Herpes simplex virus type 1-infected human embryonic lung cells studied by optimized immunogold cryosection electron microscopy.

Herpes simplex virus type 1 (HSV-1) is a common human pathogen of skin and mucous membranes and is potentially dangerous when the infection is disseminated. Viral morphogenesis, especially the mechanism of viral envelopment and the exact pathway for processing and transport of HSV-1 glycoproteins, is still unclear. We report the results of optimized immunogold-labeled cryosection electron microscopy of HSV-1-infected cultured human fibroblasts (MRC-5). The simplified method presented has proved necessary to obtain reproducible results on cellular distribution of viral glycoproteins. It is now possible to demonstrate the viral glycoprotein gD-1, but not gC-1, in the nuclear membranes and to demonstrate gD-1- and gC-1-labeled viral particles in the perinuclear space, and to show the fate of the viral particles in the endoplasmic reticulum and Golgi area in infected cells.

A time-related study of Brefeldin A effects in HSV-1 infected cultured human fibroblasts.

Glycoprotein D (gD-1) is an essential virion envelope component of herpes simplex virus type 1 (HSV-1) normally transported to the plasma membrane of the infected cells. In the present study, the intracellular transport of gD-1 was inhibited in cultured HSV-1 infected human fibroblasts by Brefeldin A (BFA) 1 microgram/ml medium added for 12 h after virus adsorption. Immunofluorescence light- and confocal microscopy revealed abolished transport of gD-1 to the plasma membrane, juxtanuclear accumulation of gD-1, and a disorderly arrangement of the tubulin fibres. Withdrawal of BFA influence for more than 60 min resulted in incomplete transport but increasing accumulation of gD-1 in the plasma membrane and in Golgi-like areas close to the nuclei. The tubulin pattern was almost normalized 6 h after removal of BFA. The egress of infectious HSV-1 particles released 9 h post-BFA treatment was not fully reestablished. The results indicate that BFA effects were not completely reversible and caused a sort of cytotoxic influence involving the structure of tubulin.

The effects of cell passages on the cell morphology and the outcome of herpes simplex virus type 1 infection.

Because cell cultures are essential in biological research which involves the analysis of virus morphogenesis, this study focused on examining the significance of cell passages. Human embryonic lung fibroblasts (MRC-5) at passage (P) 27 were seeded twice a week to P 32, P 40, and P 48, when just at confluence and then infected with herpes simplex virus type 1 (HSV-1). The structure of the non-virus-infected (MOCK) and HSV-1 infected cells, the amount of cellular infectious virus particles and the capability to express HSV-1 glycoproteins C (gC-1) and D (gD-1) were investigated by phase-contrast and immunofluorescence light microscopy, immunogold cryosection EM, plaque assays, immunoblots, and total protein assays. Modified cell structure including fragmentation of tubulin fibers were visible in MOCK from P 38 onwards. The quantity of vimentin remained unchanged while actin accumulated and beta-tubulin decreased in HSV-1 infected late P cells compared to early P cultures. Cells of high P counts contained significantly fewer infectious virus particles, very likely of lower virulence, and their expression of gC-1 and gD-1 were concordantly reduced. These observations indicate that the number of cell P must be considered in order to reproduce results of cell biology and viral morphogenesis. The MRC-5 cells ought not to be passaged more than ten times beyond P 27 in the laboratory.

The use of monetary incentives in a community survey: impact on response rates, data quality, and cost.

OBJECTIVES: To assess the effect of incentive size on response rates, data quality, and cost in a digestive health status mail survey of a community sample of health plan enrollees. DATA SOURCES/SETTING: The study population was selected from a database of enrollees in various health plans obligated to receive care at Park Nicollet Clinic-HealthSystem Minnesota, a large, multispecialty group in Minneapolis, Minnesota, and the nearby suburbs. STUDY DESIGN: A total of 1,800 HealthSystem Minnesota enrollees were randomly assigned to receive a survey with an incentive of $5 or $2. The response rates for each incentive level were determined. Data quality, as indicated by item nonresponse and scale scores, was measured. Total cost and cost per completed survey were calculated. PRINCIPAL FINDINGS: The response rate among enrollees receiving $5 (74.3 percent) was significantly higher than among those receiving $2 (67.4 percent); differences were more pronounced in the first wave of data collection. Data quality did not differ between the two incentive groups. The total cost per completed survey was higher in the $5 condition than in the $2 condition. CONCLUSIONS: A $5 incentive resulted in a higher response rate among a community patient sample with one mailing than did a $2 incentive. However, the response rates in the $2 condition approached the level of the $5 incentive, and costs were significantly lower when the full follow-up protocol was completed. Response rates were marginally increased by follow-up phone calls. The incentive level did not influence data quality. The results suggest if a survey budget is limited and a timeline is not critical, a $2 incentive provides an affordable means of increasing participation.

The impact of managed care on physicians.

This article examines the forces that led to managed care and considers its two main thrusts: traditional indemnity, with controls and constraints through utilization review, and controlled access and reimbursement, as seen in the HMO model. The strategies available to the physician are discussed, suggesting how managed care can itself be managed. Finally, the evolution of managed care is shown to be in a final phase, wherein efficient and effective physicians are identified by computer data for selective contracting, leaving a sizeable minority of (potentially) unemployed physicians.

Easy and reliable double-immunogold labelling of herpes simplex virus type-1 infected cells using primary monoclonal antibodies and studied by cryosection electron microscopy.

Cell biology concerns the interactions between different cellular compartments and between the cell and the environment. The mechanisms of herpes simplex virus type-1 (HSV-1) envelopment and the transport of virus particles and HSV-1 glycoproteins have not been completely investigated. It is of interest to examine the formation of complete virus particles and the cellular distribution of viral glycoproteins correlated with microtubules. The illustration of these conditions by immunocytochemistry is best done by multiple labelling techniques in the same cell. Single-staining of neighbouring serial sections or two-face double-immunolabelling methods are not technically compatible with ultrathin cryosections. The results are reported here of a simultaneous, simple and reliable immunogold double-staining technique using primary antibodies of the same species in ultrathin cryosections. Compared to other inactivation procedures, phosphate-buffered 3% paraformaldehyde plus 2% glutaraldehyde for 2 h at room temperature is an excellent and gentle method to destroy free anti-IgG binding sites on the antibodies and to prevent cross-labelling, which has proven necessary for obtaining reproducible results on cellular distribution of tubulin and viral glycoproteins gD-1 and gC-1.

Interstitial cystitis provoked by tiaprofenic acid.

A patient developed clinical and histological interstitial cystitis as an adverse effect of treatment with tiaprofenic acid for rheumatism. After cessation of the drug, full symptomatic recovery was obtained. Bladder biopsies 6 months later demonstrated normalization of detrusor muscle histology.

[Comparative studies of Fc receptors for IgG on resting and activated mouse T lymphocytes using different methods]. / Vergleichende Untersuchungen über Fc-Rezeptoren für IgG auf ruhenden und aktivierten T-Lymphozyten der Maus mit Hilfe unterschiedlicher Methoden.

Fc-receptors for IgG (Fc gamma R) on resting (i.e. freshly prepared) and mitogen (Con A) or alloantigen-activated mouse spleen T cells were compared using binding of different markers such as 125J-labelled immune complexes, 125J-labelled anti Fc gamma R monoclonal antibody, FITC-labelled aggr. IgG and sheep erythrocytes covered with specific antibody (EA rosetting). C3b receptors were detected by rosetting with sheep erythrocytes covered with antibody and complement (EAC rosetting). The electrophoretic mobility of the cells without or after binding of aggr. IgG was also tested. A number of differences between resting and activated T cells were found: After activation of T cells by mitogen or alloantigen, a proportion of Fc gamma R-positive cells increased two to four times. Fc gamma R number per Fc gamma R-positive cell seemed to be higher on activated then on resting cells. Fc gamma R-positive resting cells did not shed their Fc gamma R upon incubation at 4 degrees C followed by incubation at 37 degrees C, but Fc gamma R-positive activated cells shed a remarkable proportion of their Fc gamma R on the same conditions. Binding of aggr. IgG caused a decrease of electrophoretic mobility of activated but not resting cells. Fc gamma R-positive resting cells were also C3b receptor-positive, whereas Fc gamma R-positive activated cells had no detectable C3b receptors.