Konrad C. Hsu

ASSOCIATION BETWEEN POLYARTERITIS AND AUSTRALIA ANTIGEN

Abstract Four of eleven patients with biopsyproven polyarteritis nodosa were also found to have Australia (Au) antigenaemia. The four Au-antigen-positive patients exhibited a typical polyarteritis syndrome, but differed from the seven antigen-negative patients in that they also had evidence of mild hepatic damage. The presence of circulating immune complexes in the sera of three of the four Au-antigen-positive patients was demonstrated by serological, ultracentrifugal, and electro-microscopic studies. These complexes were further shown to be composed of Au antigen and immunoglobulin. Immunofluorescent studies of tissue from one of the patients revealed deposition of Au antigen, IgM, and β 1 C in blood-vessel walls.

Immunofluorescence Demonstration of a Muscle Binding, Complement-Fixing Serum Globulin Fraction in Myasthenia Gravis.

Summary 1. A muscle binding, complement fixing component has been demonstrated in the crude globulin fraction of a pool of serum from 10 patients with myasthenia gravis of recent onset and progressive character, by means of the immunofluorescence technic. This component could not be demonstrated in a similarly prepared normal serum globulin pool. Untagged myasthenic globulin was shown to block competitively adherence of fluorescein tagged myasthenic globulin to skeletal muscle striations; whereas prior treatment of muscle sections with normal serum globulin intensified staining with fluorescein tagged myasthenic globulin. Individual myasthenia gravis serums included in the pool also blocked staining with fluorescein conjugated myasthenic globulin. Normal serums did not block adherence of the fluorescein tagged myasthenic globulin to skeletal muscle. 2. The myasthenia gravis globulin fraction was shown to fix guinea pig complement to human skeletal muscle, by successive treatment of muscle sections with myasthenic globulin, guinea pig complement, and fluorescein conjugated rabbit anti-guinea pig complement. Normal serum globulin failed to fix complement by this technic. 3. Thirty-one myasthenia gravis serums were screened, with 11 normal serums, and 5 serums from patients with other generalized myopathies, for their ability to fix guinea pig complement to skeletal muscle. Thirteen myasthenic serums gave unequivocal evidences of complement fixation, using the immunofluorescence technic. No normal serums fixed complement. Serum from one patient with paroxysmal myoglobinuria also fixed complement. Serum from one patient with acute dermatomyositis gave rise to fluorescence in the sarcolemma when layered onto skeletal muscle followed successively by guinea pig complement and fluorescein conjugated rabbit anti-guinea pig complement.

THE UPTAKE AND DEVELOPMENT OF REOVIRUS IN STRAIN L CELLS FOLLOWED WITH LABELED VIRAL RIBONUCLEIC ACID AND FERRITIN-ANTIBODY CONJUGATES.

Abstract Reovirus possesses an outer capsid and a subjacent shell, presumably necessitating the rupture of two separate protective coats to release the genome. This may occur intracellularly within phagocytic viral inclusions where whole particles are sequestered and gradually lose their coats. Cells infected with purified virus particles containing labeled RNA were sampled at intervals during a 12-hour experimental period, and then subjected to light and electron microscopic autoradiography. The label was conserved in a macromolecular form throughout this period. Because of asynchrony in the release of parental genomes and lack of identifiable, partially degraded forms of the virus outside the phagocytic inclusions, it was not possible to reconstruct the sequence of stages during the penetration of inoculum RNA to its site of function. However, about 14% of the parental label was finally transferred to foci where progeny virus was being formed. Evidence, derived from experiments with infected cells and tagging by means of Ferritin-antibody conjugates specific for reovirus protein, is presented which relates virus morphogenesis with the presence of mitotic-spindle tubules and a cytoplasmic filamentous component.

The effect of hydroxyurea on virus development. II. Vaccinia virus.

Abstract Hydroxyurea in appropriate concentration inhibits the formation of mature infectious particles of vaccinia virus without preventing the synthesis of viral protein. Development of the virus is arrested at the “immature” or single-membrane stage, and the viral particles lack the internal body or “nucleoid” that is normally present. Hydroxyurea appears to act by inhibiting the synthesis of viral DNA.

IMMUNOCHEMICAL STAINING FOR ELECTRON MICROSCOPY

The successful al)plication of imntunofluorescent staining in so many branches of biological science has prompted a search for a comparable antibody label which can utilize the higher resolution afforded by electron microscopy. Such a label must have sufficient size and electron-scattering power to be readily discernible against the usual background of biological specimens. A number of methods for employing the specificity of the antibody-antigen reaction for electron microscopic cytology have been proposed and tried. By means of unmodified antibody alone (13) and even more effectively by antibody substituted with heavy metals (1 1 , 12, 22) the presence of antigen may be recognized by virtue of a diffuse increase in electron-scattering due to the bound antibody. Individual antibody molecules and their specific sites of attachment cannot be readily discerned, however, by these techniques. In 1959 Singer proposed the use of the ironcontaining protein, ferritin, a.s a label which would permit the recognition of single antibody molecules (18). Coupling of antibody and fermitin may be accomplished by means of a number of small, bifunctional molecules including nz-xylylene diisocyanate (18), toluene diisocyanate (19) and p ,p’-difiuoro-nu ,m’-dinitrophenylsulfone (14). In each case one active group is available for reaction with ferritin and the other with globulin. The chemical aspects of these reactions and some of the properties of the conjugates have been described (3, 14, 17, 19, 21). Itis the purpose of this communication to elaborate upon certain aspect-s of Singer’s original conjugation procedure which appear to be responsible for a relatively high rate of successful coupling and a satisfactory yield of conjugated antibody. In addition, examples of

Murine Influenza Virus Encephalomyelitis

The development and maturation of neurotropic WS-N strain of influenza virus in encephalitic mice was shown to occur preferentially along the “free” surface of ependymal and choroid plexus cells where it developed from the cell membrane. Virus maturation and “budding” was generally absent along the other surfaces of the same cells where they were apposed to other cells. This may suggest an inhibitory effect of adjacent cell membranes. Electron microscopic observations of ependymal surfaces may be of importance in demonstrating some viruses in human and animal tissues. Most of the virions were rounded in shape with surface spikes but a considerable number of filamentous forms were seen developing at the tips of villi. Filamentous forms had previously been described in tissue culture but not in animal encephalitic tissues. Intracellular virus within vacuoles was seen en masse and a few virus-like structures appeared to form from endoplasmic reticulum. Intracytoplasmic inclusions were demonstrated. The virus appeared ultrastructurally at a time preceding and overlapping the time at which virus replication reaches its maximum. The electron microscopic results were also considered in relation to immunofluorescence evidence in a companion paper, that virus antigen is present in deeper cells even though few mature virions appear in those sites. The combined data suggests that virus initially has a predilection for ependymal cells where mature virions develop at the surface of the cells. Later there is extension of virus antigen to deeper cells with a presumed transfer from cell to cell without the same degree of maturation of virions at cell surfaces.

Fluorescent, Electron Microscopic, and Immunoelectrophoretic Studies of Labeled Antibodies

Antibodies, produced in rabbits, to each of three bacterial species have been doubly labeled with fluorescein and ferritin. Irrespective of which label was conjugated to the antibody first, immunologic activity was maintained. Moreover, these preparations gave as high a degree of specificity in fluorescent and electron microscopic studies as did singly labeled antibodies. Immunoelectrophoretic analyses and other immunologic tests further confirmed that the antibodies were conjugated to both labels without loss of specific activity. The technique thus permits the relatively simple method of immunofluorescence to be used as an aid in selecting optimtum ferritin antibody conjugates for localizing of antigen at the molecular level by means of electron microscopy.

Murine influenza virus encephalomyelitis. I. Neuropathological and immunofluorescence findings.

The direct inoculation of neurotropic NWS strain of the influenza virus into the brains of Swiss albino white mice results in an acute meningoencephalomyelitis. The myelitis is described for the first time. The cerebral lesions are marked by a severe ventriculitis, characterized by a necrotizing ependymitis and a lesser involvement of the choroid plexus. During the first three to four days of the infection, increasingly frequent and prominent paraventricular inflammatory and degenerative lesions are seen in the brain and paracanalicular ones in the spinal cord. There is a gradual decrease in the number and severity of the lesions from the fifth to the seventh days. Intranuclear inclusions and fewer cytoplasmic ones are described for the first time in this experimental condition and are seen in both cerebral and spinal cord lesions. They are present in nerve cells but not in glial or other cells. Segmental demyelination is encountered sparingly and is most notable in the corpus callosum. Astrocytosis is a prominent and early feature of experimental murine influenzal meningoencephalitis and is probably reactive in character.

Common Antigen in Meningioma-Derived Cell Cultures

Serums of patients with intracranial meningiomas reacted in immunofluorescence assays with cell cultures and tumor imprints prepared from human meningiomas. Antibody in these serums appears to be specific for antigens in meningioma tissue and shows some cross-reactivity with neoplastic tissue of glial origin.

Tubulo-interstitial (TI) renal disease associated with chronic lymphocytic choriomeningitis viral infection in mice.

Abstract The evolution of the renal tubulo-interstitial (TI) lesions of SWR/J mice neonatally infected with lymphocytic choriomeningitis (LCM) virus was studied by immunofluorescence, light, and electron microscopy. By immunofluorescence, viral antigens were observed in the cytoplasm of TI cells and in the walls of interstitial vessels throughout the experimental period, i.e., from 20 days to 1 year after infection. In contrast, comparable deposits of mouse IgG and C3 were not demonstrable in TI structures. By light and electron microscopy, interstitial cellular infiltration, tubular cell atrophy and degeneration, and, in the late stages, peritubular, periglomerular, and perivascular fibrosis were observed. The severity of TI lesions did not correlate with that of immune complex glomerulonephritis. The immunopathologic data presented here are consistent with the hypothesis that the persistence of LCM antigen in TI structures throughout the course of the disease elecits a cell-mediated immune reaction.