five monoclonal antibodies revealed that three of the monoclonal antibodies were specific for the Mr 200,000 neurofilament protein and two, for both the Mrs 200,000 and 155,000 neurofilament proteins. None of the antibodies reacted with the Mr 68,000 band. The Mr 200,000 band could be resolved into doublet bands. Individual monoclonal antibodies reacted with either one or both of the Mr 200,000 doublets. The immunocytochemical staining of the neurofilament triplets on electroblots was compared to that of adult rat cerebellar paraffin sections. Each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes. Correlation of the staining patterns in cerebellar tissue sections with those of neurofilament polypeptides on electroblots suggested that different neurofilament polypeptides can be localized to different structures and subpopulations of neurons and that molecular heterogeneity ("neurotypy") may be revealed within the Mrs 200,000 and 155,000 neurofilament polypeptides.

The reaction of Lewy bodies in the substantia nigra and the sympathetic ganglia was studied in 10 cases of Parkinson's disease and other Lewy body-related conditions, using monoclonal antibodies to non-phosphorylated (02-40) and phosphorylated (03-44 and 07-5) neurofilaments. Although many Lewy bodies were unstained, a peripheral ring of positive immunoreactivity was frequently observed. This reaction was more common and more intense with antibodies to phosphorylated neurofilaments, suggesting a posttranslational phosphorylation of elements in the Lewy body. Although Lewy bodies appear to have some antigenic sites in common with neurofilaments, certain differences between neurofilaments and Lewy body filaments are emphasized.

Intrathecal administration of aluminum salts induces accumulation of neurofilaments in axons and perikarya of motor neurons and is associated with impaired axonal transport of neurofilament proteins. Because phosphorylation of the 200-kilodalton (kd) neurofilament protein, thought to be a major component of the sidearms, seems to be important in interactions of neurofilaments with other cytoskeletal elements, we have postulated that aluminum may produce neurofibrillary pathology by altering patterns of neurofilament phosphorylation. To test this hypothesis, antibodies against phosphorylated and non-phosphorylated neurofilament epitopes were used for immunocytochemical analysis of spinal cord sections from aluminum-injected rabbits. In control animals, phosphorylated 200-kd neurofilament proteins were not demonstrable in perikarya of motor neurons. In experimental rabbits, perikarya and proximal axons of affected motor neurons showed striking accumulations of immunoreactivity of one phosphorylated epitope. The presence of phosphorylated 200-kd neurofilament proteins in these regions may have important consequences for the organization of the cytoskeleton and for the transport of neurofilaments. A similar, but not identical, pattern of accumulation of phosphorylated neurofilament immunoreactivity has recently been observed in neurofibrillary tangles in Alzheimer's disease.

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease (AD), and their presence correlates with the presence of dementia. A major constituent of NFT is the insoluble paired helical filament which shares some antigenic relationships with normal cytoskeletal elements, particularly neurofilaments. If neurofilament proteins (200, 145-160, and 68 kilodaltons [kd]) participate in the formation of NFT, the distribution of these constituents might be expected to be abnormal. To examine this issue, we used immunocytochemical methods to localize phosphorylated and nonphosphorylated epitopes of neurofilament proteins in hippocampal neurons of controls and patients with AD. Normally, the 200-kd neurofilament protein is not phosphorylated in the perikarya of neurons. However, in AD, many pyramidal neurons contained immunoreactive phosphorylated neurofilaments. Patterns of immunoreactivity (linear, flame-shaped, or skein-like within perikarya) greatly resembled the appearance of silver-stained NFT. This pattern of immunoreactivity was not present in hippocampal pyramidal neurons in controls, except in one aged patient in whom adjacent silver-stained sections revealed a few NFT. Patterns of immunoreactivity with antibodies for nonphosphorylated neurofilament proteins were similar in control and AD neurons. Our results indicate that some NFT are associated with abnormal distributions of high molecular weight phosphorylated neurofilament proteins. One domain of the 200-kd protein is believed to be a component of the side arms which link neurofilaments and interact with microtubules. Abnormal interactions of perikaryal neurofilaments could play a role in the genesis of NFT, and this abnormality of the cytoskeleton could contribute to the dysfunction of neurons at risk in AD.

An immunocytochemical method for detecting myelin basic protein in oligodendrocytes and myelin of newborn rat C.N.S. is described. C.N.S. tissue is perfused and fixes in HgCl2--formaldehyde and 20 micron Vibratome sections are treated with antibodies to myelin basic protein using the peroxidase--antiperoxidase method. Oligodendrocytes in the newborn rat are intensely stained by antiserum to basic protein and multiple stained processes extend from the perikaryon to myelin sheaths. With this procedure it is possible to demonstrate the geometric relationships between a single oligodendrocyte and multiple myelin sheaths. Stained oligodendrocytes and myelin are present in newborn cervical spinal cord, medulla oblongata, pons and midbrain. By 25 days of age, staining in oligodendrocytes is less intense than in newborn rats and differences in amount of staining can be detected in areas that are myelinating at different rates. With anticerebroside serum, cerebroside, of newborn and developing rat C.N.S. tissue is localized only in myelin. In the developing P.N.S., myelin basic protein is localized in Schwann cell cytoplasm and myelin sheaths of the trigeminal ganglion. Cerebroside is found only in myelin.

Two-dimensional immunoblots revealed a spectrum of 200 kDa neurofilament polypeptides (Nf-H) of apparent molecular weights ranging from 200 to 170 kDa. The entire spectrum was stained immunocytochemically by three monoclonal antibodies specific for nonphosphorylated neurofilaments, while more restricted staining was revealed by four monoclonal antibodies specific for phosphorylated neurofilament epitopes. Treatment with increasing amounts of phosphatase suggested the existence of various forms of partially phosphorylated neurofilaments that posses phosphoepitopes that differ in their ease of dephosphorylation. Immunoprecipitation in low detergent concentration confirmed the existence of microheterogeneous forms of Nf-H that differed in extent of phosphorylation or in distribution of phosphorylated sites.

One-micron plastic sections of spinal cords from SJL/J mice with chronic relapsing experimental allergic encephalomyelitis (EAE) were reacted immunocytochemically with antiserum to myelin basic protein and myelin-associated glycoprotein. The distribution of myelin basic protein and myelin-associated glycoprotein in myelin sheaths was compared in acute and chronic areas of demyelination. No difference in the size of the lesion was seen with the two antisera. Myelin-associated glycoprotein was seen periaxonally in both normal myelin sheaths and sheaths which showed extensive splitting and ballooning as seen with toluidine blue stain and myelin basic protein antiserum. At least at the level of the light microscope, myelin basic protein antiserum gave intense staining of myelin while antiserum to myelin-associated glycoprotein showed little or no affinity to stain the myelin sheath itself, in contrast to other recent electron microscope observations. A few myelin basic protein or myelin-associated glycoprotein-containing oligodendrocytes were seen in lesion areas and remyelination by oligodendrocytes was rare. These observations are in agreement with findings from other models of EAE and multiple sclerosis where a primary loss of myelin has been implicated.

An IgG1 mouse monoclonal antibody produced in response to immunization with rat brain homogenate reacted with endothelial cells in the central and peripheral nervous system. Because antibody reactivity was associated with endothelia that have a selective permeability barrier, the antibody was called anti-endothelial-barrier antigen (anti-EBA). Paraffin sections of Bouins'-fixed rat tissue were used for initial screening and subsequent characterization of antibody reactivity. The antibody was generally unreactive with endothelial cells in other organs and with nonendothelial cells in or outside of the nervous system. Antibody binding was greatly reduced or absent in endothelia of the area postrema and choroid plexus, sites known to possess fenestrated blood vessels. In developing rat brain, anti-EBA binding to some microvessels was seen at 3 days postnatally. Anti-EBA reactivity outside the nervous system occurred in spleen and skin. Patchy reaction with portions of some spleen blood vessels and binding to some cells in the spleen were observed. In the skin, small cells, tentatively identified as Langerhans cells, which participate in Ia presentation, were stained. On immunoblots of rat brain microvessel preparations electrophoresed in Na-DodSO4/polyacrylamide gels, anti-EBA reacted with a protein triplet of Mr 30,000, 25,000, and 23,500 components.

Specific antibodies have been developed against P1, P2, and P0 myelin proteins and were used to study the localization of these proteins in the rat peripheral nervous system. Both peripheral and central nervous system myelin sheaths contain P1 protein. P0 and P2 proteins are found exclusively in peripheral nervous system myelin sheaths. Antisera to P1 and P0 proteins stain all peripheral nervous system myelin sheaths uniformly. P2 protein is not a component of all peripheral nervous system myelin sheaths. In sheaths that do contain P2 protein, it is concentrated in the area of the Schmidt-Lanterman incisures.

During incubation with phosphatase, the 200 kDa neurofilament protein in cytoskeletal preparations is degraded extensively. Degradation, which is divalent cation-independent, does not occur when inhibitors of phosphatase are added. The 160 kDa chymotryptic fragment of neurofilaments or affinity-purified 200 kDa protein are not degraded by phosphatase. The results suggest that (1) phosphorylated neurofilaments are protected against proteolysis, and (2) dephosphorylated neurofilaments are degraded by a calcium-independent, endogenous proteinase which is associated with assembled neurofilaments or with other cytoskeletal components, and not with the phosphatase used.

Allografts of embryonic (E14-E15) rat cerebellum in adult brain were compared using the intraparenchymal and intraventricular transplantation techniques. We studied the expression and distribution of phosphorylated neurofilament (PNF) epitopes, nonphosphorylated neurofilament (nPNF) epitopes, synapse-associated antigens, glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP). Both intraventricular and intraparenchymal grafts developed a clear trilaminar organization. Intraparenchymal grafts were much smaller and showed a large GFAP-positive glial scar and demyelination of host tissue. Nevertheless, myelinated fibers were present more often crossing the host-transplant border in intraparenchymal grafts. PNF and nPNF epitopes were present in both types of grafts. Staining patterns characteristic of normal rat cerebellum were seen. nPNF epitopes were present in Purkinje cell bodies and dendrites and PNF epitopes in basket cell axons surrounding Purkinje neurons. The appearance and distribution of PNF epitopes resembled that seen in normal postnatal cerebellar development and both PNF and nPNF epitopes were present at the same times in early development in both intraventricular and intraparenchymal grafts. In contrast to the situation in trauma and disease, PNF epitopes never appeared in perikarya of transplanted cerebellar neurons. The expression of synapse-associated antigens in grafted tissue was also similar to that seen in normal cerebellum.

The effect of an inhibitor of N-glycosylation of glycoproteins, tunicamycin, on synthesis of PNS myelin proteins was investigated in vitro by using chopped sciatic nerves or spinal roots of 21-day-old Wistar rats. Tunicamycin when incubated with these nerves in the presence of 3H-labeled fucose, mannose, or glucosamine inhibited the uptake of radioactivity into myelin proteins including some high-molecular-weight proteins, P0, 23K protein, and 19K protein by amounts ranging from 42 to 79%. Uptake of 14Camino acid mixture was inhibited much less by tunicamycin, but a new radioactive protein peak appeared when the protein mixtures had been separated by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. This protein ran directly in front of the P0 peak, did not correspond to any bands stained by Fast green, and was not labeled by fucose. This peak appeared in increasing larger proportions with progressive time of incubation of nerves with 3H amino acids in the presence of tunicamycin. The new protein, which cross-reacts with P0 antiserum, was tentatively identified as a nonglycosylated P0 protein that appears to be almost as well incorporated as P0 into the subcellular fraction containing myelin. At this time it is not possible to determine whether the unglycosylated P0 is actually assembled into a site and configuration like that of P0.

Recent immunocytochemical studies indicated that the myelin-associated glycoprotein (MAG) is localized in the periaxonal region of central nervous system (CNS) and peripheral nervous system (PNS) myelin sheaths but previous biochemical studies had not demonstrated the presence of MAG in peripheral nerve. The glycoproteins in rat sciatic nerves were heavily labeled by injection of [3H]fucose in order to re-examine whether MAG could be detected chemically in peripheral nerve. Myelin and a myelin-related fraction, WI, were isolated from the nerves. Labeled glycoproteins in the PNS fractions were extracted by the lithium diiodosalicylate (LIS)-phenol procedure, and the extracts were treated with antiserum prepared to CNS MAG in a double antibody precipitation. This resulted in the immune precipitation of a single [3H]fucose-labeled glycoprotein with electrophoretic mobility very similar to that of [14C]fucose-labeled MAG from rat brain. A sensitive peptide mapping procedure involving iodination with Bolton-Hunter reagent and autoradiography was used to compare the peptide maps generated by limited proteolysis from this PNS component and CNS MAG. The peptide maps produced by three distinct proteases were virtually identical for the two glycoproteins, showing that the PNS glycoprotein is MAG. The MAG in the PNS myelin and Wl fractions was also demonstrated by Coomassie blue and periodic acid-Schiff staining of gels on which the whole US-phenol extracts were electrophoresed, and densitometric scanning of the gels indicated that both fractions contained substantially less MAG than purified rat brain myelin. The presence of MAG in the periaxonal region of both peripheral and central myelin sheaths is consistent with a similar involvement of this glycoprotein in axon-sheath cell interactions in the PNS and CNS.

Schober R., Itoyama Y., Sternberger N.H., Trapp B.D., Richardson E.P., Asbury A.K., Quarles R.H. & Webster H. deF. (1981) Neuropathology and Applied Neurobiology 7, 421–434 Immunocytochemical study of P0 glycoprotein, P1 and P2 basic proteins, and myelin-associated glycoprotein (MAG) in lesions of idiopathic polyneuritis To investigate the mechanism of myelin breakdown in idiopathic polyneuritis, paraffin and Epon sections of lesions were immunostained with antisera to four proteins in myelin sheaths. Three of these (P0, P2, and BP) are constituents of compact myelin whereas myelin-associated glycoprotein (MAG) is restricted to membranes near Schwann cell cytoplasm in periaxonal and paranodal regions and in Schmidt-Lanterman clefts. In early lesions, there were focal abnormalities in P2, P0, and BP immunostaining of paranodal and intemodal myelin. No single protein was affected selectively and lesions occurred in fibres of all sizes, not just in larger fibres selectively stained by P2 antiserum. Early changes in MAG immunostaining occurred only in regions where myelin immunostaining also was abnormal. More severe, later changes in the distribution of P0, P2, BP and MAG were consistent with the sequence of myelinated fibre alterations seen in segmental demyelination and Wallerian degeneration. In regenerating fibres, MAG antiserum stained periaxonal regions intensely; thin regenerating myelin sheaths were stained by P0 and BP antisera, but not by P2 antiserum. The results show that myelin sheath changes are identified more easily in immunostained sections than in conventional histological preparations. Our data also suggest that in idiopathic neuritis, myelin sheaths are the primary target and that the breakdown of myelin and its proteins is not secondary to Schwann cell damage.

A specific antibody to myelin basic protein has been used to localize the protein in developing rat oligodendroglia and myelin. Basic protein is found in the oligodendroglial cytoplasm of anterior commissures of 5- and 7-day old rats before the beginning of myelination. Staining of basic protein in oligodendroglia increases, becoming most intense during early myelination; it decreases during rapid myelination. Staining intensity of oligodendroglia is dependent upon age, brain region, and nervous tract studied. In myelin, reaction of basic protein with antibody decreases when large compact sheaths are present, unless tissue sections are first treated with alcohol.

We used a library of monoclonal antibodies (Mab) that distinguish phosphorylated (P+) and non-phosphorylated (P-) neurofilament (NF) epitopes to examine phosphorylation of NF in lower motor neurons of patients with amyotrophic lateral sclerosis (ALS), of neurologically normal controls of different ages, and of patients with central chromatolysis due to injuries to motor root axons. Monoclonal antibodies directed to P+ NF immunostained five to ten times more neuronal perikarya in ALS than in age-matched controls. Spheroids, which are NF containing axonal enlargements, found in significantly greater number in proximal axons in ALS, were also intensely immunostained with Mab to P+ NF. Moreover, anterior root axons in five of eleven cases of ALS reacted only with the Mab to P+ NF, while both P- and P+ NF were present in motor roots from controls. In control groups, the number of neuronal perikarya and spheroids that immunoreacted with the Mab to P+ NF increased moderately with age. Chromatolytic lower motor neurons were recognized by Mab to P+ NF. Our results show that the process of phosphorylation is altered in ALS. We propose that phosphorylation of NF in ALS occurs prematurely and that it is more likely to be associated with an impairment of NF transport than to be part of a chromatolytic reaction of lower motor neurons.

Until recently, antibodies used in a variety of immunochemical techniques have been raised in laboratory animals by repeated injections of purified antigen and adjuvant. These antisera are a mixture of antibodies that may recognize unique or common antigenic sites and have different affinities. Such polyclonal antisera can vary considerably from animal to animal or even with different bleeds from one animal. The hybridoma technique of Kohler and Milstein (1975) permits the selection of a monoclonal antibody with a specific affinity that recognizes a specific antigenic determinant. Monoclonal antibodies to a variety of neural antigens have been produced by injection of "impure" antigen. Immunocytochemical findings with these monoclonal antibodies demonstrate some of the advantages and problems of hybridoma technology. Studies with these antibodies in normal and pathological (B,B'-iminodipropionitrile intoxication, Alzheimer's disease) conditions illustrate some of the potential applications of monoclonal antibodies.

To study the distribution of basic protein (BP) and other myelin constituents immunocytochemically in human nervous tissue, we modified the unlabeled antibody enzyme (peroxidase-antiperosidase) method. The technique is described here. Because the availability of unfixed tissue from human central nervous system is limited, we tested the method on blocks that had been fixed in formalin and embedded in paraffin, fixed and stored in 4% formalin, or frozen at autopsy and stored. We obtained the best results with paraffin blocks. BP antiserum scained oligodendroglia and myelin sheaths in the developing human nervous system. In the adult, myelin sheaths were well stained. Also, abnormalities associated with myelin breakdown could be identified in multiple sclerosis lesions. The results suggest that this method will be useful in studying the cellular distribution of myelin components in human demyelinating diseases.

Cytoskeletal proteins have a characteristic distribution within neurons when immunocytochemical techniques are used on conventional paraffin sections. For example, phosphorylated neurofilaments are located within axons but are not normally present in the majority of perikarya of the central nervous system. This pattern can be altered in disease, and neurofilaments that accumulate within perikarya can be phosphorylated inappropriately. To determine whether retained neurofilaments were phosphorylated inappropriately, we used immunocytochemical techniques to examine several diseases in animals in which neurofilaments accumulate within neuronal perikarya. Our investigations of diseases with disparate etiologies show that, whenever neurofilaments are retained within the neuronal perikarya, they are phosphorylated. These results suggest that phosphorylation of neurofilaments in an inappropriate location, i.e. perikarya, may be a nonspecific disease-related response of neurons that can be initiated by a variety of cellular injuries.

Endothelial cells (EC) are increasingly being considered as important participants in the early evolution of inflammatory and immune responses in experimental allergic encephalomyelitis (EAE). We have found that a mouse monoclonal antibody, which reacts with the luminal plasma membrane of central nervous system endothelium, detects an alteration in the blood-brain barrier (BBB) in lesions in Lewis rats with EAE. Anti-endothelial barrier antigen (EBA) reacted with microvessels in normal rat brain and spinal cord. This reaction was abolished in ‘EAE’ microvessels surrounded by inflammatory cells. In rats that had recovered from one attack most EC reacted with the antibody, indicating that EBA was reexpressed during recovery. However, blood vessels in areas with residual inflammatory lesions were negative. The biochemical changes that lead to this absence of antibody binding and the cells or mediators responsible for producing this change are not yet known. However, anti-EBA should provide a useful tool for exploring molecular mechanisms underlying BBB breakdown.

In order to compare the apparently reactive phosphorylation of neurofilaments (Nf) observed in trauma and neurodegenerative disorders, we examined the expression and distribution of Nf in implants of embryonic rat cerebellum after 2 to 4 weeks survival. Donor cerebella were taken from embryos at gestational day E14 to E15 and implanted into brain parenchyma or ventricles. Immunocytochemistry with monoclonal antibodies to 5 different phospoepitopes of Nf (anti-PNf) and 5 different epitopes in non-phosphorylated Nf (anti-nPNf) (1) was done on Bouin-fixed paraffin sections using peroxidase- antiperoxidase complex from monoclonal antiperoxidase. Antibodies to other neural antigens such as myelin basic protein, glial fibrillary acidic protein and SAP, a monoclonal antibody that reacts with synapse-rich areas, were used to compare their appearance and distribution with the Nf antibodies. After 2 weeks survival, the earliest age examined, cresyl violet staining of some transplants showed the beginning of a trilaminar organization. Both PNf and nPNf epitopes were present. As in our studies of normal rat cerebellar development there was no lag in the appearance of PNf. Nf were seen in both the ventricular and parenchymal implants. By 4 weeks, transplants exhibited a high degree of internal organization. Staining patterns characteristic of cerebellum were seen with the antibodies: nPNf in Purkinje cell bodies and dendrites and PNf in basket cell axons surrounding the Purkinje cells. In summary, the transplants demonstrated cytoarchitectural features characteristic of the cerebellum. Appearance and distribution of Nf epitopes resembled that seen in normal postnatal cerebellar development and both PNf and nPNf were present at the same time in early development. In contrast to trauma and disease (2), PNf never appeared in perikarya. The rapid phosphorylation of newly formed Nf may not only indicate rapid assembly of neurofilaments but may also protect newly formed Nf against the known susceptibility of nPNf to rapid proteolysis.

Rabbits were immunized with the myelin-associated glycoprotein (MAG) that had been purified from isolated rat brain myelin by selective extraction with lithium diiodosalicylate (LIS) and phenol followed by preparative SDS gel electrophoresis. Antibodies to MAG were detected qualitatively by immunodiffusion and quantitatively by a double antibody assay utilizing [3H]fucose-labeled MAG as antigen. The antisera were capable of precipitating between 300 and 500 micrograms of MAG/ml of serum under the conditions of the assay. Preincubation of the anti-MAG serum with other glycoproteins or glycolipids did not inhibit the precipitation of labeled MAG. Similarly, preincubation of the antiserum with LIS-phenol extracts of non-neural tissues did not inhibit the immune precipitation of MAG. The specificity of the antiserum was also indicated by the selective double antibody precipitation of MAG from solubilized whole myelin that contained a heterogeneous mixture of [3H]fucose-labeled glycoproteins. The antibodies to MAG were not effectively absorbed by whole brain homogenate or purified myelin, indicating that the antigenic site(s) is not accessible in the intact membranes, but can be exposed by treatment with detergent or partial purification. Low levels of antibodies reacting with MAG were detected in three rabbits with experimental allergic encephalomyelitis induced by injection of purified myelin in complete Freund's adjuvant.

The immunocytochemical staining patterns of 37 neuron-specific monoclonal antibodies previously described fell into four groups: (i) anti-synapse-associated, (ii) anti-neurofibrillar, (iii) anti-perikaryonal-neurofibrillar, and (iv) a single antibody reactive with a widely distributed epitope that covered the patterns of groups ii and iii. Antibodies of groups ii, iii, and iv were shown to be specific to neurofilament triplet subunits, even though there was little overlap in staining patterns between groups ii and iii. We examined nine of these antibodies as to their ability to distinguish functional states of neurofilaments dependent upon phosphorylation. Upon digestion with phosphatase, electroblot staining of neurofilament components was abolished with the five antibodies from group ii, enhanced with the three antibodies from group iii, and unaffected with antibody iv. Immunocytochemical staining of Bouin-fixed paraffin sections of rat brain was unaffected by phosphatase pretreatment. With antibodies of group ii, digestion with trypsin also left staining unaffected, but when followed by digestion with phosphatase, staining was diminished with three out of five antibodies. In contrast, digestion with trypsin abolished all staining with each antibody from group iii. If followed by digestion with phosphatase, staining reappeared, but the group iii pattern was replaced by a group ii pattern. Staining of this pattern was again abolished upon a second treatment with trypsin. The antibody from group iv lost most of its groups ii and iii staining patterns when sections were digested with trypsin. The group ii pattern reappeared and, indeed, was enhanced upon a subsequent phosphatase treatment and was reduced again upon a second trypsin treatment. Staining by four out of five antibodies from group ii was inhibited by inorganic phosphate. The data indicate that certain nerve cell bodies, their dendrites, and at least proximal axons possess nonphosphorylated neurofilaments and that long fibers, including terminal axons, possess phosphorylated neurofilaments. We propose that phosphorylation may be a factor in stabilizing compacted forms of neurofilaments and that heterogeneity of the compacted structures may play a role in a possible multiplicity of function within individual nerve cells.

The classification of cells into types and the functions assigned to each cell type have, in the past, been dependent primarily upon morphological criteria. The functions usually associated with oligodendrocytes are: (1) myelin formation by interfascicular oligodendrocytes and (2) nutrition of neurons by satellite oligodendrocytes in the gray matter, the satellite cells being presumed not to participate in myelination (for a review, see Raine, 1981). More recently, immuncytochemistry has become a useful tool for confirming and extending (or contradicting) conclusions based on morphological observations. In addition, immunocytochemical studies have led to the discovery of new functions associated with oligodendrocytes and have shown that oligodendrocytes can no longer be considered as being solely myelin-producing cells.

After repeated exposure, acrylamide (AC) produces degeneration of distal axons. Because neurons whose axons have been injured (e.g. by axotomy) show alterations in their structural and chemical properties, the present study was designed to differentiate the direct effects of AC intoxication from neuronal responses secondary to axonal injury caused by AC. Rats were given AC as either a single high dose (75 mg/kg), or as daily intraperitoneal injections (30 mg/kg, six days per week for four weeks). Dorsal root ganglia of the fifth lumbar level, L5, were examined using a variety of monoclonal antibodies directed against nonphosphorylated (2–135) and phosphorylated (03-44, 06-17, 07-05) epitopes of 145 and 200 kilodalton neurofilament proteins. In control rats, antibody 2–135 stained axons and neuronal cell bodies; antibodies against phosphorylated epitopes of neurofilaments stained only axons distal to the glomerulus. Following chronic AC intoxication, all three antibodies directed against phosphorylated epitopes of neurofilaments (particularly 07–05) demonstrated intense immunoreactivity in 20–300% of neuronal cell bodies. In addition, the glomerular region of these axons was stained. Electron microscopy revealed many chromatolytic cells containing few neurofilaments. In contrast, a single high dose of AC produced no abnormal staining of neuronal cell bodies at a time when slow axonal transport was impaired. Our findings are compared to those observed following axotomy and to those occurring in aluminum-intoxicated rabbits, two experimental disorders in which altered distributions of phosphorylated filaments have been documented.

Monoclonal antibodies provide opportunity to purify known antigens, discover and purify new antigens, characterize their submolecular structure, collect, in favorable cases, relevant cDNA from multiple clones obtained from an expression vector, and finally study normal and pathologic interactions of antigens in situ. Our work had been initiated as a “peche a la ligne” expedition, using whole brain homogenate as bait to the immune system of mice and fishing out from the pool of fused immune B cells those that produced antibodies of special interest, while discarding antibodies that were irrelevant. Special interest imposed a limitation to include antibodies that reacted at high specificity and selectivity. The requirement was fulfilled when the antibodies detected only one antigen, previously known or unknown, to the exclusion of any other antigen in the same host. This type of analysis could be carried out when the antibodies produced by clones derived from fusions of immune spleen cells with myeloma cells were analyzed by immunocytochemistry, which does not require prior knowledge of the antigens searched for, in contrast to the more commonly performed kinds of analysis, such as enzyme-linked or radioimmunoassay, which utilize the specific antigen looked for.

Adult and developing rat cerebella were stained immunocytochemically with six neuron-specific monoclonal antibodies obtained from spleen cells of BALB/c mice immunized with hypothalamus. Monoclonal peroxidase-antiperoxidase complex was used in the staining procedure. In the adult, three different staining patterns were seen with these antibodies. The general patterns have been designated as (1) neurofibrillar, (2) perikaryal-neurofribillar, and (3) synapse-associated, in addition, each antibody within a designated group showed a different immunocytochemical distribution. Some antibodies reacted widely, for example, with many neuronal perikarya and fibers, and their distribution increased with the development of the brain. Other antibodies had a more restricted distribution and stained only some structures within an area or pathway. To account for this type of restriction of distribution, we propose that there may be microheterogneity of some of the antigens visualized and have called this microheterogeneity ‘neurotypy’. A second type of restriction was also observed. With several antibodies a loss of staining occurred in the adult cerebellum in structures that had reacted during early development. These differences in staining probably reflect developmental regulation of the antigens (neurotypes).

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called "neurotypes") rather than different antigenic determinants in single antigens. On examining the distribution in peripheral organs of staining patterns of 11 antineuronal brain-reactive antibodies, we now confirm that these antibodies are, indeed, largely brain-specific. In general, non-neuronal elements in liver, lung, heart, thymus, intestine, adrenal, and spleen remained unstained. However, most of the antibodies stained peripheral neural elements. Occasional antibodies did stain selected, non-neuronal structures. Four out of five antineurofibrillar antibodies stained nerve fibers in adrenal medulla, intestine and thymus. All of three antiperikaryonal-neurofibrillar antibodies also stained nerve fibers in the adrenal medulla, but not in other organs. Two out of three antisynapse-associated antibodies stained what appear to be nerve contacts on adrenal medullary cells, but not on any other peripheral cells examined. The non-neuronal peripheral staining patterns were restricted to selective nuclear staining exhibited by two out of five antineurofibrillar antibodies and the staining of macrophage and selected cardiac muscle nuclei by two of three antisynapse-associated antibodies. However, one antineurofibrillar antibody also stained the cytoplasm of selected liver cells. Among non-neuronally reacting antibodies, two antibodies stained nuclei of all cells except neurons in brain as well as peripheral organs. An antibody staining the ciliary epithelium of choroid plexus also stained basal bodies of ciliated bronchial epithelium. The overall data suggest that the specificity of brain-reactive antibodies is high and that their cross-reactivity with epitopes in non-nervous tissue is rare. In these cases, the antibodies seem to provide specific reagents for these additional structures as well as for their specific brain antigens.

Hybridomas from spleen cell fusions of six BALB/c mice immunized with hypothalamus were analyzed by immunocytochemistry for antibodies reactive with paraffin sections of fixed rat brain. In a total of 135 antibody producers, 60% were brain specific. Among these, 54% reacted with glial elements, pituitary cells, or basal lamina of intracerebral capillaries, with little variation among individual hybridomas in each of these groups. Forty-six percent of brain-specific antibodies reacted with neuronal structures, localizing on nerve fibers, neurofibrils, or perikarya. Neuron-specific hybridomas could be classified into groups that localized in anatomically defineable overall patterns. Within these patterns individual hybridomas exhibited extensive qualitative localization diversity ("neurotypy"). Conceivably, the genetic message for a common "proantigen" within an overall pattern may be slightly modified during differentiation of a neuron, thus leading to minor variability in antigenic expression. During antibody formation, similar minor changes occur in the differentiation of the genetic message for the antibody variable region. Apparently, minor changes in the antibody combining site among groups of hybridomas is reflected in the detectability of minor neurotypic changes among differentiated neuronal proantigens. If neurotypy proves to be the result of single-base substitutions or of variability in alignment of peptide-coding exons, the Scharrer concept of the fundamental significance of neurosecretion could also become applicable to neuronal specialization.

The unlabeled peroxidase-antiperoxidase method has been used with antiserum against "myelin-associated glycoprotein" to establish the presence of the glycoprotein in myelin and myelin-forming cells of the developing rat nervous system. Myelin-associated glycoprotein is found in oligodendroglial cytoplasm before the beginning of myelination. Staining intensity of oligodendroglia increases during early development and slowly declines during the period of rapid myelination. Myelin staining is confined to the periaxonal region of the myelin sheath and does not increase as large, compact sheaths are formed. Antiserum to central nervous system myelin-associated glycoprotein also stains Schwann cells in developing trigeminal ganglia and the periaxonal region of peripheral myelin sheaths.

Alzheimer tangles, despite their location in neuronal perikarya, react immunocytochemically with monoclonal antibodies to phosphorylated epitopes of neurofilaments. Normal perikarya do not contain phosphorylated neurofilaments. The aberrant phosphorylation in both plaques and tangles seems to be largely restricted to individual phosphorylation sites among the many sites available in neurofilaments. It is suggested that the Alzheimer lesion involves an imbalance within specific kinases responsible for phosphorylation of different sites in neurofilaments.

To study how viruses interact with oligodendroglia and produce demyelination, we immunostained paraffin and epon sections of lesions from patients with progressive multifocal leukoencephalopathy (PML) with antisera to papovaviruses, oligodendroglial myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) according to the peroxidase-antiperoxidase method. In paraffin sections from a rapidly progressive case of PML, hyperimmune JC virus antiserum stained single oligodendroglia which were located in white matter that appeared normal histologically and stained normally with MAG and MBP antisera. In zones surrounding areas of demyelination, virus containing oligodendroglia were most numerous and MAG staining of periaxonal regions was decreased, but there was little change in MBP staining. In demyelinated regions, both MAG and MBP staining were severely altered; also there was much less JC virus staining. In tissue from three other chronic cases, viral antiserum stained fewer oligodendrocytes and the differences in MAG and MBP staining were much less striking. In epon sections from two biopsies of central nervous system tissue, we studied the electron microscopic appearance of oligodendroglia that also had been stained by JC virus antiserum. Virions were present in all nuclei and in some cytoplasmic regions. The results suggest that changes in MAG distribution are useful indicators of early oligodendroglial abnormalities which can cause myelin breakdown.

The reaction of Lewy bodies in the substantia nigra and the sympathetic ganglia was studied in 10 cases of Parkinson's disease and other Lewy body-related conditions, using monoclonal antibodies to non-phosphorylated (02-40) and phosphorylated (03-44 and 07-5) neurofilaments. Although many Lewy bodies were unstained, a peripheral ring of positive immunoreactivity was frequently observed. This reaction was more common and more intense with antibodies to phosphorylated neurofilaments, suggesting a posttranslational phosphorylation of elements in the Lewy body. Although Lewy bodies appear to have some antigenic sites in common with neurofilaments, certain differences between neurofilaments and Lewy body filaments are emphasized.

We studied the spongy myelinopathy of glycine encephalopathy in five patients by using specific antisera. The walls of the vacuoles were stained with the myelin basic protein but not with the myelin associated glycoprotein or the glial fibrillary acidic protein immunostains. The pattern suggested that the vacuoles originated in compact myelin and not from the adaxonal portion of the sheath or from glial processes. Ultrastructural study revealed myelin vacuoles resulting from intraperiod splitting, and there were unusual intranuclear and cytoplasmic inclusions in skeletal muscle in two cases. In addition to the action of glycine as an inhibitory neurotransmitter, structural alterations of myelin may be important in the pathogenesis of the neurologic disorder of glycine encephalopathy.