Modulation of HLA class I antigen and ICAM-2 on endothelial cells after in vitro infection with human cytomegalovirus.

The aim of the present work is to investigate the expression of HLA class I antigen and intercellular adhesion molecule-2 (ICAM-2) on the surface of human umbilical cord endothelial cells (HUVEC) after infection with human cytomegalovirus (HCMV). The expression of HLA class I antigen and ICAM-2 were determined (using antibodies against HLA class I antigen and ICAM-2) by attachment inhibition assay and flow cytometric analysis. Attachment inhibition assay demonstrated that HCMV increased the expression of HLA class I antigen. This was confirmed by flow cytometric analysis, which showed an increase in HLA class I antigen expression on HCMV-infected HUVEC. The results of the expression of ICAM-2 using attachment inhibition assay revealed that ICAM-2 is involved significantly in the increased adhesion of T lymphocytes to HCMV-infected HUVEC. However, flow cytometric analysis revealed that there were no changes in the expression of ICAM-2 on HCMV-infected HUVEC. One possible explanation for this is that HCMV induces the activation of ICAM-2 on the surface of HCMV-infected endothelial cells without affecting its expression.

Alteration in the expression of endothelial cell integrin receptors alpha 5 beta 1 and alpha 2 beta 1 and alpha 6 beta 1 after in vitro infection with a clinical isolate of human cytomegalovirus.

Human cytomegalovirus infection of human umbilical vein endothelial cells reduces the ability of these cells to bind to fibronectin, collagen type IV and laminin. This suppression requires active virus, since UV-inactivated virus did not alter the binding ability of these cells to adhere to fibronectin, collagen type IV, and laminin. In an attempt to elucidate the molecular mechanism of this altered interaction, the surface expression of alpha 5 beta 1, alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 1 integrins on cytomegalovirus-infected endothelial cells was examined using attachment inhibition assay and flow cytometric analysis. The results presented here show that infection with human cytomegalovirus selectively alters the expression of integrin on human endothelial cells, with the ability to induce downregulation of alpha 5 beta 1 and alpha 2 beta 1 (p = 0.001) and p = 0.03, respectively), while significantly upregulating alpha 6 beta 1 (p = 0.03), and marginally upregulating alpha 3 beta 1 (p = 0.05).

Alterations in the expression of ELAM-1, ICAM-1 and VCAM-1 after in vitro infection of endothelial cells with a clinical isolate of human cytomegalovirus.

Human cytomegalovirus (HCMV) infection of endothelial cells resulted in increased adhesion of the cells to peripheral blood leukocytes. It was demonstrated by flow cytometry that increased adhesiveness parallels the increased expression of cell surface adhesion molecules (ELAM-1, ICAM-1, VCAM-1). The increased adhesion of PMN and T-lymphocytes was due to upregulation in the expression of ELAM-1 and ICAM-1. The upregulation of VCAM-1 resulted in the increased adhesiveness of monocytes and T-lymphocytes to HCMV-infected HUVEC. The increased adhesiveness to leukocytes was caused by HCMV replication since endothelial cells exposed to HCMV-free supernatants and UV-inactivated HCMV did not show any increase in adhesiveness to any of the leukocytes tested.

Comparison of the infectivity of the laboratory strain AD169 and a clinical isolate of human cytomegalovirus to human smooth muscle cells.

The results displayed by human cytomegalovirus (HCMV) IE/E antigen expression and virus release into the supernatant at various times post infection with a clinical isolate (C3/p5) and AD169 laboratory strain of HCMV, illustrated differences in the biology of these viruses on the various cell lines. While AD169 strain was shown to infect fibroblasts efficiently, it showed a low infectivity profile to smooth muscle cells, whereas C3/p5 displayed comparable infectivity characteristics on both cell lines. Neither virus demonstrated propensity for infecting endothelial cells, although passaging of the C3/p5 for additional 11 passages in endothelial cells provided virus capable of infecting endothelial cells efficiently. These results show that HCMV is capable of infecting smooth muscle cells which could be of relevance to the proposed role of HCMV in atherogenesis and provides further evidence on the adaptation of AD169 to fibroblasts.

Cytomegalovirus infection of vascular endothelial cells alters production of GM-CSF and G-CSF.

Infection of human umbilical vein endothelial cells with the clinical isolate of human cytomegalovirus (HCMV; at a multiplicity of infection of 2) severely suppresses the production of granulocyte-CSF and granulocyte-macrophage-CSF at late stages of infection (6 days post infection onwards). The effect was produced by actively multiplying virus which indicates that HCMV antigen expression is important for this suppression. The suppression in the production of these two cytokines was not due to their accumulation inside the cell nor to cell damage or lysis after infection.

Estimation of total DNA in crude extracts of plant leaf tissue using 4',6-diamidino-2-phenylindole (DAPI) fluorometry.

A method of estimating total double-stranded DNA in crude extracts of citrus leaf tissue by evaluating the enhancement of fluorescence intensity of 4',6-diamidino-2-phenylindole (DAPI) was assessed. For pure citrus DNA and citrus leaf tissue crude extract each in the presence of 100 ng/ml DAPI, excitation spectral response curves converged at excitation wavelength of 360 nm. At this excitation wavelength, maximum fluorescence intensity occurred across a range of emission wavelengths from 445 nm to 460 nm. The appropriate excitation and emission wavelengths were shown to be 360 nm and 450 nm, respectively. Fluorescence intensity increased linearly with DNA concentration and non-DNA components of the tissue homogenates had negligible effect on fluorescence at these wavelengths. Sodium dodecyl sulfate (SDS) in the incubation solution resulted in some suppression of DAPI-DNA fluorescence and produced a non-linear response to changing DNA concentration. The method should be applicable to DNA quantitation from crude tissue extracts of any plant species.

Interleukin-1 production and cell-activation response to cytomegalovirus infection of vascular endothelial cells.

Human cytomegalovirus (HCMV) is a source of major complications in immunosuppressed individuals, and endothelial involvement in HCMV infection is well documented. Traditionally laboratory strains of HCMV have been used in experimental investigations in vitro; however the continuous propagation of these strains in fibroblasts have attenuated the virus making it unsuitable for infecting other cell systems such as endothelial cells. In this study a recent clinical isolate of HCMV was propagated through several passages in endothelial cells and was used to investigate the effect of HCMV infection of human umbilical vein endothelial cells (HUVEC) on IL-1 production and cell proliferation. Infection of HUVEC with the clinical isolate of HCMV (at multiplicity of infection 5:1) suppressed the production of IL-1 alpha (82%) and IL-1 beta (99%) at 5 h post infection; the levels returned to that of the control within 24h post infection. Ultraviolet inactivated (but not heat killed) virus produced similar suppression confirming that a thermolabile viral structural protein or intact virion were responsible for this suppression. Infection of HUVEC with the clinical isolate increased the number of these cells and the rate of their proliferation. An increase of infected HUVEC number under quiescent growth conditions continued as the infection progressed (6-10 days post infection), exhibiting, at 3 days post infection, 5 times the number of uninfected HUVEC (control) which did not tolerate the quiescent culture conditions for more than 4 days. Live virus is responsible for this increase because UV-inactivated virus did not maintain the proliferation of HUVEC. These studies suggest that while infection of HUVEC with a recent clinical isolate of HCMV suppressed the production of IL-1 at early hours after infection, it increased the proliferation of these cells at later stages of infection.

Increased cytomegalovirus infection of human fibroblast and endothelial cells by electroporation.

Electroporation of fibroblasts and endothelial cells in the presence of HCMV caused an increase in the infection of these cells by the virus. This method could be applied to cells that are difficult to infect by ordinary laboratory methods and also in cases when synchronized infection of the cells by the virus is needed.

The effect of human cytomegalovirus on the production and biologic action of interleukin-1.

The effect of mycoplasma-free human cytomegalovirus (HCMV) on the production and biologic activity of interleukin-1 (IL-1) from peripheral blood monocytes was examined. The use of biologic thymocyte assays revealed a time-dependent decrease in the IL-1 activity of both HCMV-challenged and control monocytes after initiation of culture. A decrease in the amount of IL-1 beta secreted as measured by ELISA was also detected. The amount of IL-1 beta secreted by HCMV-challenged cells was always greater than that produced by control cultures at similar times. Despite containing higher levels of IL-1 beta, supernatants from challenged cells were markedly less effective in supporting thymocyte proliferation. It is proposed that this is due to the concomitant production of an inhibitor of IL-1 activity from HCMV-challenged monocyte cultures.

The possible involvement of immunosuppression caused by a lentivirus in the aetiology of jaagsiekte and pasteurellosis in sheep.

A South African isolate of ovine lentivirus was shown to cause a mild immunosuppression in sheep, reflected by a reduced delayed hypersensitivity reaction. This effect, measured in terms of skin swelling after intradermal inoculation with tuberculin, showed a positive linear relationship with the latency period before the appearance of jaagsiekte symptoms in animals co-infected with JSRV, as well as with the activity of monocytes. In a parallel study, increased susceptibility of lentivirus-infected sheep to infection with Pasteurella haemolytica was demonstrated. It is concluded that the lentivirus may play an enhancing role in both viral and bacterial infections of sheep by compromising the host's cellular immune response.

Altered polyamine concentrations in cytomegalovirus-infected human cells.

Polyamines have a regulatory effect on DNA and RNA synthesis and their levels are elevated in rapidly growing cells, including lymphoblasts. However, as shown in the current experiments, exposure to cytomegalovirus (CMV) reduces the polyamine levels in these cells, suggesting that the virus interferes with their metabolism. Studies have shown that the activity of ornithine decarboxylase is increased in CMV-infected fibroblasts and that there is an increased conversion of putrescine to spermidine and spermine. Thus it may be expected that the concentration of these molecules would increase in the infected cell. However, the results presented here demonstrate that only the concentrations of putrescine and spermidine are increased, the spermine concentration decreasing with infection.

Proliferative activity of vervet monkey bone marrow-derived adherent cells.

Vervet monkey bone marrow-derived adherent cell population cultured in Fischer's medium supplemented with 12.5% fetal calf serum and 12.5% horse serum consists of two cell shapes: fusiform (type I) and polygonal (type II). Limiting-dilution cloning of the cells suggested that the two morphologically distinct cell types belong to the same cellular system even though they differ in their proliferative capabilities. The labeling index of type II cells, as measured by autoradiography, was found to be consistently lower than that of type I cells. It is probable that these two phenotypes represent different stages of differentiation, where progenitor type I gives rise to type II cells. The bone marrow-derived adherent cells were found to be cytokinetically at rest in vivo, using the thymidine suicide test, and relatively radioresistant with a D0 = 2.1 Gy and ñ = 2.36 at the time of explantation from the bone. Furthermore, in culture these cells are characterized by a relatively long cell cycle of 60 h, where the length of the S phase is 30 h, G2 is 12 h, M is 6 h, and G1 is 12 h. Thus the vervet monkey bone marrow-derived adherent cells represent a cell population with a low turnover rate both in vivo and in vitro.

Demonstration of growth-inhibitory as well as growth-stimulatory factors in medium conditioned by lung lavage cells stimulated with a chemotactic factor secreted by jaagsiekte tumour cells.

Both growth-inhibitory and growth-stimulatory factors were detected in vitro in medium from chemotactically stimulated cultures of lung lavage cells. The macrophage component of the lavage cells was found to produce a growth stimulatory factor that was replaced by a growth inhibitory factor following chemotactic factor stimulation.

Interaction of strain AD169 and a clinical isolate of cytomegalovirus with peripheral monocytes: the effect of lipopolysaccharide stimulation.

The effect of human cytomegalovirus (CMV) infection on lipopolysaccharide (LPS)-stimulated and unstimulated monocytes from seronegative donors was studied by using the laboratory-adapted strain AD169 and a recent clinical isolate. LPS-stimulated and unstimulated monocytes infected with the isolate showed expression of immediate-early CMV antigens and were significantly more suppressive for lymphocyte proliferation than were strain AD169-infected monocytes, which rarely expressed detectable viral protein. Human CMV infection of LPS-stimulated and unstimulated monocytes resulted in abrogation of interleukin-1 activity, with the effect being marked in LPS-stimulated monocytes infected with the clinical isolate of CMV. Addition of interleukin-1 to infected, stimulated monocytes completely restored lymphoproliferative responses to phytohemagglutinin, whereas addition of this leukokine to infected, unstimulated cells could not restore this response.

Production of a macrophage chemotactic factor by cultured jaagsiekte tumour cells.

The increase of alveolar macrophages in jaagsiekte sheep lungs is not caused by excessive surfactant production but is due to a chemotactic factor secreted by the tumor cells. This factor has a molecular mass in the region of 13 kilodaltons, is stable at 56 degrees C but labile at 100 degrees C and, being sensitive to proteases, indicates that it is a small protein molecule.

The localization of a Mason-Pfizer monkey virus-related antigen in jaagsiekte tumour tissue and cell lines.

Mason-Pfizer monkey virus-related antigen was detected in 3 out of 5 jaagsiekte lungs examined using a direct immunoperoxidase staining technique with anti-MPMV p27 serum. Most of the antigen was localized in the alveolar lumina of the lesions. The reaction was further characterised on immune blots and found to involve a protein with a molecular mass of 29 000 daltons (JSRV p29). JSRV p29 antigen was also detected in 2 jaagsiekte cell lines.

Plasma membrane proteins and glycoproteins induced by human cytomegalovirus infection of human embryonic fibroblasts.

An analysis of the plasma membrane proteins of human embryonic fibroblasts (HEF) infected with human cytomegalovirus strain AD169 (HCMV) was performed using in vitro radioactive labelling techniques followed by PAGE. Of the 12 virus-induced proteins detected in infected cells, glycoproteins of mol. wt. 34000 (34K), 53K to 55K, 60K to 63K, 70K to 72K, 98K to 103K and 145K to 150K and proteins of 130K to 133K and 260K to 270K were considered significant. The 60K to 63K, 70K to 72K and 130K to 133K components were detectable at early stages of infection (8 h), although only the latter two were labelled by surface iodination. The others only appeared in the membrane from 48 h to 80 h after infection. Serological studies indicated that the 34K, 70K to 72K, 98K to 103K and 145K to 150K components may be HCMV-specified virion constituents, as these glycoproteins reacted with antibodies raised against virions and extracted envelope glycoproteins. Of immunological importance was the exposure on the cell surface of the protein moieties of 70K to 72K and 130K to 133K proteins at 8 h and 53K to 55K, 60K to 63K, 70K to 72K and 145K to 150K components at 80 to 90 h after infection. Pooled human immune sera contained antibodies which reacted with these exposed proteins, as well as with three other virus-induced membrane components of 230K to 240K, 98K to 103K and 78K to 80K.

Transfer of label from 3H-glucose in Digitaria eriantha leaves to the rust fungus Puccinia digitariae Pole Evans.

Digitaria eriantha pentzii was fed 3H-glucose prior to inoculation with uredospores of Puccinia digitariae Pole Evans. Twenty-one hours after inoculation, uptake of label from 3H-glucose by the primary infection structures of P. digitariae was demonstrated employing autoradiography. These results indicate that an exchange of nutrients between host and pathogen occurs very early on in the infection process, during the formation of the primary infection structures. Despite contrary reports that obligate parasites receive no nutrition before establishment of haustoria, this study supports the work of Andrews (Can J Bot 53:1103, 1975), who demonstrated uptake of 3H-glucose label from lettuce cotyledons into the primary and secondary infection vesicles, appressoria, and germ tubes of Bremia lactucae.