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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.

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.

The use of immunoperoxidase technics has spread dramatically in recent years to almost daily use in certain clinical and surgical pathology laboratories. A wide variety of cell products, including enzymes, polypeptide and steroid hormones, immunoglobulins, oncodevelopmental antigens, viral antigens, and other cell-specific proteins, have been demonstrated in frozen or conventionally fixed and embedded tissue sections by the use of these methods. Major applications of these technics at present include demonstration of various immunoglobulin classes in tumors of lymphoreticular origin and atypical lymphoproliferative processes, identification of polypeptide hormones in endocrine tumors, and demonstration of various tumor markers such as carcinoembryonic antigen and alpha fetoprotein in neoplastic and preneoplastic conditions. The indirect peroxidase conjugate and peroxidase antiperoxidase procedures are presented and reviewed in detail with emphasis on tissue preparative procedures, evaluation of tissue and immunologic controls, background staining, and evaluation of results. The danger of false-negative and false-positive results should be dealt with in a precise manner so that results from different laboratories are both reliable and reproducible. The application of immunoperoxidase technics represents a major advance for the surgical pathologist, but additional evaluation, development, and standardization are required. To accomplish this, comparative studies of the same material, including known positive and negative tissue controls, by different laboratories should be encouraged.

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.

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.

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.

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.

Immunospecific staining in 100-micron-thick Vibratome section by the unlabeled antibody method resembles Golgi staining and reveals an abundance of luliberin- (luteinizing hormone releasing hormone, LHRH) positive cells and fibers in close contact with the surface of the third ventricle. The polarity of LHRH cells can be seen and processes can be traced for several millimeters. In the medial preoptic and suprachiasmatic areas bipolar LHRH neurons send short stout processes to the ventricular surface and long processes toward the organum vasculosum laminae terminalis. These cells resemble the receptor cells contacting the cerebrospinal fluid that have been described by Vigh and Vigh-Teichmann [Vigh, B. & Vigh-Teichmann, I. (1973) Int. Rev. Cytol. 35, 189-251]. In the septal region some bipolar neurons send both of their processes towards the ventricular surface. LHRH neurons in the nucleus of the anterior commissure and the bed nucleus of the stria terminalis project over the anterior commissure to form a dense plexus of fibers in the subfornical organ. The proximity of LHRH perikarya and fibers to the ventricular surface supports the hypothesis [Knigge, K.M., Joseph, S.A., Scott, D.E. & Jacobs, J.J. (1971) in The Neuroendocrinology of Human Reproduction, eds. Mack, H.C. & Sherman, A.J., (Thomas, Springfield, IL), pp. 6-22.] that the cerebrospinal fluid functions in neuroendocrine control mechanisms.

1.1. As shown by the previous results of other workers, compound 4880 is a potent histamine liberator and depletor in many animals, in vivo and in vitro. 2.2. In the guinea pig the injection or inhalation of the compound fails to produce asthma or emphysema as is commonly seen in anaphylactic or histamine intoxication. 3.3. The symptoms in the guinea pig are itching of the skin, prostration, convulsions, and sometimes embarrassed respiration. 4.4. Autopsy findings show a hemorrhagic, contracted lung and a congested liver. 5.5. Repeated administration of this compound intraperitoneally or by aerosol fails to make the animals refractory to this drug. 6.6. The concept that release of histamine is the major toxic action of this drug is weakened by the poor inhibiting effects of antihistamines. 7.7. These findings do not detract from the establishment of this compound as a substance which releases or depletes histamine in other animals, or in the guinea pig under different conditions. It must be concluded, however, that in the living guinea pig other toxic effects, at present undetermined, predominate. These toxic effects present an obstacle to the use of this compound as a histamine liberator and depletor.

Brief exposure of serum to alkali in the cold seems to convert non-precipitating antibody to a precipitin-like material. This was disclosed by the following lines of evidence. 1. Immune sera, after addition of antigen and removal of the ensuing precipitates, yielded after alkali treatment more antibody-antigen complex than did sera to which no antigen was added. 2. When immune sera were depleted of precipitating antibody, treated by alkali, and antibody-antigen complex was allowed to precipitate, the resulting supernates formed additional prescipitates upon admixture of specific antigen. No precipitation occurred upon addition of non-specific protein to the treated supemates or the addition of specific antigen to depleted sera not treated by alkali. 3. Alkali-treated immune sera precipitated more antigen than untreated sera.

Soluble horseradish peroxidase-antihorseradish peroxidase (rabbit) complex was used to localize the actomyosin-like contractile protein, thrombosthenin, in both intact human platelets (Epon-embedded) and ultrathin sections (methacrylate-embedded). Antibody staining of ultrathin sections showed the presence of membrane-associated and cytoplasmic thrombosthenin. Antibody staining of intact cells showed that membrane-associated thrombosthenin was localized, at least in part, in the exterior, "fluffy" coat of the platelet. Even after prolonged fixation and/or incubation with the various antisera, we were unable to demonstrate antibody penetration of intact, fixed platelets. Brief treatment with Pronase removed the surface-localized proteinaceous material and completely abolished all antibody staining.

Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 Å. s20, w, 11.98 x 10–13; d20, w, 2.48 x 10–7; molecular weight by sedimentation velocity, 429,000, and equilibrium, 413,000. Sensitivity and specificity of immunohistochemical staining of spirochetes was about 100- to 1000-fold that of immunofluorescence. The unexpected ratio of PO to anti-PO is presumed to be due to stabilization by the pentagonal shape in which three corners are suspected to be PO and two antibody fragment Fc.

Sheep antibody to rabbit antibody was immunospecifically protected during extensive chelation with uranium in a procedure which involved insolubilization of rabbit-γ-globulin (RGG) by coupling with diazotized p -aminobenzylcellulose, specific absorption of antibody, exposure of antibody-RGG- p -azobenzylcellulose to uranyl acetate solution, and elution of antibody by alkali. The antibody was 40–75% pure and contained 29–90 atoms of uranium per molecule. Antigen first localized by specific rabbit antiserum became electron dense upon treatment with immunospecifically purified and protected, uranium-conjugated sheep antibody to rabbit antibody. Staining after sectioning for electron microscopy was facilitated by pretreating sections with benzene-saturated water. Alkali treatment of unprotected, uranium-chelated areas in antibody and γ-globulin appeared to affect irreversible structural changes leading to increase in size and solvation of the molecule. Protected sites did not seem to participate in these changes appreciably.

The number of discrete unit stains on the surface of sheep erythrocytes enumerated by electron microscopy after preembedding staining using 125I antierythrocyte antibody (anti-E) and the unlabeled antibody enzyme method was correlated with enzyme activity and radioactivity of lysed cell suspensions. Purified anti-E was applied in a concentration range resulting in the binding of 10-2,600,000 molecules/cell. Sheep serum antirabbit immunoglobulin and peroxidase-antiperoxidase complex were used in excess. With a new light-scattering assay for peroxidase the sensitivity of measurement of enzyme activity on suspensions of cell stromata exceeded that of radioactivity assay allowing measurement of as little as 5 x 10–13 mmole antibody/ml. Discrete unit stains on cell sections were visualized to 1.5 x 104 anti-E/cell. Above this number, staining occurred in patches of increasing size but was still discontinuous to ∼3 x 104 anti-E bound/cell. Above 2-3 x 104 anti-E bound/cell continuous staining of the membrane was observed. Assuming random distribution of bound anti-E, the number of discrete unit stains visualized per cell section was formulated as a probability function. The observed numbers of unit stains per section agreed well with the values so formulated to ∼8,000 anti-E/cell. Thus, enzyme activity of peroxidase-antiperoxidase, in suspensions or by enumeration of unit stains by electron microscopy, served as a sensitive quantitative measure of specific antibody bound per cell. The data permit the interpolation that staining of sparsely distributed cell surface antigens by the unlabeled antibody enzyme method with excess antibody permits the enumeration of antigen sites. In addition, mobility of surface antigens may be quantitated by comparison of observed and predicted numbers of discrete unit stains per section.

A dialyzable and a nondialyzable principle were obtained from alternaria grown on a synthetic medium. The antigen in the nondialyzable fraction was purifed by deproteinization, precipitation as the uranyl salt, and low-temperature centrifugal ultrafiltration. The nondialyzable fraction reacted by skin test in allergic individuals and was precipitated with rabbit antiserum. The dialyazble fraction also reacted on skin tests. It failed to precipitate with antiseurm but acted as a hapten in specifically inhibiting precipitation of the nondialyzable fraction.

Reinjection of antigen into immunized rabbits usually results in an immediate increase of circulating antibody-antigen complex. This immediate formation of complex seems to depend upon the presence of non-precipitating antibody before reinjection. The complex disappears by the 2nd day after reinjection and reappears between the 4th to 7th day. This is earlier than its first appearance after a primary injection and may indicate an anamnestic response of the non-precipitating antibody responsible for the formation of circulating antibody-antigen complex.

The labeling procedure described involves three steps: removal of antibody from antiserum by agglutination with bacteria; exposure of the washed agglutinate to relatively large amounts of U salts, the specific combining regions of the antibody in the agglutinate now being protected by antigen; and dissociation of antibody from antigen by brief treatment with alkali in the cold, yielding thereby specifically purified antibody containing U in its nonspecific areas but possessing specific combining sites free to react. In application of the procedure, anti-B. bronchiseptica antibody was removed from serum by absorption with B. bronchiseptica cells. The washed agglutinate was exposed to recovered from this agglutinate by brief treatment with 0.1N NaOH at 1 deg C was 80 to 100% pure and contained 18 to 312 atoms of U per unit of 156,000 molecular weight. The U stained the walls of living or formalin-killed, and the cytoplasm of living B. bronchiseptica. Escherichia coli and mouse spleen cells were not stained. Contrast was diminished when B. bronchiseptica was exposed to unlabeled antiB. bronchiseptica antiserum prior to U-antibody conjugate. Absorption with B. bronchiseptica, but not with E. coli, abolished the staining capacity of purified U-antibody conjugate. Whole antiserum labeled with U without specific protection,more » even though retaining some antibody activity, was not suitable as a stain since it deposited a large amount of debris and apparently stained both E. coli and B. bronchiseptica. (BBB)« less

1. 1. The modifying effect of sodium nucleate injections on asthma in the guinea pig produced by antigen aerosols has been confirmed. 2. 2. A similar inhibiting effect in asthma produced by a histamine aerosol is questionable. It is possible that sodium nucleate does not alter tissue sensitivity to histamine, but perhaps interferes with the availability of the histamine or other mediator released in the antigen-antibody reaction. 3. 3. Bilateral adrenalectomy does not appear to increase the sensitivity of the guinea pig to either an antigen or histamine aerosol. 4. 4. The absence of adrenals in the guinea pig does not prevent the inhibiting effect from asthma produced by sodium nucleate. It is concluded that such a “stress” is not mediated by pituitary adrenocortical stimulation. 5. 5. It is emphasized that the preceding findings apply to one “stress,” sodium nucleate, and to one animal, the guinea pig. Whether similar results will be obtained with other stresses and in other species, including man, will be the subject of further investigation.

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.

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.

A new peptide with LHRH-like immunoreactivity has been isolated from rat hypothalamus and pituitary using an immunosorbent prepared with monoclonal antibody to luteinizing hormone-releasing hormone (LHRH). This peptide contains part of the C-terminal sequence of LHRH and has a molecular weight of about 2000. It comprises over 20% of hypothalamic immunoreactivity recognized by the monoclonal antibody. Chromatographic and amino acid analysis data confirm the distinction between this peptide and LHRH. Immunocytochemical evidence is consistent with its being a normal constituent of the LHRH system, while its presence in the pituitary suggests that it may play a role in the regulation of this gland.

In Araldite sections of male rat pituitaries, stained after embedding by the unlabeled antibody enzyme method with antisera to native luteinizing hormone-releasing hormone (LH-RH) or LH-RH azo-conjugated to bovine serum albumin, localization is confined mainly to the interior of the large, and to a lesser extent to that of the small, secretion granules of the gonadotrophic cells. Plasma membranes are not demonstrated. Except for weak staining in the granules of corticotrophs, no other pituitary cell is stained. Pretreatment of sections with LH-RH (to dilutions of 4 pg/mul) increases staining intensity in the gonadotrophic granules. Other cells are unaffected. The lesser the gonadotroph staining intensity without pretreatment, the greater the increase (more than 23-fold reactivity). Augmented staining is measurable (P less than 0.001) to antiserum dilutions of 1:240000. Pretreatment with des-Glu-1-LH-RH, porcine corticotropin or rat prolactin has no effect. LH-RH-Gly-10(des-amide) inhibits. Rat glycoprotein hormones enhance staining with anti-azo-conjugated LH-RH. With antinative LH-RH these hormones enhance weak staining, but inhibit strong staining. Thick vibrotome sections of male rat or rabbit pituitaries stained before embedding reveal specific localization on plasma membrane and gonadotrophic secretion granules provided the sections have been pretreated with LH-RH (250 pg/mul). The data show that LH-RH after reaction with receptor is not sterically hindered from binding specific antibodies. Receptor may be found in secretion granules, both in the free state or combined with LH-RH. Plasma membrane receptor, on the other hand, was free under the conditions of the experiments. Immunization with LH-RH elicits not only heteroimmune antibodies specific for LH-RH, but also a group of still ill defined autoimmune antibodies, some of which may conceivably be reactive with glycoprotein hormone alpha-chains.

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.

Affinity-purified anti-LHRH, affinity-purified LHRH-anti-LHRH complex, as well as antisera depleted of anti-LHRH by solid phase immunoabsorption were used to test the specificity of the immunocytochemical approach for detection of receptor-bound LHRH. The solid phase immunoabsorbent was prepared by attaching LHRH to Sepharose via an RNase spacer. Purified anti-LHRH was eluted from the immunoabsorbent by acidification. Unabsorbed antisera as well as purified antibody conferred moderate immunocytochemical staining to the secretion granules of rat pituitary gonadotrophs. Staining intensity became greatly increased upon treatment of the electron microscopic sections with LHRH before immunocytochemical staining. All gonadotroph staining was abolished when absorbed antisera were used. The specificity of the immunoabsorbent was tested on mixtures of anti-LHRH and anti-ACTH17-39. On absorption of such mixtures with insolubilized LHRH, all gonadotroph staining disappeared, but the optical density indices of ACTH staining remained unaffected. It is concluded that gonadotroph secretion granules contain, in addition to an LHRH receptor reacting with LHRH in vitro, stable complexes of endogenous LHRH with receptor.

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.

Using 100-micron thick Vibratome sections and a modification of the peroxidase--antiperoxidase method of immunocytochemical staining we achieve a Golgi-like image of luteinizing hormone releasing hormone (LHRH) cells and fibers in mouse brain. Five LHRH pathways are described: 1) A dense projection of fibers from LHRH cells in the medial preoptic and septal areas to the wall of the third ventricle; 2) a projection of fibers from neurons in the bed nucleus of the stria terminalis and the nucleus of the anterior commissure to the subfornical organ; 3) projections of fibers from neurons in the medial septal nucleus and the diagonal band of Broca to the olfactory bulb; 4) fibers which travel within or just lateral to the wall of the third ventricle from the organum vasculosum laminae terminalis to the median eminence; 5) cells and fibers located just dorsal to the optic tracts which project rostrally to the preoptic area and caudally to the level of the median eminence where they course medially to converge and enter the median eminence.

Paraffin sections of mouse adrenals processed with antiserum to luteinizing hormone-releasing hormone (LHRH) in the unlabeled antibody enzyme method reveal moderate staining in the cytoplasm of cells of zona fasciculata and reticularis. The stain is intensified upon pretreatment of sections with LHRH. Pretreated sections processed with solid phase immunoabsorbed LHRH are unstained. Analogues of LHRH deficient in the C-terminal glycine amide inhibit staining, while analogues deficient in the N-terminal pyroglutamic acid have no effect. It is concluded that the adrenal contains receptors for a ligand resembling LHRH in receptor and immunoreactivity. The possibility is considered that the ligand may be an inhibitor of pineal origin.

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.

Adjacent paraffin sections of rat hypothalami fixed in Bouin's fluid were treated either with buffer or with luteinizing hormone-releasing hormone (LHRH) before immunocytochemical staining with anti-LHRH. Upon buffer pretreatment, pituitary gonadotrophs were unstained and hypothalamic fibers were stained. Upon LHRH pretreatment, pituitary gonadotrophs were stained (receptor reaction) and hypothalamic fibers were unstained. Extension of washes and use of series of neutralizing antisera between LHRH application and immunocytochemical staining, as well as the absence of inhibiting concentrations of LHRH in the later washes and neutralizing antisera removed from the sections, excluded the possibility that the disappearance of visualization of hypothalamic fibers was due to blockage of anti-LHRH in immunocytochemical staining. The results suggested that LHRH removed from the sections an immunocytochemically stainable but as yet unknown analog of LHRH and replaced it with LHRH, which in turn became lost during subsequent immunocytochemical processing. This idea was confirmed by the isolation by high-pressure liquid chromatography of a peak, distinct from LHRH, upon treatment of hypothalami with LHRH. It is suggested that the new substance may be carrier-held and that this substance, rather than LHRH, is normally detected by immunocytochemistry with anti-LHRH. Added LHRH binds not only to high-affinity pituitary receptors but also to low-affinity hypothalamic carriers.

Peripheral cells talk to each other via hormones. The message, although diffused into the bloodstream, is picked up selectively by those groups of cells that possess specific receptors for it. Neurons also talk to each other via hormones. By virtue of the axonal networks, they are able to direct a message to receptors on specific synapses with heightened selectivity and specificity. Thus, anatomical structure in the nervous system has added a relay system to the hormonal communication system found in the periphery. Immunocytochemistry provides simple and rapid means to differentiate axons as to the neurotransmitters carried, thereby permitting distinction among dopaminergic, serotonergic, and noradrenergic fibers.

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.

Estrogen receptor activity was preserved in fixed, paraffin-embedded tissue and demonstrated by binding of estrogen which, in turn, was detected immunocytochemically. Estrogen was added to rat endocervial epithelium to protect specifically receptors during fixation. The protective estrogen was apparently lost during embedding and had to be resupplied before staining. Estradiol-mediated immunocytochemical staining was inhibited by diethylstilbestrol and nafoxidine hydrochloride.

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.

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.