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.

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.

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.

Growth and morphological characteristics of vervet monkey bone marrow-derived adherent cells.

Monkey bone marrow-derived adherent cells were maintained in culture for six months. Phase contrast and scanning electron microscopy showed two cell shapes: fusiform and polygonal. No difference was observed in the cyto-chemical staining of the two shapes. Both stained positively for alpha-napthyl acetate esterase, acid phosphatase, collagens I and III, and lipids and negatively for peroxidase and factor VIII antigen. A small proportion (1.5%) were alkaline phosphatase-positive. An average of 14% of the cells were phagocytic in the primary culture, but this proportion decreased progressively with passage. Fc receptors were not detected, while C3 receptors were detected in 1% of primary cultured cells in primary culture, but were not detected in subcultured cells. Adherent cells were not evident in cultures supplemented with 10 mM ammonium acetate. These findings indicate that monkey marrow-derived adherent cells are fibroblastoid in nature.

Influence of a fibroblastoid cell line derived from murine bone marrow (H-1 cells) on stem cell proliferation.

A murine fibroblastoid cell line (H-1) with properties similar to those of adventitial reticular cells can support granulopoiesis and the development of mononuclear phagocytes in vitro. In the current study the effect of these cells on stem cell maintenance in vitro was assessed. The H-1 cells were unable to support CFUs replication in liquid culture, while treatment of some stem cells with H-1 conditioned medium appeared to inhibit their proliferation.

The morphology and morphogenesis of jaagsiekte retrovirus (JSRV).

Jaagsiekte retrovirus ( JSRV ) was recently shown to be the aetiological agent of jaasiekte (ovine pulmonary adenomatosis). The morphogenesis of JSRV was studied in jaagsiekte tumour tissue. Intracytoplasmic particles, often associated with centrioles, were found in tumour cells. JSRV budded from tumour cells with a complete core which appeared to mature during the budding process. Extracellular particles were found in the alveolar lumen. Immature extracellular particles were rare. Mature extracellular JSRV was membrane-bound and had a slightly eccentric nucleoid with an electron-dense perinucleoidal space. In negatively stained preparations of JSRV the envelope was covered with spikes. JSRV is morphologically distinct from all known retroviruses.

Effect of in vivo exposure to benzene on the characteristics of bone marrow adherent cells.

The effect of benzene on the adherent cell population, cultured from the bone marrow of exposed mice was investigated in the presence and absence of hydrocortisone. The adherent CFUs from exposed animals did not differ either in numbers or self-replicate ability to those derived from shown exposed animals. Adherent layers from mice exposed to 100 or 400 pp-benzene were devoid of fat cells regardless of the presence or absence of hydrocortisone. Hydrocortisone was shown to influence the proportion of acid phosphatase-positive cells derived from benzene-exposed animals. Those results suggest that benzene exposure may influence the bone marrow stromal cells.

Regulation of the colony-stimulating activity produced by a murine marrow-derived cell line (H-1).

The production of molecular species that stimulate growth of granulocyte or macrophage colonies (GM-CSF) by the fibroblastoid H-1 cell line is unaffected by either native or iron-saturated lactoferrin, although some inhibition is detected with 10 microM prostaglandin E1. The H-1 GM-CSF is able to support the formation of macrophage, neutrophil, and mixed colonies. Feeder layers of H-1 cells are also able to support the development of colony-forming units stimulated by GM-CSF (GM-CFUc) although the number of colonies produced when the optimal H-1 cell concentration is plated (2.5 x 10(3) cells) is only 30% of the number with conditioned medium alone. This inhibitory effect is observed irrespective of the presence of an additional agar layer between the feeder cells and plated bone marrow cells, implying that diffusable substances are involved. Addition of indomethacin (10 microM) to feeder layers derived from 2.5 x 10(3) H-1 cells increases the number of GM-CFUc detected to 50% of that seen with conditioned medium alone. This result suggests that released prostaglandin may be responsible for some, but not all, of the observed inhibition of colony formation. In the presence of the H-1 feeder layers, only macrophage colonies are detected and hence it appears that the H-1 cells produce, in addition to prostaglandin, a diffusible inhibitory substance that preferentially inhibits granulopoiesis.

Characterization of a murine cell-line derived from cultured bone marrow stromal cells.

The properties of a cell line (H-1) derived from the adherent layer of a murine marrow liquid culture system are described in detail. Morphologically the cells are of a fibroblastic nature, having none of the specialized structures found in endothelial cells or macrophages. They have neither Fc receptors nor peroxidase activity confirming that they are not mononuclear phagocytes. However, they possess alkaline phosphatase and acid phosphatase, synthesize collagen, and are weakly phagocytic. Adventitial reticular cells in bone marrow, which have a close association with all developing hemopoietic cells, possess these properties. It is suggested that the H-1 cell line, which produces colony stimulating factor (GM-CSF), is derived from the adventitial reticular cell of the marrow.

Decreased expression of receptors on monocytes from cancer patients.

The expression of crystallizable fragment and complement receptor sites was significantly decreased on the peripheral blood monocytes from 10 patients with advanced carcinoma. In addition the phagocytic activity of these cells was depressed, although no differences in the content of lysosomal enzymes could be demonstrated by staining techniques. These results suggest that the functions of monocytes from such cancer patients may be severely depressed.

Energy-dependent accumulation of Ca2+ by human embryonic lung fibroblasts.

Human embryonic fibroblasts accumulate Ca2+ in the presence of extracellular ATP and Mg2+, the uptake being maximal at 3 mM ATP. Iodoacetic acid, oligomycin and temperatures of 2 degrees C all inhibit the ATP-potentiated uptake suggesting that an active process may be involved in the transport of Ca2+ into these cells under certain conditions.

Inhibition of energy-dependent Ca2+ accumulation in fibroblasts by smooth muscle antibodies.

An antibody prepared against smooth muscle myosin interferes with active Ca2+ accumulation of fibroblasts. This provides further evidence for the existence of myosin at the cell surface.

(Ca-2+ + Mg-2+)-Activated ATPase in the plasma membrane mouse liver cells.

1. Purified plasma membranes from dissociated adult mouse liver cells posses a (Ca-2+ + Mg-2+)-stimulated ATPase (EC 3.6.1.3) activity. 2. Enzyme activity is at a maximum with the addition of 0.3 mM Ca-2+ and 3 mM Mg-2+. 3. Using medium devoid of alkali metal ions (Ca-2+ + Mg-2+)-ATPase enzyme activity was observed with Km1 = 0.35 - 10-3 M at a substrate concentration of 1 mM or less and an apparent Km2 = 0.88 - 10-3 M at higher substrate concentrations. 4. In the presence of Na+ and 4 mM ATP, an increase in activity was seen, suggesting the presence of a (Ca-2+ + Mg-2+ + Na+)-activated ATPase. 5. In the presence of both Na+ and K+ the (Ca-2+ + Mg-2+)-dependent enzyme activity was further increased, indicating that a (Ca-2+ +Mg-2+ + K+)-stimulated ATPase may also be present.