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R B Banati

Publications and source records attributed to R B Banati.

48 records · Page 3Linked to original sources
PubMed1993

Early and rapid de novo synthesis of Alzheimer beta A4-amyloid precursor protein (APP) in activated microglia.

Upon acute activation, microglia, the immuneffector cells of the brain parenchyma, express the amyloid precursor protein (APP) that is otherwise prominent in pathological structures related to Alzheimer's disease. In this disease complex amyloid-bearing neuritic plaques contain beta A4-amyloid protein, the APP, and numerous inflammatory proteins. The accompanying activation of microglia has mostly been viewed as a secondary reaction to amyloid deposits. Activation of microglia was performed in a graded fashion. Transection of peripheral nerves such as the facial or sciatic nerve causes a microglial reaction within hours in the nucleus of origin or in projection areas of the CNS. A predominantly glial up-regulation of APP mRNA and protein could be detected as early as 6 h post lesion not only at the site of affected neuronal cell bodies but also in corresponding projection areas. Its time course suggests rapid transneuronal signalling to glial cells in the projection area. Light and electron microscopy demonstrate that microglia, which are cells of mononuclear phagocyte lineage and comprise up to 20% of all glial cells, are the dominant source for non-neuronal APP expression. Ultrastructurally, brain perivascular cells within the basal lamina constitutively express APP and thus are a possible source of vascular amyloid. Additionally, microglia express leukocyte-derived (L)-APP mRNA and protein that have recently been described in mononuclear cells of the immune system. Increased L-APP expression may serve as a potential marker for glial/microglial activation. Such immune-mediated amyloidogenesis initiated by microglia might have implications for the treatment of neurodegenerative diseases.

Amyloid beta-Protein Precursor↗
PubMed1993

Cell adhesion molecule expression in the regenerating rat facial nucleus.

Transection of the rat facial nerve leads to an intrinsic microglial reaction in the facial nucleus. In the present study, we have examined immunocytochemically the expression of cell adhesion molecules during this process. Resting microglia constitutively expressed the LFA-1 alpha and beta chain (CD11a and CD18) in the white matter, rather than in the intact, control facial nucleus. From 24 h after facial nerve transection onward, activated microglia showed an increased expression of LFA-1 alpha and beta. The immunoreactivity reached its peak around day 7 following axotomy, i.e. at a time when activated microglia are found in a close perineuronal position. In contrast, the expression of the principal ligand of LFA-1, i.e. ICAM-1, remained unchanged following axotomy; the immunoreactivity being constitutively found on cerebral blood vessels. Increased LFA-1 expression thus appears to be a general marker for microglial activation and might further be involved in the rearrangement of the microglial cytoskeleton upon activation of this cell.

Animals↗
PubMed1993

Membrane properties of ameboid microglial cells in the corpus callosum slice from early postnatal mice.

Microglial cells in culture are distinct from neurons, macroglial cells, and macrophages of tissues other than brain with respect to their membrane current pattern. To assess these cells in the intact tissue, we have applied the patch-clamp technique to study membrane currents in microglial cells from acute, whole brain slices of 6-9-d-old mice in an area of microglial cell invasion, the cingulum. As strategies to identify microglial cells prior to or after recording, we used binding and incorporation of Dil-acetylated low-density lipoproteins, binding of fluorescein isothiocyanate-coupled IgG via microglial Fc-receptors, and ultrastructural characterization. As observed previously for cultured microglial cells, depolarizing voltage steps activate only minute if any membrane currents, while hyperpolarizing voltage steps induced large inward currents. These currents exhibited properties of the inwardly rectifying K+ channel in that the reversal potential depended on the transmembrane K+ gradient, inactivation time constants decreased with hyperpolarization, and the current was blocked by tetraethylammonium (50 mM). This study represents the first attempt to assess microglial cells in situ using electrophysiological methods. It opens the possibility to address questions related to the function of microglial cells in the intact CNS.

4-Aminopyridine↗
PubMed1992

Alzheimer beta A4-amyloid protein precursor in immunocompetent cells.

The mechanism of proteolytic breakdown of the beta A4-amyloid protein precursor (APP) has attracted much attention because of its relevance for Alzheimer's disease. Apart from the pathological role of APP in the amyloidogenesis, many efforts have been made to identify the functional significance of this widely expressed protein in various biological processes. Employing biochemical techniques, we demonstrate that APP is involved in the initiation of the immune response. Upon stimulation, it is expressed by the major functional types of T-lymphocytes, i.e. CD4+ and CD8+ cells. As was demonstrated for the CD4+ lymphoid cell line H9, APP is predominantly secreted. The remaining COOH-terminal fragments generated upon secretion were highly unstable. Of the APP produced by immunocompetent cells, considerable amounts were shown to be leukocyte-derived APP (L-APP). In addition, we were able to identify the KPI-containing L-APP isoform, L-APP733, as the major expressed L-APP isoform in immunocompetent cells, including rat microglial cells and astrocytes. The L-APP expression pattern of these cells showed high similarity. These findings seem to be indicative of an important function of APP within the immune system. Therefore, APP may be involved in various immunopathogenic conditions of the periphery and in the central nervous system.

Amyloid beta-Peptides↗
PubMed1992

Flow cytometric analysis of protease activities in vital cells.

The analysis of lysosomal proteases in cell lysates is complicated by pH-dependent and oxidative changes of their activity and complex formation with cytosolic inhibitors. Therefore, new flow cytometric methods were developed for the intracellular measurement of protease activities in viable cells. Intracellular cleavage of substrates such as Z-Arg-Arg-4-trifluoromethylcoumarinyl-7-amide to green fluorescent 7-amino-4-trifluoromethylcoumarin (AFC) in viable neutrophils and monocytes was only detected following phagocytosis of Escherichia coli. A measurement of the cysteine or serine proteinase activities in resting human leukocytes was, however, not possible with AFC derivatives as the overlapping blue fluorescence of the substrates reduces sensitivity. Nonfluorescent bis-substituted peptide derivatives of rhodamine 110 (R110), which are intracellularly cleaved to green fluorescent mono-substituted R110 and free R110 proved to be more sensitive substrates. The activity of the lysosomal cysteine proteinases of human monocytes or rat macrophages, i.e. cathepsin B and L, was specifically measured with (Z-Arg-Arg)2-R110, (Z-Phe-Arg)2-R110, or (Z-Ala-Arg-Arg)2-R110. Fluorescence generation was completely inhibited by Z-Phe-Ala-diazomethane or E-64. The serine proteinases of human neutrophils were analyzed with Elastase-substrates such as (Z-Ala-Ala)2-R110 or (Z-Ala-Ala-Ala)2-R110. Specificity was shown by inhibition with diisopropylfluorophosphate.

Amino Acid Sequence↗
PubMed1991

A subpopulation of bone marrow-derived macrophage-like cells shares a unique ion channel pattern with microglia.

Rat microglia share a number of antigenic, functional, and morphological similarities with macrophages from other tissues, but are characterized by a distinctly different pattern of ion channels in the cellular membrane (Kettenmann et al., J Neurosci Res 26:278-287, 1990). Macrophages typically express outward and inward K+ currents. In contrast, microglia lack outward currents and only show inwardly rectifying K+ currents, regardless of the isolation or cultivation method employed for microglia. In this study we demonstrate that a subpopulation of bone marrow-derived macrophage-like cells possesses inward rectifier K+ currents, but no outward currents and thus with regard to the electrophysiological characteristics closely resembles microglia. A second population of bone marrow-derived macrophage-like cells shows the usual channel pattern described for other body macrophages. Our results strengthen the hypothesis that in the bone marrow distinct pools of precursor cells exist, possibly reflecting an early differential lineage determination for body and brain macrophages, i.e., microglia.

Animals↗
PubMed1991

HIV-1 Nef protein exhibits structural and functional similarity to scorpion peptides interacting with K+ channels.

The persistent infection of human glial cells with HIV-1 is characterized by prominent expression of the Nef protein. In order to evaluate the possible role of Nef in the development of HIV-1-associated neurological disorders, we compared Nef with known neuroactive proteins. We found that HIV Nef shares sequence and structural features with scorpion peptides known to interact with K+ channels. Sequence similarity encompasses two distinct regions of scorpion peptides. Based on crystallography data, both regions in scorpion peptides cooperate in forming a common domain stabilized by ion pairs between charged amino-acid residues. Recombinant Nef protein, as well as a synthetic part of a scorpion channel active peptide (M10), reversibly increased the total K+ current of chick dorsal root ganglions in patch-clamp experiments without killing the cells. These results indicate that a region conserved in HIV Nef and scorpion peptides concurs in both structure and electrophysiological activity and suggest that Nef, like scorpion peptides, may affect neuronal cell function.

Amino Acid Sequence↗
PubMed1991

Respiratory burst activity in brain macrophages: a flow cytometric study on cultured rat microglia.

A new flow cytometric method for the investigation of the respiratory burst of macrophages/microglia isolated from neonatal rat brain has been established. Respiratory burst activity was measured quantitatively in single viable cells by the intracellular oxidation of non-fluorescent dihydrorhodamine 123 (DHR) to fluorescent rhodamine 123. Cultured microglia exhibited high spontaneous respiratory burst activity already before stimulation. After maximal stimulation with phorbol myristate acetate, DHR oxidation rose by 40-95%. The respiratory burst activity in resident or inflammatory, i.e. thioglycolate elicited, peritoneal macrophages was significantly lower than in cultured brain macrophages suggesting a high potential of microglia for oxidative tissue destruction.

Animals↗
PubMed1989

Immunophenotypic characterization of rat brain macrophages in culture.

Five monoclonal antibodies specific for rat monocytes/macrophages were used to characterize macrophages/microglia bulk isolated from neonatal and adult rat brain. The majority of brain macrophages was positive for all antibodies tested with minor differences between cultures derived from developing and mature central nervous tissue. These results contrast in vivo findings indicating that most antigens of peripheral macrophages are absent from resting, activated and phagocytic microglia in situ. We conclude that brain macrophages/microglia newly express antigens of the myelomonocytic lineage when in culture and that cultured brain macrophages may be derived from different types of precursor cells normally present within the CNS.

Age Factors↗
PubMedDate not supplied

Microglia in the immune surveillance of the brain: human microglia constitutively express HLA-DR molecules.

The degree of MHC class II expression in histologically normal human brain biopsy or autopsy tissue is still controversial. According to the generally held view MHC class II expression is rather low in the normal brain with the exception of the white matter. In the present study, HLA-DR expression was examined immunocytochemically in different brain areas obtained from three autopsy cases with short post-mortem times (i.e. 6 h). Based on standard histological evaluation, the brain areas studied appeared as histologically normal tissue. In all brain areas there was a strong constitutive HLA-DR expression on ramified microglia. The number of HLA-DR-immunoreactive microglia was strongest in the white matter (the corpus callosum and the capsula interna for example). The border zone between white matter and grey matter, however, revealed a sharp contrast between a high density of HLA-DR-immunoreactive microglia in the white matter and a rather low number in the grey matter. In the grey matter, HLA-DR-immunoreactive microglia were much less frequent than in the white matter and more pronounced on perivascular cells. The staining and distribution pattern of HLA-DR-immunoreactive microglia was confirmed by immunocytochemistry with a panel of different anti-HLA-DR monoclonal antibodies as well as by quantitative analysis of the immunostaining. Unlike the HLA-DR immunoreactivity, HLA-ABC immunoreactivity (detecting MHC class I antigens) was confined to endothelia and not observed on microglia. In the choroid plexus stromal macrophages expressed both class I and II antigens (i.e. at a location which could provide the peripheral immune system access to CNS antigens). Constitutive HLA-DR expression by microglia qualifies them as the main resident antigen-presenting cell of the brain. The pronounced overall HLA-DR expression by resting microglia questions a central dogma of the brain as an immune-privileged site and further points to the key role of the microglia in brain immune surveillance.

Adult↗
PubMedDate not supplied

Antibodies against microglia/brain macrophages in the cerebrospinal fluid of a patient with acute amyotrophic lateral sclerosis and presenile dementia.

This is the report of a case of a 41-year-old male patient with a rapidly progressing amyotrophic lateral sclerosis (ALS) combined with a severe presenile dementia. Screening for antibody binding on fresh-frozen human brain sections, we found that the patient's cerebrospinal fluid contained antibodies against microglia, the dominant and potentially cytotoxic immuneffector cell of the brain. This finding extends previous observations of CSF autoantibodies against microglia in neurodegenerative diseases. It demonstrates that in addition to antigens of neuronal origin (e.g. anti-Purkinje cell antibodies in paraneoplastic cerebellar dysfunction), glial antigens also appear to be under the surveillance of the CNS and/or the peripheral immune system. Our data provide evidence for a close link between neurodegeneration and the activation of microglia; a possible local antigen production against microglia/brain macrophages, which might regulate the concomitant glial activation in neurodegenerative diseases; and the presence of microglia-binding cerebrospinal antibodies as a marker for the acuity of the disease process underlying the acute deterioration of the neurological and cognitive performance in patients with, e.g., ALS. Future therapeutic strategies could therefore include the modulation of the possibly disease-promoting activation of microglia/brain macrophages.

Acute Disease↗