Sites of cytoplasmic ribonucleoprotein-filament assembly in relation to helical body formation in axenic trophozoites of Entamoeba histolytica.

Axenic trophozoites of Entamoeba histolytica showed increased logarithmic growth but absence of "chromatoid" material (stacked helical arrays of ribonucleoprotein [RNP]) when grown in an all-liquid monophasic culture. Organisms grown in a liquid overlay on a semisolid slant (biphasic medium) showed slow logarithmic growth and the presence of chromatoid material. Chromatoid material accumulated in the rapidly growing trophozoites from monophasic culture during treatment with the Vinca alkaloid, vinblastine. Many of the glycogen-free regions of vinblastine-treated trophozoites as well as, to a lesser degree, of normal cells grown in monophasic and biphasic cultures, contained free ribosomes and randomly oriented 60 A filaments. As ribonucleoprotein assumed the packed helical configuration, areas consisting of parallel, packed filaments could be detected adjacent to and continuous with the ordered RNP arrays. This arrangement could be visualized most frequently in vinblastine-treated trophozoites grown in monophasic cultures. Depending on the tilt of the section with respect to the longitudinal axis of individual helices, 60 A filamentous material could be demonstrated associated with the RNP helices. Localization of ribonucleoprotein precursors was followed by means of high resolution radioautography with uridine-(3)H and cytidine-(3)H. With a short (30-min) pulse, label could be visualized only over the glycogen-free areas containing free ribosomes and filaments. With 60-min pulses, label could also be seen over the packed helical arrays. With 30-min pulses followed by a 60-min cold chase, label was seen chiefly over RNP helices. It is postulated that the areas containing ribosomes and filaments represent sites of assembly of the RNP helices possibly on a filament protein column. The possibility that the final helical configuration may be due to a property of this protein is suggested.

cAMP-dependent protein kinase phosphorylations on tau in Alzheimer's disease.

To elucidate the role cAMP-dependent protein kinase (PKA) phosphorylations on tau play in Alzheimer's disease, we have generated highly specific monoclonal antibodies, CP-3 and PG-5, which recognize the PKA-dependent phosphorylations of ser214 and ser409 in tau respectively. The present study demonstrates by immunohistochemical analysis, CP-3 and PG-5 immunoreactivity with neurofibrillary pathology in both early and advanced Alzheimer's disease, but not in normal brain tissue and demonstrates that cAMP-dependent protein kinase phosphorylations on tau precede or are coincident with the initial appearance of filamentous aggregates of tau. Studies using heat-stable preparations demonstrate that neither site appears to be phosphorylated to any appreciable extent in normal rodent or human brain. Further analysis demonstrates that the beta catalytic subunit of PKA (Cbeta), the beta II regulatory subunit of PKA (RIIbeta), and the 79 kDa A-kinase-anchoring-protein (AKAP79), are tightly associated with the neurofibrillary pathology, positioning cAMP-dependent protein kinase to participate directly in the pathological hyperphosphorylation of tau seen in Alzheimer's disease.

Mitochondrial abnormalities in Alzheimer's disease.

The finding that oxidative damage, including that to nucleic acids, in Alzheimer's disease is primarily limited to the cytoplasm of susceptible neuronal populations suggests that mitochondrial abnormalities might be part of the spectrum of chronic oxidative stress of Alzheimer's disease. In this study, we used in situ hybridization to mitochondrial DNA (mtDNA), immunocytochemistry of cytochrome oxidase, and morphometry of electron micrographs of biopsy specimens to determine whether there are mitochondrial abnormalities in Alzheimer's disease and their relationship to oxidative damage marked by 8-hydroxyguanosine and nitrotyrosine. We found that the same neurons showing increased oxidative damage in Alzheimer's disease have a striking and significant increase in mtDNA and cytochrome oxidase. Surprisingly, much of the mtDNA and cytochrome oxidase is found in the neuronal cytoplasm and in the case of mtDNA, the vacuoles associated with lipofuscin. Morphometric analysis showed that mitochondria are significantly reduced in Alzheimer's disease. The relationship shown here between the site and extent of mitochondrial abnormalities and oxidative damage suggests an intimate and early association between these features in Alzheimer's disease.

Detection of HIV in fetal central nervous system tissue.

Neurological disease is a common finding in children with AIDS and in others without signs of disease but with evidence of congenital HIV-1 infection. To investigate the possibility that HIV-1 can infect fetal central nervous system (CNS) tissue and therefore possibly serve as the substrate for the abnormal neurodevelopment characteristic of pediatric AIDS, eight abortus CNS samples (one set of twins) from seven HIV-1-seropositive intravenous drug users (IVDUs) and eight control abortus CNS samples from eight HIV-1-seronegative IVDUs were analyzed for HIV-1 infection. HIV-1 nucleic acid was detected only after the use of polymerase chain reaction (PCR) in three of eight CNS samples from HIV-seropositive IVDUs but not in samples from seronegative subjects. In situ hybridization confirmed that HIV-1 DNA sequences were in cells in the CNS parenchyma of two of the three positive samples. This study demonstrates that HIV-1 can infect human fetal CNS tissue in vivo, but that the use of PCR may be necessary for its detection.

Further studies on the inflammatory response induced by zinc wire implants in the central nervous system of rats.

Implantation of zinc wires into the central nervous system of adult Lewis rats initiates an inflammatory reaction with a predominantly mononuclear cell profile. Extensive cuffs of small lymphocytes, mature plasma cells, macrophages, and histiocytes are found around the site of the wire implant and around adjacent blood vessels. The inflammatory response persists for at least 35 weeks but, with time , is gradually replaced by extensive fibrosis, collagen deposition, and proliferation of glial cells in the adjacent neuropil. The Lesion is not necrotic, nor is it characteristic of a foreign body granuloma, since neither giant cells nor organized epithelioid cells are found. No obvious clinical effect is observed. The fact that the implantation of zinc wire initiates an inflammatory response which resembles certain immune-mediated reactions is consistent with the reports of others that zinc affects immunological responses both in vivo and in vitro. Other metals (Be, Co, Mg, Pt, and Ni), implanted to control for the effect of zinc, initiated responses that were both distinct for the metal used and reproducible, and thus underscore the significance of studying the role of metals in both acute and chronic diseases.

Nickel-induced changes and reappraisal of Rosenthal fibers in focal CNS lesions.

Nickel (Ni) wire implants into the CNS of Lewis rats induce the formation of structures morphologically similar to Rosenthal fibers (RF) seen in human conditions. We describe in detail the time courses of Ni-induced changes in rat astrocytes and compare the Ni-induced RF with RF occurring in focal lesions. Immunocytochemistry at the level of electron microscopy suggests that the Ni-induced RF are largely made up of plasma proteins. We suggest that the mechanism of RF formation and possibly the protein composition of RF depends on the condition in which they are found.

Ubiquitin-immunoreactive dystrophic neurites in Down's syndrome brains.

Ubiquitin-immunoreactivity was studied in Down's syndrome brains ranging in age from two days to sixty years. Numerous randomly distributed ubiquitin-immunoreactive dot-like structures in the white matter were shown to correspond to granular degeneration of myelin. Granular degeneration of myelin was first detected at age 21 and increased thereafter with age. Other larger and more coarsely granular ubiquitin-immunoreactive structures, most numerous in the middle and upper cortical layers, were consistent with dystrophic neurites. Immunoelectron microscopy demonstrated that the dystrophic neurites contained non-filamentous, membranous, dense bodies. In Down's syndrome, ubiquitin-immunoreactive dystrophic neurites were first detected at age six in the hippocampus, and were consistently more numerous in comparison to age-matched control subjects. In the presence of amyloid, either as diffuse or as compact deposits, ubiquitin-immunoreactive dystrophic neurites frequently formed aggregates consistent with senile plaques. Although apparently independent events, these data suggest that amyloid deposition is associated with local accentuation of ubiquitin-immunoreactive neuritic dystrophy. In addition, since dystrophic neurites appeared substantially earlier in the grey matter in Down's syndrome than in age-matched normals, this may be further evidence that selective aspects of aging are accelerated in Down's syndrome.

Early myelination in the human fetal lumbosacral spinal cord: characterization by light and electron microscopy.

Myelination in the human central nervous system is well documented after 20 weeks of gestation (WOG). However, earlier stages of this process have not been described in detail, although it is assumed that human myelinogenesis is similar to that observed in other animals. We used light and electron microscopy to study myelination in the human lumbosacral spinal cord during the second trimester of gestation. The kinetics of myelin-associated gene expression were analyzed by immunocytochemistry using antibodies to the myelin markers myelin basic protein (MBP) and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase). These studies show that in 12-13 WOG specimens, occasional MBP-positive processes are found in developing white matter in areas distinct from the root entry zones. At this time, ultrastructural study revealed early investment of axons by glial processes and rare compacted myelin. CNPase staining was qualitatively and quantitatively less than that of MBP. The numbers of MBP- and CNPase-positive myelin sheaths increased with time, and by 24 WOG many were evident in all areas of the spinal cord except in the corticospinal tracts. Ultrastructural study of corresponding areas revealed many thin lamellae of compact myelin. This study provides initial normative data for early human myelination in the lumbosacral spinal cord and may serve as a baseline for future developmental and pathological studies.

Effects of persistent lymphocytic choriomeningitis virus infection on cholinergic neurons in the mouse.

We have examined the effects of persistent infection with the Armstrong E350 strain of lymphocytic choriomeningitis virus (LCMV) on choline acetyltransferase activity in several regions of Balb/c mouse brain. Despite the presence of high titres of virus in brain for as long as 6 months, and a widespread distribution of virus antigen, no decreases in choline acetyltransferase activity could be demonstrated. The enzyme activity was increased in some regions of brain, showing an effect of the persistent virus infection on a differentiated cell function. Although these data do not suggest a role for LCMV in human neurologic disease, similar studies may allow useful animal models to be conveniently and reproducibly generated.

Conformational change as one of the earliest alterations of tau in Alzheimer's disease.

Paired helical filaments (PHFs) found in Alzheimer's disease (AD) are mainly comprised of an abnormal form of tau (PHF-tau) that has undergone several post-translational modifications. Previous studies have shown that the monoclonal antibody MCI identifies a distinct conformation of tau in AD. We have assessed the temporal and spatial occurrence of the tau conformation recognized by MC1, and found its appearance in hippocampal neurons vulnerable to neurofibrillary tangle (NFT) formation in Braak Stage I and II cases. Electron microscopy has clearly demonstrated that this conformation precedes the formation of PHF. MC1 immunoaffinity chromatography also has identified a nonfilamentous, soluble pool of this abnormal tau. ELISA and immunoblotting have shown that this material is indistinguishable from that found in NFTs. This soluble component has the ability to self-assemble into PHFs in a concentration-dependent manner. Because the conformational change recognized by MCI appears before the assembly of and is found in PHF, but is not present in the normal brain, we suggest that the formation of the MCI epitope is one of the earliest pathological alterations of tau in AD.

Mitotic phosphoepitopes precede paired helical filaments in Alzheimer's disease.

We have shown previously that the TG-3 and MPM-2 antibodies recognize phosphoepitopes common to mitosis and degenerating neurons of Alzheimer's disease(AD) brain. Here, we have evaluated their occurrence in human brain biopsy tissue, and confirm that they are absent in mature neurons of adult brain, but reappear during neurodegeneration in AD. The TG-3 epitope appears ahead of the MPM-2 epitope and is distributed throughout the neuronal soma. Tau is the major TG-3 antigen in AD brain. The initial localization of MPM-2 immunoreactivity in primary dendrites, it's robust occurrence in granulovacuolar bodies, and the increased immunoreactivity with 300-350-kDa proteins, suggest MAPI B as a candidate MPM-2 antigen in AD. Production of mitotic phosphepitopes in more than one type of human neurodegenerative lesion implicates mitotic kinases as common mediators of neuronal death. Because mitotic phosphoepitopes appear before paired helical filaments, it is suggested that mitotic kinase activation triggers neurofibrillary tangle formation. Future studies will need to focus on factors influencing mitotic kinase activity, a point with potential for early diagnosis and disease abrogation.

Alz-50 immunoreactivity in the hypothalamus of the normal and Alzheimer human and the rat.

Alz-50 is a monoclonal antibody recognizing a 68 kilodalton protein that is abundant in Alzheimer's disease (AD) but not detectable by immunoblotting methods in normal brains. When used for immunohistochemistry in AD cortex, Alz-50 recognizes large numbers of neurofibrillary tangles (NFT), neuritic plaques, and some neurons that show no evidence of neurofibrillary degeneration by conventional histopathological staining methods. Alz-50 immunoreactivity is described at the light and electron microscopic levels in the hypothalamus of brains obtained at autopsy from normal and AD subjects. Alz-50 immunoreactivity in the rat hypothalamus is also described. A well-defined population of Alz-50 immunoreactive hypothalamic neurons was identified in both the normal human and rat. At the light microscopic level in the normal human, immunoreactive neurons were most concentrated in the periventricular region, but were also scattered throughout the arcuate nucleus (ARC), lateral hypothalamic area, and tuberal region. Immunoreactive fibers were seen in the periventricular region, dorsal division of the ventromedial nucleus (VMNd), ARC, and external layer of the median eminence (ME). In the rat, reactive neurons were seen only in the periventricular region, and reactive fibers were seen in the periventricular zone, medial preoptic nuclear complex, suprachiasmatic nucleus, VMNd, ARC, and external layer of the ME. Ultrastructurally, all immunoreactivity in the normal human and rat hypothalamus was associated with intraneuronal vesicles. In the AD hypothalamus, Alz-50 identified numerous senile plaques and NFT in addition to the cells and fibers that were stained in the normal brains. Immunoreactive plaques and NFT were most numerous in regions previously reported to undergo neurofibrillary degeneration. At the ultrastructural level, the immunoreactivity in the AD hypothalamus was associated with filaments as well as vesicles. The significance of the selective staining of a specific population of vesicles by Alz-50 is unknown; however, the present results suggest that it is independent of AD pathology.

Proliferative membranopathy and human immunodeficiency virus in AIDS hearts.

In order to determine if cardiac tissue from AIDS patients or patients with seropositivity to HIV-1 might be infected by HIV-1, portions of myocardium obtained postmortem were evaluated for HIV-1 DNA sequences. Cellular DNA was extracted and digested with EcoR1 and Southern blots were performed. One of three AIDS hearts was positive for HIV-1 DNA sequences without amplification, whereas two additional hearts were positive for HIV-1 DNA after amplification. Accordingly, other tissue from the heart positive for HIV-1 without amplification was studied by electron microscopy to localize HIV virions. Unexpectedly, large numbers of proliferating multilamellated membrane bodies were identified in myocytes, predominantly associated with mitochondria. Identical membrane bodies were found in two additional AIDS hearts, and in one heart from a patient with seropositivity to the AIDS virus, but in none of three similarly fixed controls. Immunocytochemistry for HIV core (p24) and envelope (gp120) antigens did not localize gold-labeled antibodies to the membrane bodies. We believe this membranopathy may be an HIV-1- or AIDS-specific abnormality of unknown etiology that may be related to the ultimate development of cardiomyopathy. In addition, our studies provide further support that HIV-1 may be present in AIDS hearts, although as yet we cannot state with certainty where the HIV-1 is located in these tissues.

HIV-1 infection of human fetal thymocytes.

Some neonates with congenital human immunodeficiency virus type 1 (HIV-1) infection exhibit immune dysregulation. This suggests that fetal CD4+ cells, possibly thymocytes, may be infected during gestation. If thymocytes are infected, this may result in a disruption of T-cell differentiation. To examine this hypothesis, normal human fetal thymocytes were established in tissue culture, characterized, and then exposed to HIV-1. On initial isolation, fetal thymocytes were analyzed for phenotypic markers by flow cytometry and assessed for T-cell function by mitogen-stimulated thymidine incorporation. The thymocytes comprised greater than 70% double positive (CD4+, CD8+) cells and responded to T- but not to B-cell mitogens. Thereafter, thymocytes were incubated in either tissue culture medium containing infectious HIV-1 or in control (HIV-free) medium. Infection of fetal thymocytes was determined by light and electron microscopy in combination with immunocytochemistry, molecular hybridization, and an infectious cell center (ICC) assay. After 1 week in culture, the thymocytes exposed to HIV-1 were positive by immunocytochemistry for the HIV-1-associated protein gp41. In addition, the presence of HIV-1 DNA was detected by molecular hybridization confirming infection of these cells. The ICC assay demonstrated the production of infectious HIV-1 particles and budding of mature virions was observed by electron microscopy. These studies demonstrate that human fetal thymocytes can be infected with HIV-1 in vitro and that this infection results in production of infectious virions. These results support the hypothesis that vertical transmission of HIV-1 in vivo may result in the infection of fetal thymocytes, which may contribute to postnatal HIV-1-associated pathologic conditions.

Hippocampal degeneration differentiates diffuse Lewy body disease (DLBD) from Alzheimer's disease: light and electron microscopic immunocytochemistry of CA2-3 neurites specific to DLBD.

Immunocytochemistry with antibodies to ubiquitin is currently the most sensitive method for detecting cortical Lewy bodies, which are a sine qua non for the diagnosis of diffuse Lewy body disease (DLBD), an increasingly recognized form of primary degenerative dementia. In the systematic application of ubiquitin immunocytochemistry to sections of hippocampus from control subjects and patients with a wide spectrum of neurodegenerative diseases, we noted the frequent occurrence of ubiquitin-immunoreactive neurites in the CA2-3 region in DLBD. The nature of these neurites was investigated with immunocytochemistry in DLBD, Alzheimer's disease (AD), normal elderly subjects, and Parkinson's disease (PD). Although the number of neurites varied from case to case, they were virtually always detected in DLBD but not in normal, AD, or PD brains. Double immunolabeling studies with anti-ubiquitin demonstrated a small fraction of double-stained neurites with antibodies to neurofilament or Alz-50, but no double staining with an antibody to Alzheimer neurofibrillary tangles. These results are different from those for neurites in AD, which are rarely seen in CA2-3 and which are immunoreactive with all these antibodies. Neuritic degeneration in the CA2-3 region of the hippocampus appears to be a specific histopathologic feature of DLBD.

Visceral myogenic tumors. A manifestation of HIV infection in children.

We report a primary smooth-muscle tumor of undetermined malignant potential of the liver in a child with acquired immune deficiency syndrome (AIDS). This patient represents the eighth child infected with the human immunodeficiency virus who developed a mesenchymal tumor other than Kaposi's sarcoma. All these children were younger than 10 years of age. These tumors often were histologically or clinically malignant and all but one were smooth-muscle tumors. These tumors arose exclusively in visceral organs, and the hepatobiliary, gastrointestinal, and tracheopulmonary systems were involved. Transmission of the virus occurred both vertically (in six children) and via blood transfusion (in two). Given the rarity of smooth-muscle tumors in uninfected children, the unusual frequency of these tumors suggests that immunosuppression induced by the virus permits the unregulated proliferation of a primitive mesenchymal cell disposed to myogenous differentiation, a situation not unlike that observed in the development of AIDS-related Kaposi's sarcoma in adults.

Human microglia mediate anti-Cryptococcus neoformans activity in the presence of specific antibody.

The interaction of the opportunistic fungus Cryptococcus neoformans with human microglia was studied in vitro in the presence and absence of capsule binding antibody. In the absence of capsule binding antibody there was little or no phagocytosis. Addition of the murine monoclonal antibody (mAb) 2H1 (IgG1, kappa) to the capsular glucuronoxylomannan (GXM) produced a dose-dependent enhancement of C. neoformans phagocytosis by microglia. Phagocytosis resulted in marked inhibition of fungal proliferation. Microglial antifungal activity was studied by colony forming unit assay, L-[3H]leucine incorporation assay, and phase contrast microscopy. At microglia: C. neoformans ratios of 10:1 to 80:1 fungal growth was reduced by 61-95%. Inhibitors of nitric oxide synthase and reactive oxygen intermediates did not prevent antifungal activity mediated by human microglia. Transmission electron microscopic studies revealed that although some internalized yeast cells were killed, the majority were intact consistent with fungistasis. Human microglia cells are potent effector cells against C. neoformans in vitro in the presence of specific antibody. Enhancement of microglial activity in vivo by opsonins may be a useful therapeutic strategy.

Non-plaque dystrophic dendrites in Alzheimer hippocampus: a new pathological structure revealed by glutamate receptor immunocytochemistry.

Alzheimer's disease is a progressive dementia characterized by a pronounced neurodegeneration in the entorhinal cortex, hippocampal CA1, and subiculum. Excitatory amino acid receptor-mediated excitotoxicity is postulated to play a role in the neurodegeneration in Alzheimer's disease. The present study investigated immunocytochemical localization of excitatory amino acid receptor subunits in the hippocampus of twelve Alzheimer's disease and eleven controls, matched for age, sex and post mortem interval. Immunocytochemistry with antibodies specific for glutamate receptors GluR1, GluR2(4) (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid), GluR5/6/7 (kainate) and NR1 (N-methyl-D-aspartate) receptor subunits demonstrated that virtually all projection neurons in all subfields contained subunits from each receptor class. However, regional differences in immunoreactivity were apparent in Alzheimer's disease vs normal human brain. In the vulnerable regions (i.e. CA1) immunolabelling of GluR1, GluR2(4), GluR5/6/7 and NR1 was reduced, presumably due to cell loss. In contrast, GluR2(4) immunolabelling appeared to be increased in the inner portion of the molecular layer of the dentate gyrus. In addition to cellular labelling, GluR1, GluR2(4) and NR1 immunolabelling revealed a novel pathological structure in 12 of 12 Alzheimer's disease, but none of the control brains. The lesions were juxtacellular clusters of granular immunoreactivity in the neuropil of the pyramidal cell layer. Ultrastructural analysis revealed these to be cellular processes containing dense vesicles and flocculent material with immunolabelling localized to plasma and vesicular membranes. They were not specifically associated with amyloid fibrils and did not contain paired helical filaments and they were also distinct from granulovacuolar degeneration. Several structures contained Hirano body filaments indicating that the dystrophic processes were most likely dendritic in origin. Additional studies are needed to determine the pathogenesis of these lesions, which could provide an additional index of dendritic deterioration in Alzheimer's disease.

Primary fixation in osmium-potassium ferrocyanide: the staining of glycogen, glycoproteins, elastin, an intranuclear reticular structure, and intercisternal trabeculae.

Primary fixation in an osmium-potassium ferrocyanide (K4Fe(CN)6) mixture combines selective fixation, staining, and extraction of various cellular components; membranes, glycogen, glycoproteins, and elastin are preserved and stained. An intranuclear reticular structure that is composed of 3-6 nm fibers and permeates the entire nucleus, except for the nuclear pores, is demonstrated by electron microscopic examination of tissues prepared in an osmium-potassium ferrocyanide fixative. Condensations of the reticulum parallel the distribution of heterochromatin in interphase nuclei. This preparative procedure also reveals a network of trabeculae that are associated with the cisternae of rough endoplasmic reticulum and connect the parallel cisternae in hepatocytes, plasmacytes, neurons, and pancreatic ancinar cells. The intercisternal trabeculae are associated with both free and bound ribosomes.

Experimental approaches to mechanisms of protection and pathogenesis in M. tuberculosis infection.

It has, for many years, been widely assumed that the fundamental mechanism of protection in tuberculosis infection is a CD4 T cell response producing lymphokines that activate macrophages to kill or restrict the intracellular growth of M. tuberculosis. Just as certain cytokines, e.g. IFN-gamma, are currently perceived to be important for protection, others, particularly tumor necrosis factor (TNF), are thought to be responsible for much of the tissue destruction associated with the disease. Yet there are remarkably few critical experimental or clinical data that have defined the immunological requirements for protection and pathogenesis. One of the initial stimuli to the work we have undertaken has been careful reflection on the results of the many prospective trials of BCG against tuberculosis. Two aspects have impelled us to reconsider conventional wisdom. The first, of course, is the wide discrepancy in the degree of protection imparted, ranging from 0% in South India to 77% in the British MRC trial (1, 2). The second is that, in all trials that examined them, skin test conversions to tuberculin positivity were 85% or greater, indicating a disparity between the presence of delayed hypersensitivity to tuberculin and protection. We and others have argued (1, 2) that there are multiple possible explanations for this discrepancy, the principal one being protection caused by infection with environmental mycobacteria. But, the general point raised is whether cell mediated immunity as manifested by CD4+ cell production of lymphokines and macrophage activation is a sufficient mechanism for protection against M. tuberculosis infection.