The assembly of HIV within the Golgi apparatus and Golgi-derived vesicles of JM cell syncytia.

Chronic infection of the T-lymphocyte cell line JM with HIV-1 isolate GB8 results in the formation of multinucleate cells (syncytia). Transmission electron microscopy of these syncytia showed the presence of HIV particles both at the cell surface and within cytoplasmic vesicles. HIV particles were observed in dilated Golgi cisternae and Golgi-derived vesicles and in large vacuoles near the periphery of the syncytia. Immunolabelling was performed using an affinity-purified antiserum to the Golgi enzyme galactosyltransferase. This enzyme was consistently localized within both the Golgi apparatus and within virus-containing vesicles of JM syncytia, indicating that these vesicles originated from the Golgi apparatus.

Immunization with class I human histocompatibility leukocyte antigen can protect macaques against challenge infection with SIVmac-32H.

OBJECTIVE: To evaluate the efficacy of immunopurified class I human histocompatibility leukocyte antigen (HLA) to protect against SIV infection. METHODS: HLA class I antigens were immunopurified from a human B-lymphoblastoid cell line. Groups of four macaques were vaccinated subcutaneously with four doses of the immunogen in adjuvant, or with adjuvant alone and subsequently challenged intravenously with 10 median monkey infectious doses of cell-free SIVmac-32H. Infection was determined by polymerase chain reaction for SIVmac proviral DNA and by virus isolation. Antigen-specific humoral and cellular immune responses were monitored. RESULTS: Macaques immunized with the HLA molecules produced anti-HLA class I antibodies that inhibited SIV replication in vitro and downregulated autologous T-cell proliferation against irradiated C8166 cells. They were partially protected (two out of four) from virus infection for at least 33 weeks when challenged with SIV grown in human cells. All four control animals were infected. CONCLUSIONS: This demonstration of partial protection, together with our previous work reporting that vaccination with allogenic cynomolgus lymphocytes can protect against challenge infection with SIV grown in simian cells, suggests that allogenic immune response induced before or during establishment of HIV infection may have important implications for AIDS disease progression.

Further evidence of icosahedral symmetry in human and simian immunodeficiency virus.

Specimens of HIV and SIV have been examined by electron microscopy, using the techniques of conventional thin sectioning, freeze-substitution, cryosectioning, and cryomicroscopy of frozen hydrated specimens. In addition freeze-drying and critical point drying were used for both shadowed replicas and scanning electron microscopy. Thin sections revealed hexagonal, pentagonal, or near-spherical profiles. Angular particles were seen in shadowed replicas and also by scanning electron microscopy. The images observed were consistent with an icosahedral shape of the virus. It is proposed that mature HIV (SIV) is an icosadeltahedron with flat triangular facets. Size measurements of the specimens showed a wide range of values for conventional embedding, but a narrow range for specimens prepared by low-temperature techniques.

Antiviral properties of Ro 31-8959, an inhibitor of human immunodeficiency virus (HIV) proteinase.

Ro 31-8959 inhibits the spread of HIV infection and the production of cytopathic effects in cultures of acutely infected cells. IC50 values for these effects are in the range 0.5-6.0 nM and IC90 values are in the range 6.0-30.0 nM. This inhibitor is effective even when added to cultures at a late stage of infection, after syncytia have started to form. Virus antigen, virus particles and virus cytopathic effects can largely be cleared from cultures treated with compound from 3 days until 6 days post infection. In chronically-infected cells, inhibition of virus maturation can be detected after 24 hours' treatment with 10 nM Ro 31-8959. In addition, a significant reduction of the proteolytic processing of p56 to p24 can be demonstrated in these cells with compound at picomolar concentrations. These properties indicate that Ro 31-8959 is highly effective against HIV with the potential to inhibit acute, established acute and chronic infections.

A morphological and immunolabelling study of freeze-substituted human and simian immunodeficiency viruses.

Rapid freezing, freeze substitution and low temperature embedding were used to obtain resin-embedded specimens of HIV and SIV for morphological and immunolabelling studies, with particular emphasis on the 'lateral bodies' and p6 protein. HIV- or SIV-infected cells were fixed in 3% paraformaldehyde and cryoprotected with 0.5 M sucrose. Cells were applied to pieces of Whatman No 1 filter paper and impact-frozen onto a liquid nitrogen cooled copper block. Specimens were freeze-substituted at -90 degrees C using one of three different media: (a) absolute methanol, (b) methanol containing 0.5% uranyl acetate, and (c) methanol containing glutaraldehyde, osmium tetroxide and uranyl acetate. Specimens substituted in methanol and uranyl acetate showed both good structural preservation and retention of antigenicity. We found that the use of filter paper for supporting the specimen was an important factor in obtaining good freezing rates and was more practical than freezing mixtures of cells and gelatin. When compared with specimens prepared by conventional fixation and embedding, freeze-substituted virus particles showed a greater uniformity of shape and size and were more dense in appearance. Distinct 'lateral bodies' were not observed in freeze-substituted viruses. The viral protein p6 was widely distributed in the centre of mature virus particles.

Ultrastructural localization of the RNA of immunodeficiency viruses using electron microscopy in situ hybridization and in vitroinfected lymphocytes.

Cells infected in vitro with immunodeficiency viruses have been examined by electron microscopy in situ hybridization (EM ISH) methods for localization of viral RNA. Techniques used for preparation of specimens and probes are described. Unambiguous positive results were obtained using a mixture of two or three single negative strand DNA oligonucleotides complementary to regions of the gag, env and nef genes, each 200-300 bases and labelled with dig-11-UTP. Positive strand probes were used as a negative control. Cells were fixed with a mixture of formaldehyde and glutaraldehyde, dehydrated in ethanol with progressive lowering of temperature and embedded in Lowicryl K4M or HM20 at -35 degrees C. Permeabilization or pre-treatment of sections with proteinase K was not essential. The hybridization mixture was applied for 3-4h at 37 degrees C and probe was visualized by direct immuno-staining with sheep anti-digoxigenin antibodies conjugated to 10nm gold. This method would be suitable for future studies of the pathogenesis of retroviral infections and as a basis for further development of the EM ISH technique. EM ISH of in vitro infections of immunodeficiency viruses has shown the location of viral RNA in immature and mature viruses and its relationship to multimerized Gag protein during viral budding. The label for RNA has also been found in the cytoplasm of infected cells; it was mainly located adjacent to the plasma membrane and unassociated with visible Gag proteins. This may indicate that viral RNA migrates to the plasma membrane independently of the Gag protein and may, in some instances, arrive at the plasma membrane prior to the Gag protein. Viral RNA has also been found in the nucleus of peripheral blood mononuclear cells (PBMC) that were showing no morphological evidence of infection. The RNA was typically located in the nucleolus and in peripheral dense chromatin. These cells, which displayed morphological features of macrophage lineage, may have been the initial cell type to be infected in the PBMC.

Ultrastructural and immunocytochemical study on the infection of enterovirus 71 (EV 71) in rhabdomyosarcoma (RD) cells.

Rhabdomyosarcoma monolayers were inoculated with enterovirus 71 (EV 71) at 73 degrees C, sampled at intervals during the replicative cycle, and examined in thin sections by electron microscopy, using routine and immunoelectronmicroscopy with polyclonal antibodies against EV 71. The location of EV 71 or its precursors was followed during the viral replicative cycle. The earliest samples (3 h postinoculation) showed a cell shape change, from elongated to rounded. At 6 h postinoculation, the presence of early virus-induced vesicles developing within the cytoplasm was pointed out, although no virus particles were observed at these stages. At 12 and 20 h postinoculation, virus particles appeared in the cytoplasm. They were found free or in clusters in the cytoplasmic matrix, between the virus-induced vesicles. EV 71 particles were also occasionally observed in the form of paracrystalline arrays situated in the vesiculated areas. The immunolabel (gold beads) was found, initially, over dense cytoplasmic areas and in more advanced process at the vesiculated area and over the virus particles. Therefore the main cellular alterations observed in this infection were the shape change of the cell and the appearance of electron-dense areas (viroplasm) and smooth walled vesicles which are probably the site of the virus replication.

Patterns of ED use and perceptions of the elderly regarding their emergency care: a synthesis of recent research.

INTRODUCTION: The research literature reveals that the emergency care rendered to elderly patients may be of poor quality. Research examining elderly patients' ED use and their perceptions of their ED experiences was analyzed and synthesized, revealing gaps in the research and identifying areas for future research. METHODS: A computerized search was made of 3 databases (medline, HealthSTAR, and CINAHL). Each of the studies was systematically evaluated with use of the Nursing Practice Research Analysis Tool. RESULTS: Despite increased length of stay, more diagnostic tests, and higher expenses, the elderly have a higher rate of recidivism and are dissatisfied with their outcomes upon leaving the emergency department. Several areas that need to be explored further include quality of services rendered from the elderly ED patient's perspective; whether ageism exists within the emergency department; and the consequences of that ageism on the quality of care provided. DISCUSSION: The elderly are being cared for by ED personnel who have limited geriatric education within an environment that is antithetical to their needs. Research and endeavors that concentrate on improving the care of the elderly ED patient must be given top priority.

Emergency nurses' moral evaluation of patients.

Introduction Judgments by nurses concerning patients' moral fitness and the appropriateness of their visits to the emergency department are made constantly. The treatment rendered to patients is affected by these judgments. This study explored which patient characteristics emergency nurses used to morally evaluate patients, determined reasons the nurses gave for this behavior, and identified some factors that contributed to the evaluative behavior. Methods An exploratory, descriptive study with a mailed questionnaire was conducted on nurses working in emergency departments in hospitals throughout Alberta. Results Eighty-three questionnaires were completed and returned, representing a response rate of 55%. A hierarchy emerged from the data, validating previous research. Nature of the illness and diagnosis, in addition to certain patient characteristics, were critical in determining emergency nurses' attitudes. Nurses appeared to be unaware that they were acting prejudicially. Discussion When patients are aware of being disliked by the nurses caring for them, nurse-patient communication and relationship are jeopardized. This may affect care and, ultimately, recovery. Recommendations included encouraging nurses to recognize the dynamics, consequences, and inherent dangers of labeling patients and to improve their understanding of patients identified as unfavorable.

Assembly of cell-wall glycoproteins of Chlamydomonas reinhardii: Oligosaccharides are added in medial and trans Golgi compartments.

A series of monoclonal antibodies and a polyclonal antiserum have been used to investigate the localisation and pathway of biosynthesis of the cell-wall hydroxyproline-rich glycoprotein 2BII in the alga Chlamydomonas reinhardii. Glyco-protein precursors were detected within the endoplasmic reticulum using a polyclonal antiserum raised to the deglycosylated 2BII. Monoclonal antibodies which are known to recognise different carbohydrate epitopes of 2BII were found to label two distinct regions of the Golgi stack. The immunolabelling results demonstrate that there is compartmentation of protein synthesis and glycosylation steps for these O-glycosidically linked glycoproteins. Newly synthesised glycoproteins are transported from the Golgi apparatus to the cell surface via two distinct routes. They then undergo assembly into a cell wall, the inner wall layer being formed first and probably functionaing as a template within which the outer crystalline wall layers are assembled.

The zygote cell wall of Chlamydomonas reinhardii: a structural, chemical and immunological approach.

The zygote cell wall of Chlamydomonas reinhardii has been studied using structural, chemical and immunological methods. Monoclonal antibodies and polyclonal antisera that were originally raised to the major hydroxyproline-rich glycoproteins of the vegetative cell wall were used to probe the zygote wall for common antigenic components. These antibodies cross-reacted strongly and specifically with components of the zygote cell wall, and were used to show the origin, route of transport, and the location of these antigens within the zygote cell wall. The zygote cell wall contained about 10% protein, with hydroxyproline accounting for 22.5 mol % of the total amino acids present. Glucose was the most abundant sugar residue, and accounted for 56% of the total sugar present. Gas liquid chromatography-mass spectrometry showed the presence of a (1-3)ß-D-glucan as the major structural polysaccharide within the zygote cell wall. The (1-3)ß-D-glucan was detected and localised within the zygote cell wall by immunogold labelling of thin sections. Using an antiserum directed against (1-3)ß-D-linked glucose units, this polysaccharide was found to be consistently present within the non-staining layer of both young and mature zygote cell walls. (1-3)ß-D-Glucan was also detected in other wall layer using higher concentrations of antiserum. No intracellular labelling was found, indicating that the plasmamembrane is the site for the synthesis of this polysaccharide within the Chlamydomonas zygote.

The morphology of simian immunodeficiency virus as shown by negative staining electron microscopy.

Negative staining electron microscopy was used to study sucrose gradient-purified preparations of the simian immunodeficiency virus (SIVmac251). Both isolated and aggregated virus particles were observed together with some free-lying virus cores. The cores were 110 nm long and 25 to 50 nm wide and were mainly conical or wedge-like in shape. Surface projections were seen on the envelope membrane of many of the virus particles; the knobs were approximately 6 nm in length, 10 nm wide and from an end-on view they had a Y or triangular-shaped morphology.

Comparative morphology of Gag protein structures produced by mutants of the gag gene of human immunodeficiency virus type 1.

Six mutants that differ in the extent of their carboxyterminal sequences and two deletion mutants of the gag gene of HIV-1 have been characterized morphologically following their expression in Spodoptera frugiperda cells using recombinant baculoviruses. Electron microscopy has revealed distinct morphological forms of the Gag protein that can be classified as either (i) particulate, three-dimensional, spherical or tubular shells or (ii) non-particulate, two-dimensional, flat, curved or convoluted sheets. Progressive truncation of the carboxy terminus of Gag was accompanied by changes in the morphology and formation of spherical particles from predominantly C-type assembly and budding at the plasma membrane, through B-type intracytoplasmic assembly, to A-type assembly with budding mainly into cytoplasmic vacuoles. Deletions within the Pr24 CA domain of Gag abolished particle formation but retained association of the protein with the plasma membrane. All of the observed morphologies of the mutant Gag proteins could be accommodated within an icosahedral model for the organization of spherical particles and a basic hexagonal arrangement of assembled Gag protein monomers.

Intracellular localisation of dengue-2 RNA in mosquito cell culture using electron microscopic in situ hybridisation.

Non-isotopic in situ hybridisation was used at the electron microscope level to determine the localisation of viral RNA in dengue-2 infected mosquito cells at 14, 24, 48 and 72 h post-infection. In situ hybridisation was carried out on sections of dengue-2 infected mosquito cells using a digoxigenin-labelled DNA probe to the envelope protein gene sequence of the virus. Viral RNA was consistently localised over the rough endoplasmic reticulum and the virus-induced smooth membrane structures which form within the endoplasmic reticulum. During the later stages of infection electron-dense areas were observed to develop in close proximity to the smooth membrane structures. Electron microscopic in situ hybridisation showed that these denser areas contained both viral RNA and virus particles. Our results show that in dengue-2 infected mosquito cells the smooth membrane structures are an important site for the concentration of dengue viral RNA and its possible subsequent encapsidation into virus particles.

Cell wall glycoproteins: structure and function.

Hydroxyproline-rich glycoproteins form an important, but little understood, structural component of most cell walls. Their occurrence, chemistry, synthesis, secretion, cross-linking and functions in higher plant cell walls will be briefly reviewed. Similar molecules also occur in other groups of plants; in particular, in the algae. In many of these they form highly ordered cell surface arrays, and we have studied these by high-resolution electron microscopy and computer image reconstruction. Some resulting three-dimensional models of these are presented. One particular glycoprotein, the major structural component of the cell wall of Chlamydomonas reinhardii, has been investigated in some detail. The chemistry and structure of this glycoprotein, which we have called volvin, has been studied and a family of monoclonal antibodies has been raised against it. Some of these antibodies appear to be specific to oligosaccharide side-chains and allow the localization of these substituents and their sites of synthesis. Immunofluorescence studies have shown that the expression of some of these antigenic determinants is developmentally regulated or cell-cycle-dependent. Immunogold labelling of thin sections has enabled the sites of synthesis and the method of secretion to be determined. These results will be discussed in the context of other cell wall glycoproteins, their relation to other glycoproteins, such as the mating agglutinin, and to their possible functions.

Expression of meningococcal epitopes in LamB of Escherichia coli and the stimulation of serosubtype-specific antibody responses.

The class 1 outer membrane protein (OMP), a major variable surface antigen of Neisseria meningitidis, is a component of novel meningococcal vaccines currently in field trials. Serological variants of the protein are also used to serosubtype meningococci. Most of the amino acid changes that give rise to antigenic variants of the protein occur in two variable regions (VR1 and VR2) that are thought to form loops on the cell surface. The polymerase chain reaction (PCR) was used to amplify the nucleotide sequences encoding VR1 and VR2 from the chromosomal DNA of N. meningitidis strain M1080. These were cloned in frame into the lamB gene of the Escherichia coli expression vector pAJC264. Whole-cell enzyme-linked immunosorbent assays (ELISAs), using monoclonal antibodies, and SDS-PAGE confirmed that, upon induction, strains of E. coli carrying these constructs expressed hybrid LamB proteins containing the N. meningitidis surface loops. These strains were used to immunize rabbits and the resultant polyclonal antisera reacted specifically with the class 1 OMP of reference strain M1080 (P1.7). Immunogold labelling of meningococcal cells and whole-cell dot-blot analyses with these antisera showed that the variable epitopes were exposed on the cell surface and confirmed that this approach could be used to obtain serosubtype-specific antisera. The binding profiles of the antisera were determined from their reactions with overlapping synthetic peptides and their reactivity compared with that of relevant serosubtype-specific monoclonal antibodies. This approach was used successfully to raise antisera against two other class 1 OMP VR2s. A fourth antiserum raised against a VR2, including the P1.1 epitope, was not subtype specific.