Luxol Fast Blue MBSN-Levafix Red Violet E-2BL. A combined stain for myelin sheaths and glia fibers.

During investigations of reactive dyes, Levafix Red Violet E-2BL was found suitable for staining of glia fibers. Experiments were carried out on 37% formaldehyde-fixed human autopsy material. Paraffin sections were treated with Luxol Fast Blue MBSN as usual, differentiated until glia fibers were decolorized, and counter-stained in a 0.25% solution of Levafix Red Violet E-2BL in 0.25% acetic acid. Myelin sheaths were colored blue. Gila fibers, smooth muscle cells, and nuclei were stained red violet. Axons and connective tissue remained unstained; occasionally, coarse bundles of collagen showed patchy coloration. Polarization microscopic studies proved that Levafix Red Violet E-2BL is bound to well-oriented fibrous proteins in glia fibers. The similar staining and polarization microscopic properties of glia fibers and smooth muscle support previous findings that glia fibers contain a myosin-like protein.     

Silver impregnation of myelin sheaths

A method is described, based on mordantiation with phosphotungstic acid and tannic acid plus impregnation with silver nitrate solution (pH 10.4) and a physical developing technique. Water glass is used as a protective colloid in the developer.     

Hidden IgG antiglobulins in normal human serum.

Hidden antiglobulins reacting with whole rabbit immunoglobulin were found by radioassay or indirect haemagglutination after gel filtration or ultracentrifugation of normal serum. Suprisingly, both the IgM and IgG antiglobulins were present in the same macroglobulin fractions. The IgG antiglobulins could be dissociated into 7S components by separating the serum under acid conditions. Antiglobulin activity was a function of the Fab region.     

Ballooning of myelin sheaths in normally aged macaques

In aged animal brains, a variety of holes are formed in the neuropil. One type of hole, here designated as the myelin balloon, is an abnormality of the myelin sheath and is found in a number of diverse sites in the brain. Profiles of myelin balloons display rather smoothly rounded peripheral contours and typically range up to 10 m in diameter, although exceptionally large examples may be twice this size. The balloons are bounded by lamellae of myelin, and to accommodate the contents of the balloon, the myelin sheath becomes split at the intraperiod line. Since the intraperiod line is formed by the apposition of the outer faces of the myelin-forming plasma membrane, the contents of the myelin balloons are, in effect, in continuity with the extracellular space, and it is suggested that the contents of the balloons are fluid, with the fluid exerting an outward pressure on the walls of the balloons to produce their spherical shapes. Myelin balloons are not only produced during aging but also occur in a number of genetic strains of mice and in a number of human disease states. They thus represent a non-specific, though distinctive and common, alteration of the myelin sheath and are a reflection of the fact that under a variety of conditions, including normal aging, oligodendrocytes are unable to maintain the integrity of their sheaths.     

Differentiation of the human NT2 cells into neurons and glia

A method underlying a strategy for differentiation of the NT2 human teratocarcinoma cell line into neuronal and glial cells is described. The aim of this work is to provide a human model to study the relationships between neurons and glia in vitro during developmental or degenerative events. NT2 cells are seeded on polylysine precoated plastic or glass and differentiated by all-trans retinoic acid; persistent undifferentiated cells are eliminated by cytosine--D-arabinofuranoside; then cell cultures are maintained during four weeks until the appearance of glutamatergic receptors. Along the differentiation procedure, we have followed the expression of neuronal and glial phenotypes as well as the excitotoxic response to N-methyl-D-aspartate treatment taken as an indication of neuronal maturation. The procedure described leads to the development of a mixed population of neurons and glia sensitive to glutamate exposure.     

Neurons,glia, and plasticity in normal brain aging

Early manifestations of brain aging have received much less attention than the drastic degeneration of AD and MID. During nonpathological changes of normal aging, brain systems differ in the involvement of neuron loss. Spatial learning can become impaired without evidence for neuron loss, whereas eye-blink conditioning deficits are well correlated with Purkinje neuron loss. Glial activation, in particular the increased expression of glial fibrillary acidic protein (GFAP), may be a factor in impaired synaptic plasticity. Lastly, it is discussed how developmental variations in the numbers of Purkinje cells and ovarian oocytes can be factors in outcomes of aging that are not under strict genetic control.     

Staining in normal and ischemic human myocardium. A study of myoglobin, IgG, glycogen, and diastase-PAS

Using the indirect immunofluorescence technique, we studied the distribution of myoglobin in normal and ischemic human myocardium obtained at autopsy and at surgery. Glycogen, diastase-PAS staining of the sarcoplasm, and IgG were also studied and compared with the structure of the lesions and the distribution of myoglobin. The surgical material we used was largely free of autolysis and was the most satisfactory. Prolonged fixation of tissues in formaldehyde solution or perfusion fixation of autopsy specimens both proved to be unsatisfactory as myoglobin was absent from the myocardium. This loss presumably represents diffusion of myoglobin due to autolysis and the method of fixation. Another group of autopsy specimens that was briefly fixed by immersion in formaldehyde solution prior to processing was more satisfactory. Although they showed some extracellular diffusion of myoglobin, the autolyzed normal areas could still be clearly differentiated from the autolyzed ischemic areas.     

Cytotoxicity of human lymphocytes induced by rabbit antibodies to chicken erythrocytes. Inhibition by normal IgG and by human myeloma proteins of different IgG subclasses

Normal IgG preparations of human, rabbit or guinea-pig origin (IgG2) were tested for their capacity to inhibit the cytotoxicity of purified human lymphocytes, as induced by rabbit IgG antibodies to chicken erythrocytes. All IgGs were found to be about equally efficient inhibitors. Human F(ab′)₂ used for control, gave no inhibition. Human myeloma proteins of subclasses IgG1, IgG2 and IgG3, were about equally efficient inhibitors. In contrast, the inhibitory action of myeloma proteins belonging to subclass IgG4 was weak and more irregular. In this assay system, a large excess (∼ 10⁶ ×) of normal IgG over antibodies had to be added in order to achieve ≥50 per cent inhibition. Heating of the inhibitors to 63° for 30 minutes did not significantly enhance their inhibitory capacity.For comparison, the same human IgG preparations and myeloma proteins were also tested for their capacity to inhibit phagocytosis by human blood monocytes of chicken erythrocytes sensitized with rabbit IgG antibody. As was to be expected in this system, only HGG, IgG1 and IgG3 caused inhibition whereas F(ab′)₂, IgG2 and IgG4 were completely negative.     

Binding and internalization of human IgG by living cultured endothelial cells

Interactions between circulating IgG and endothelial cells (EC) in humans have been described only in conditions associated with pathologic immunoglobulins and/or activated or damaged EC. In this study we provide evidence that normal human IgG includes one/some antibody species that bind to and are internalized by living EC in culture. This novel function of EC and natural autoantibodies is of potential importance for the understanding of physiologic interactions between vessels and the immune system and for the clarification of pathogenesis of vasculitis and mechanisms of action of pooled IgG used in the therapy of such conditions.     

Binding of Fibronectin to Clq; Inhibition of Binding by Aggregated IgG

Fibronectin was shown to bind to Clq using alkaline phosphatase conjugated fibronectin and Clq coated polystyrene tubes. The binding of the alkaline phosphatase conjugated fibronectin to Clq was dose dependent and inhibited by fibronectin and by the sulfated polymers heparin and chondroitin sulfate. The fibronectin interaction was inhibited only lightly by gelatin indicating that the fibronectin-gelatin interaction was different from that with Clq. Heat aggregated IgG blocked the binding of fibronectin to Clq and fibronectin inhibited the binding of aggregated IgG to Clq. These results suggest that fibronectin may be a factor affecting the determination of immune complexes in serum specimens by Clq binding assays.     

Binding of Fibronectin to Clq; Inhibition of Binding by Aggregated IgG

Fibronectin was shown to bind to Clq using alkaline phosphatase conjugated fibronectin and Clq coated polystyrene tubes. The binding of the alkaline phosphatase conjugated fibronectin to Clq was dose dependent and inhibited by fibronectin and by the sulfated polymers heparin and chondroitin sulfate. The fibronectin interaction was inhibited only lightly by gelatin indicating that the fibronectin-gelatin interaction was different from that with Clq. Heat aggregated IgG blocked the binding of fibronectin to Clq and fibronectin inhibited the binding of aggregated IgG to Clq. These results suggest that fibronectin may be a factor affecting the determination of immune complexes in serum specimens by Clq binding assays.     

IgE levels in normal human sera and IgG preparations

Serum IgE levels were determined in 318 Hungarian blood donors by PRIST technique. Age-dependent decrease of IgE and higher values in males than in females were found. Commercial IgG preparations contained variable amounts of IgE related to the procedures for purification. The normal IgE level in healthy Hungarian adults were compared to the reported normal values of other countries.     

Synapses between NG2 glia and neurons

NG2-expressing glia are precursors to oligodendrocytes and subpopulations of astrocytes. They are unique among glial cells in that they enter into synaptic specialisations with neurons throughout all areas of grey and white matter and at all ages. To date, the NG2 cells appear to represent a postsynaptic compartment, and synapses are formed with axons. With differentiation to oligodendrocytes, NG2 is downregulated and myelin antigens upregulated: this coincides with a loss of the synaptic contacts between neurons and NG2 glial cells. The functional roles of this glial-neuron synapse in regulation of differentiation into myelinating oligodendrocytes or additionally responding to and modulating neuronal network activity remain to be elucidated.     

Solid-phase radioimmunoassay of IgA, IgG, and IgM antibodies to human rotavirus.

A solid-phase radioimmunoassay (RIA) has been developed for the detection of human rotavirus-specific IgA, IgG, and IgM antibodies. Nebraska calf diarrhea virus grown in LLC-MK2 cell cultures in the presence of trypsin was directly adsorbed onto polystyrene balls, and antibodies that attached to the virus-coated balls were detected by subsequent binding of 125I-labeled antibodies specific to human alpha, gamma or mu chains of human Iga, IgG, or IgM immunoglobulins. A total of 116 serum specimens from 58 adult patients were tested. Binding ratios between the positive and the negative serum varied between 5 and 15, occasionally being 20 or more in the IgA and IgG assays, but rarely exceeding 3 in the IgM assay. The RIA was found to be more sensitive in detecting antibodies to rotavirus than the complement fixation (CF) test, the RIA titers obtained being 50--100 times as high as the CF titers. The method described offers a possibility of evaluating the immune response to human rotavirus and of detecting recent infection.     

18. Determination of Insulin Binding to Human IgG by Quantitative Radioimmunoelectrophoresis

Summary. The quantitative radioimmunoelectrophoresis for the determination of insulin binding to IgG is described. In 95% of 267 Danish diabetics treated with insulin, insulin-binding IgG has been demonstrated. The method has the advantage of being specific, sensitive, and simple. A number of precautionary measures to be taken when using isotopes in electrophoresis are mentioned.     

Antibodies of IgA and IgG class in normal human urine

Urine collected and concentrated from three individuals after tetanus toxoid immunization has been studied for antibody activity. Gel-filtration on Sephadex G-200 was used to fractionate the urine concentrates and antibody activity in the eluates was assessed by a passive haemagglutination test. Haemagglutinating activity was demonstrated by inhibition experiments in the IgG and IgA classes of immunoglobulin but not in the IgM or IgD classes. In no case could antibody activity be ascribed to molecules smaller than intact IgG.