Experimental hepatitis E: pathogenesis in cynomolgus macaques (Macaca fascicularis).

The pathogenesis of experimental hepatitis E has not been thoroughly investigated. The purpose of this study was to more accurately document the events in this disease. Cynomolgus macaques were inoculated intravenously with bile or feces containing hepatitis E virus (HEV). Serum, bile, and liver specimens were evaluated with light microscopy, immune electron microscopy, immunofluorescence microscopy, EIA, and polymerase chain reaction. In the third week, there were histopathologic changes and HEV antigen (HEVAg) in liver, HEV in bile, and alanine aminotransferase (ALT) elevations. Widespread pathologic changes were detected during the fourth week and antibody to HEV (anti-HEV) and peak ALT values in the fifth or sixth week. By the sixth week, HEVAg had disappeared but pathologic changes persisted. This study supports the concept that experimental hepatitis E has an initial phase in which hepatic HEV replication is accompanied by the onset of hepatitis and a later phase in which the appearance of anti-HEV is accompanied by progression of the hepatitis.

Infection of owl monkeys (Aotus trivirgatus) and cynomolgus monkeys (Macaca fascicularis) with hepatitis E virus from Mexico.

Owl and cynomolgus monkeys were inoculated with hepatitis E virus (HEV) to compare disease models and produce antibody and virus. By immune electron microscopy (IEM), all six owl monkeys were shown to have serologic responses manifested by unusually high levels of anti-HEV at 6 months, but only three developed hepatitis. Virus-related antigen in liver (HEV Ag) was detected by immunofluorescence microscopy of biopsies from two of four owl monkeys; one with HEV Ag also had HEV in acute-phase bile (detected by IEM) and feces (detected by infecting another owl monkey). In contrast, cynomolgus monkeys propagated HEV to higher levels and all five had hepatitis. Moderate-to-high levels of HEV Ag correlated with detectable HEV in bile for both species. Thus, the value of using HEV-infected cynomolgus was confirmed. Owl monkeys were shown to be HEV-susceptible and sources of high-level anti-HEV; Sustained anti-HEV in these monkeys may also be useful for understanding immune responses.

Identification of Mycoplasma incognitus infection in patients with AIDS: an immunohistochemical, in situ hybridization and ultrastructural study.

Monoclonal antibodies (Mabs) were developed against antigens from a pure culture of Mycoplasma incognitus grown in modified SP-4 medium. All the Mabs obtained were shown to react only with M. incognitus, and not with other species of human mycoplasma. The Mabs identified M. incognitus immunohistologically in thymus, liver, spleen, lymph node, or brain from 22 patients with AIDS, as well as in 2 placentas delivered by patients with AIDS. Using an 35S-labeled DNA probe specific for M. incognitus and in situ hybridization technique, we also identified M. incognitus-specific genetic material in these tissues. Furthermore, ultrastructural studies of the specific areas of tissues which were highly positive for M. incognitus antigens revealed characteristic structures of mycoplasma organisms. These mycoplasma-like particles could be identified intracellularly and extracellularly. Histopathology of the tissues infected by M. incognitus varied from no pathological changes to fulminant necrosis with or without an associated inflammatory reaction. M. incognitus, a novel pathogenic mycoplasma, was cytopathic and cytocidal.

Association of the virus-like infectious agent originally reported in patients with AIDS with acute fatal disease in previously healthy non-AIDS patients.

We studied 6 patients from 6 different geographic areas who presented with acute flu-like illnesses. The patients developed persistent fevers, lymphadenopathy or diarrhea, pneumonia, and/or heart, liver, or adrenal failure. They died in 1-7 weeks. These patients had no serological evidence of HIV infection and could not be classified as AIDS patients according to CDC criteria. The clinical signs as well as laboratory and pathological studies of these patients suggested an active infectious process, although no etiological agent was found despite extensive infectious disease work-ups during their hospitalization. Post-mortem examinations showed histopathological lesions of fulminant necrosis involving the lymph nodes, spleen, lungs, liver, adrenal glands, heart, and/or brain. No viral inclusion cells, bacteria, fungi, or parasites could be identified in these tissues using special tissue stains. We report that immunohistochemistry using rabbit antiserum raised against VLIA, the virus-like infectious agent previously identified in patients with AIDS and shown to cause fatal systemic infection in primates, revealed VLIA antigens in these necrotizing lesions. In situ hybridization using an 35S labeled VLIA-specific DNA probe also detected VLIA genetic material in the areas of necrosis. Furthermore, virus-like particles closely resembling VLIA were identified ultrastructurally in these histopathological lesions. VLIA was associated with the systemic necrotizing lesions in these previously healthy non-AIDS patients with an acute fatal disease.

A comparative study of morphometric measurements of nucleoli in uveal melanomas from electron micrographs and plastic-embedded and paraffin-embedded sections.

Morphometric measurements of nucleoli were done on uveal melanomas from surviving and nonsurviving patients. The melanomas were embedded in paraffin and plastic, and measurement data from Papanicolaou-stained paraffin-embedded sections, toluidine blue-stained plastic-embedded sections and scanning transmission electron micrographs (STEM) of plastic-embedded sections were compared. The results showed that one parameter, the coefficient of variation (CV) of nucleolar area, correctly classified 80% of the cases as to survival when plastic-embedded material was used and 70% of the cases when paraffin-embedded material or STEM micrographs were used. The inverse standard deviation of the nucleolar area was a better predictor of outcome than was the CV of nucleolar area only in the paraffin-embedded sections. The nucleolar measurements were most easily and rapidly performed in the plastic-embedded sections.

Cytologic diagnosis and ultrastructure of fine-needle aspirates of ganglion cysts.

This report describes 28 ganglion cysts in 21 patients. The presence of a colorless to pale-yellow gelatinous material in the aspirate is pathognomonic of ganglion cyst. The smears are fairly monotonous, and show abundant mucoid material, single cells resembling histiocytes, a few tight clusters of cells, some collagen fibers, and some red blood cells with altered shapes. Ultrastructural studies performed on five specimens reveal the fibroblastic and/or histiocytic nature of the cells in the aspirates.

Instrumentation for the three-dimensional reconstruction of cellular organelles by electron microscopy.

Instrumental additions to the Zeiss EM-10 electron microscope are described as well as a flat-bed scanning densitometer specifically intended for the tomographic assessment of the three-dimensional structure of chromosomes. The additions to the electron microscope consist of two ion-getter pumps, reducing contamination to 0.0004 A/s. Further, a device was attached that made it possible to indicate through markers in the final negative the direction of the axis of tilt of the goniometer stage, i.e. markers indicating the tomographic axis in the digitized image. The flat-bed scanner permits correction of image rotation caused by refocusing. The preparation itself contains polystyrene latex spheres, i.e. objects the shape of which is independent of rotation (tilt). These can be used for computational corrections of the digitized image.

Reconstruction of a chromosome model from its projections.

As a pilot experiment towards the reconstruction of human chromosomes from their electron microscopic projections, a chromosome model was photographed and several cross-sectional planes successfully reconstructed. Some practical constraints and conditions for this type of work are defined.

Association of centrioles and chromosomes observed in preparations of whole-mounted human chromosomes.

Pairs of centrioles are a frequent finding in whole-mounted, critical-point-dried chromosome preparations from normal and irradiated human lymphocyte cultures. Most frequently they are found in association with a group C chromosome, although apparently free diplosomes are not uncommon. Fibers that in every respect are the morphologic equivalent of those seen in the body of the chromatid connect to the thick-walled open-ended part of the parent centriole. These features argue for the possibility that fibers connecting centrioles are an integral part of nuclear or chromosomal chromatin. It was observed that the smaller (daughter) centriole was connected and held in the well-known angular configuration to the thick-walled opne end of the parent centriole by a few, probably only two, colchine-resistant fibers.

Recovery of reovirus type 2 from an immature dog with respiratory tract disease.

Two similar cytopathic agents were recovered from the throat and rectal swab specimens of an immature dog with upper respiratory tract disease. The reference isolate, 14-72R, was shown to be a member of the reovirus group by its physicochemical properties, cytopathic effects in cell cultures, and appearance when examined in the electron microscope. Both isolates hemagglutinated human type O erythrocytes and antigenically were closely related to reovirus type 2. The affected pup had an increase in antibody titer to reovirus types 2 and 3. The latter findings provide evidence for possible heterotypic antibody responses in dogs to reovirus infection.

Electron microscopy of negatively stained jackbean urease at three levels of quaternary structure, and comparison with hydrodynamic studies.

Electron microscopy, with sodium phosphotungstate as negative stain, has been carried out on purified jackbean urease prepared at three levels of quaternary structure: (a) A1 urease, Mr = 240 000, S20,W = 11.5 S (b) alpha urease, Mr = 480 000, S20,w = 18.3 S (c) polymers of alpha urease above the tetramer stage. The compatibility of the images from level to level leaves no doubt that the enzyme itself is being visualized, and the following geometry is suggested by electron microscopy: A1 molecules are cyclic trimers, which pair up in eclipsed position across a 1-nm cleft to form the hexameric alpha, which displays D3 (or 32) symmetry of a trigonal prism. Polymers consist of alpha molecules aligned with their clefts coplanar and an angle of 120 degrees between each triplet of 3-fold axes. These features correspond reasonably well with sedimentation and electrophoretic studies of the solvated enzyme, which have indicated a hemispherical A1, a spherical alpha, and string-of-beads polymers. Sedimentation constants of the urease polymers up through the pentamer level were found to be compatible with the rosette, straight-chain, and zig-zag forms seen in the electron microscope, and with the suggested protomer arrangement in A1 and alpha urease.

Determination of the mass of viruses by quantitative electron microscopy.

The photometric method of quantitative determination of dry mass by electron microscopy has been applied to the study of various types of viruses: animal, plant, insect, and bacterial. The method is applicable to all viruses having a mass of 1 x 10-18g or greater. The molecular weight of viruses can be calculated from the mass value by multiplying it by Avogadro's number. In comparison to other methods of determining the molecular weight of viruses, sedimentation and diffusion, sedimentation equilibrium, light scattering, and electron microscopy counting, the method of quantitative electron microscopy is competitive. In some ways quantitative electron microscopy is superior to other methods for the determination of molecular weight: There is no limitation to the size of the virus, no experimental time involved and no concentration and purity of virus preparations required, and finally it is independent of the geometry of the virion. In one important aspect it is unique when compared to other methods; namely, it affords one the capacity to analyse individual virus particles.