Alopecia in mice infected with murine cytomegalovirus (MCMV).

When mouse cytomegalovirus was injected subcutaneously into 4-12 day old CDI mice there was infection of dermal cells and the dermal papillae of hair follicles. Infected cells were never seen in the epidermis nor in the epithelium of hair follicles. When larger doses of virus (5 X 10(4) pfu) were given, dermal infection led to gross necrosis of the skin, ulceration, scabbing and healing with alopecia. Smaller doses (10(4) pfu) did not cause gross necrosis but damage to follicles resulted in alopecia or sparse hair growth. Skin lesions were not seen after infection of 4-8 week old mice, even when the inoculated skin area had been epilated, or when hyaluronidase was mixed with the virus inoculum. These experiments show that cytomegalovirus, in contrast to herpes simplex and varicella-zoster viruses, infects dermal but not epidermal cells, and that dermal tropism is age-restricted.     

Alopecia in mice infected with murine cytomegalovirus (MCMV)

When mouse cytomegalovirus was injected subcutaneously into 4-12 day old CDI mice there was infection of dermal cells and the dermal papillae of hair follicles. Infected cells were never seen in the epidermis nor in the epithelium of hair follicles. When larger doses of virus (5 X 10(4) pfu) were given, dermal infection led to gross necrosis of the skin, ulceration, scabbing and healing with alopecia. Smaller doses (10(4) pfu) did not cause gross necrosis but damage to follicles resulted in alopecia or sparse hair growth. Skin lesions were not seen after infection of 4-8 week old mice, even when the inoculated skin area had been epilated, or when hyaluronidase was mixed with the virus inoculum. These experiments show that cytomegalovirus, in contrast to herpes simplex and varicella-zoster viruses, infects dermal but not epidermal cells, and that dermal tropism is age-restricted.     

Inflammatory and immunological responses to murine cytomegalovirus in resistant CBA mice

Summary The resistance of CBA mice to MCMV is associated with the resistance of H-2^k cells to infection in vitro, high NK and virus-specific DTH responses, and minimal accumulation of cytostatic peritoneal macrophages. This study investigates the functional capacity of lymphoid cells from infected CBA mice, using this strain as a model for successful control of CMV. Splenic viral replication was high 1–3 days p.i. and cell numbers were depressed, but T and B cells frequencies were maintained. The remaining spleen cells were hyporesponsive in culture and accessory cell function was marginally deficient. By 7 days p.i., virus titres declined, responsiveness increased and the residual defect was associated with cytostatic macrophages. The lymph nodes did not atrophy, exhibited low levels of viral replication and proliferative capacity was retained. The beige mutation did not affect the local response to intraperitoneal infection, but spleen cell numbers and responsiveness declined progressively. The results suggest the spleen may contribute to CMV disease by the replication of virus in susceptible cells until the NK response reduces virus titres and hence limits acute virus-induced immunosuppression. Macrophage-mediated suppression persisted in the spleen during recovery so clonal expansion of protective virus-primed T cells may occur predominantly in the lymph nodes.     

Restricted infection of murine cytomegalovirus (MCMV) in neonatal mice with MCMV-induced sensorineural hearing loss

BackgroundCongenital infection with human Cytomegalovirus (HCMV) is known to be a causative agent of sensorineural hearing loss (SNHL).ObjectivesTo clarify the nongenetic etiology of SNHL by identifying the Cytomegalovirus (CMV)-infected region in the cochleae.Study designWe established an animal model of SNHL by injecting neonatal Balb/c mice with intracerebral murine Cytomegalovirus (MCMV) within 24 h after delivery.ResultsAt 3 weeks of age, unilateral and bilateral SNHL were observed in 24% (5/21) and 29% (6/21) of the mice, respectively. SNHL thereafter progressed, with 79% of mice developing bilateral SNHL by 6 weeks of age. MCMV antigens and DNA were detected in the spiral ganglion, and cells surrounding the meninges and scala tympani at 1 week of age. However, both MCMV antigens and DNA had completely disappeared by 2 weeks of age. It is possible that the MCMV reached the spiral ganglion via cerebrospinal fluid as the result of meningitis, as the stria vascularis was found to be MCMV antigen negative. Myosin VI expression in the outer hair cells was lost at 3 weeks of age. MCMV and myosin VI expression disappeared before and during SNHL progression, respectively.ConclusionsThere was a definite lag time between the period in which MCMV antigens/DNA-positive cells were observed and that in which SNHL developed and myosin VI-negative hair cells were observed. Further study is needed to explore the role of MCMV in the loss of myosin VI expression in the outer hair cells.     

Modulation of T-cell subsets in asthmatic children by THF, a thymic hormone

Induction of theophylline-sensitive T-suppressor cells with THF, a thymic hormone, resulted in elevation of the low levels of these lymphocytes in non-treated asthmatic children. Evaluation, however, by monoclonal OKT8 antibodies did not show changes in the levels of the OKT8+ cells after THF incubation. In the treated asthmatic children, normal levels of theophylline-sensitive T-suppressor cells as well as OKT8+ cells were found. These normal levels were not changed with THF after in vitro incubation.     

The role of macrophages in mice infected with murine cytomegalovirus.

Quantitative studies were made of the infection of mouse peritoneal macrophages in vitro by cytomegalovirus, using virus assays and immunofluorescence. The efficiency of infection was low. Broth-induced peritoneal macrophages were about four times more resistant to infection than unstimulated macrophages and it was even more difficult to infect activated macrophages taken from mice 6 days after intravenous infection. Peritoneal macrophages (unstimulated) were infected at least 15 times more readily by tissue culture-passed (attenuated) virus than by salivary gland (virulent) virus, but macrophages prevented the spread of tissue culture virus to underlying susceptible mouse embryo fibroblasts, whereas they did so much less effectively with virulent salivary gland virus. The pathogenesis of infection was studied in intact mice by immunofluorescence, and the observations paralleled the in vitro findings. When large doses of salivary gland virus were injected intravenously, infected Kupffer cells (liver macrophages) were occasionally seen and the inoculated virus directly infected large numbers of hepatic cells. In similar experiments with tissue culture-passed virus, there was initial infection of occasional Kupffer cells, which only rarely gave rise to infected hepatic cells. Differences in the extend of Kupffer cell infection by the two strains of virus were not detected in these experiments. Salivary gland virus also usually failed to infect splenic or lymph node macrophages. Occasional infected mononuclear cells were seen in the blood, lung and bone marrow, but were not identified. Infected cells were very rarely seen in the thymus, even in suckling mice.     

Lymphoid cell necrosis, thymic atrophy, and growth retardation in newborn mice inoculated with murine cytomegalovirus.

During studies on the effect of murine cytomegalovirus on the developing retina, virus was inoculated into the eyes of newborn Swiss mice, and the animals were sacrificed at various times thereafter. Controls consisted of mice inoculated with ultraviolet-inactivated murine cytomegalovirus and uninjected mice. Marked lymphoid cell necrosis, thymic atrophy, pronounced growth retardation, bacteremia, and death occurred in the animals inoculated with live virus. this virus-induced injury resulted in a marked depletion of lymphocytes in the subcapsular and cortical areas of the thymus as well as in the spleen, lymph nodes, and Peyer's patches. Areas of necrosis with viral inclusions were present at the site of inoculation and in various other organs including the spleen and bone marrow. Since growth retardation has been associated with thymic atrophy due to other causes, the observed abnormal physical development in the present study was interpreted as a sequel to the thymic injury. An implication of this study is that some human infants with concomitant immune deficiency and viral infection may have a primary viral disease with resultant secondary lymphoid tissue alterations, rather than a thymic disorder with a subsequent viral infection.     

Neutralization of different strains of murine cytomegalovirus (MCMV)-effect ofin vitro passage

Summary Two strains of mouse cytomegalovirus (MCMV) propagated in mouse salivary glands could be distinguished in neutralization tests.Cell (mouse embryo fibroblast)-passaged virus of either strain was generally 10–20 times easier to neutralize than virus produced in mouse salivary glands. Evidence was obtained that mouse immunoglobulin was attached to virus particles in salivary gland preparations. First, antibody to mouse immunoglobulin inactivated salivary gland virus but not cell culture-grown virus. Second, virus obtained from mice early after infection, before antibody production, was as easy to neutralize as cell culture-grown virus.The attached immunoglobulins were CMV-specific, non-neutralizing, and interfered with the action of neutralizing antibody. Non-neutralizing antibodies that bind to infectious virus particles are present in mouse serum at the time salyvary glands are harvested, 3 weeks after infection.With 1 Figure     

Production of interferon and serum hyporeactivity factor in mice infected with murine cytomegalovirus.

Mice injected intraperitoneally with murine cytomegalovirus produced as many as 1,000 U of serum interferon. The response appeared biphasic, with maximum titers in the first phase detectable from 2 through 4 days after infection. A second phase peaked 10 days after infection. By carboxyhexyl-Sepharose affinity chromatography, the serum interferon behaved like lymphocyte interferon. The infected mice also produced substantial quantities of serum hyporeactivity factor (D.A. Stringfellow, E.R. Kern, D.K. Kelsey, and L.A. Glasgow, J. Infect. Dis. 135:540-551, 1977), although always in the presence of interferon. This factor was separated from the serum interferon by concanavalin A-Sepharose affinity chromatography.     

Detection of murine cytomegalovirus (MCMV) DNA in skin using the polymerase chain reaction (PCR)

We used the polymerase chain reaction (PCR) and primers for the immediate early gene of murine cytomegalovirus (MCMV) to detect MCMV DNA in skin harvested from mice during acute infection. MCMV DNA was also detected in DNA extracted from spleen and salivary gland of MCMV-infected mice, but not in the skin, salivary gland, or spleen of uninfected, seronegative mice. Detection of MCMV DNA in skin provides direct evidence that skin can serve as a vehicle for transmission of MCMV. This observation is relevant to humans, such as burn patients, who receive skin allografts that may be infected with cytomegalovirus.     

Increased weight loss with reduced viral replication in interleukin-10 knock-out mice infected with murine cytomegalovirus

The anti-inflammatory cytokine interleukin (IL)-10 plays an important role in the regulation of host-immune responses. Here we studied the role IL-10 plays in host responses to cytomegalovirus (CMV) infection. We demonstrate that manifestations of murine CMV (MCMV) disease are more severe in IL-10 knock-out mice, despite significantly reduced levels of viral replication. Cytokine analysis of serum revealed increased levels of interferon (IFN)-gamma, monocyte chemotactic protein 1 (MCP-1) and IL-6, all of which are potent stimulators of inflammatory responses. Depletion of IFN-gamma by monoclonal antibodies in IL-10 knock-out mice failed to improve the physical condition of the mice, while increasing viral replication. In contrast, serum levels of IL-6 in the knock-out animals were unaffected by IFN-gamma depletion and remained significantly elevated early in the course of infection. These data suggest that increased weight loss observed in IL-10 knock-out mice may be attributed to the uncontrolled production of proinflammatory cytokines, including IL-6.     

Characterization of a major virion envelope glycoprotein complex of murine cytomegalovirus and its immunological cross-reactivity with human cytomegalovirus

Three glycoproteins on the murine cytomegalovirus (MCMV) virion with apparent molecular weights of 150K (gp 150), 105K (gp 105), and 52K (gp52) were immunoprecipitated by two monoclonal antibodies (MAbs) 8G5.12A and 2E.12A. However, only 8G5.12A was able to neutralize MCMV infectivity in the presence of complement. The accessibility of these three glycoproteins to radiolabeling by surface-iodination reactions suggested that they were exposed on the surface of the virion. Western blot analysis of the three glycoproteins showed that gp150 shared antigenic determinants with gp105 and gp52. Briefly, the MAb 8G5.12A reacted with gp150 and gp105, whereas the MAb 2E8.12A reacted with gp150 and gp52. A third MAb 3H2.12A was also found to be reactive with gp150 and gp105 in Western blots, but was unable to immunoprecipitate these glycoproteins. Data from pluse-chase experiments suggested that all three virion glycoproteins were synthesized from a common 128K precursor, providing a partial explanation of their antigenic relatedness. Furthermore, we have demonstrated the presence of high-molecular-weight complexes formed by disulfide bonding between gp150, gp105, and gp52. Lastly, the MAb 8G5.12A was able to immunoprecipitate 84K and 99-110K glycoproteins from human CMV-infected WI-38 cells, demonstrating that conserved determinants exist between murine and human CMV envelope glycoproteins.     

Feedback inhibition of thymic secretory activity in mice treated by the thymic extract TP-1 (thymostimulin).

The ultrastructural changes occurring in the medullary epithelium of the thymus of young mice, as a result of repeated injections of thymic extract, TP-1 (thymostimulin) was investigated. After daily injection of TP-1 for 3 weeks, no changes in thymus architecture could be observed by light microscopy. However, by electron microscopy, specific changes were noticed in the epithelial cells. The secretory granules became dilated and engorged; diameter of granules in normal control thymus was approximately 200-250 nm, but reached 1000 nm in treated mice. Degenerative changes appeared in some of these granules, including myelin bodies, distorted configuration and fat droplets. Signs of involution of whole cells and presence of cellular debri within macrophages were observed. Acid phosphatase staining disclosed many lysosomes containing ingested granules. No such findings were observed in control untreated mice, or in mice treated by a heart extract similarly prepared to TP-1. All these findings can be taken as ultrastructural evidence for feedback inhibition of thymic secretory activity, in analogy to the changes occurring other feedback inhibited, peptide hormone secreting glands. The data indicate that (i) the thymus respond to feedback inhibitory stimuli, as other endocrine glands do; (ii)TP-1, the thymic extract under study, contains a physiologically significant thymic hormone, which, when introduced in high doses can exert specific feedback inhibition. This can be taken as an additional, new criterion for the definition of thymic hormones.     

Interleukin-10 repletion suppresses pro-inflammatory cytokines and decreases liver pathology without altering viral replication in murine cytomegalovirus (MCMV)-infected IL-10 knockout mice

Objective and design  

To determine the role of interleukin-10 (IL-10) in protecting against the deleterious pro-inflammatory cytokine response to murine cytomegalovirus (MCMV), we studied the impact of IL-10 repletion in MCMV-infected IL-10 knockout (KO) mice.     

Suppression of immunological responses in mice by treatment with amphotericin B.

The polyene antibiotic amphotericin B (AmB) caused a marked suppression of the cell-mediated immune response in mice. Similar treatment did not effect the humoral antibody response. The immunosuppressive property of the drug was related to its ability to inhibit the manifestation rather than the induction phase of the delayed-type hypersensitivity response. In vitro AmB suppressed mitogen-induced lymphocyte proliferation. The drug seemed to act at the proliferative phase of the response. Results presented show that the T cell response was much more sensitive to the action of AmB than was the B cell response. During AmB chemotherapy consideration must be given to the immunosuppressive properties of this drug.     

Immune responses in mice with murine leprosy

Mice infected with Mycobacterium lepraemurium showed marked histological changes in the thymus and lymph nodes. In the thymus there was a progressive depletion of lymphoid cells and replacement by macrophages, many of which contained bacilli in the advanced stages of the disease. In lymph nodes there was depletion of paracortical immunoblasts and accumulation of macrophages. Infected mice showed impaired cell-mediated immune responses, including delayed rejection of skin grafts and depression or absence of contact sensitivity. Humoral immune responses to bovine serum albumin and sheep erythrocytes in infected mice were normal. The defect in cell-mediated immune responses is thought to be secondary to the massive infection, and is discussed in relation to impaired immune responses in human lepromatous leprosy.     

Suppression of contact sensitivity by a plastic adherent T-cell,induced in mice infected with Newcastle disease virus (NDV).

Previous studies have demonstrated that lymph node cells of mice skin-sensitized 24 h before are able to transfer contact sensitivity (CS) in naive recipients. These antigen presenting cells (APC) lose the ability to induce CS when donor mice are treated with the virus of Newcastle Disease (NDV) at the time of sensitization. In this paper we demonstrate that the cell capable of suppressing CS is a virus induced plastic adherent T-cell, which inhibits otherwise normal APC. In fact, the APC in infected mice are fully competent, as demonstrated by their ability to transfer CS, if the adherent T-cell population is removed by plastic adherence. Analysis shows that the CS suppressing adherent T-cells are Thy 1.2+, Lyt 1.1+ and I-J+ subset. The inhibition of CS by the NDV induced adherent T-cell is antigen non-specific and genetically restricted. We have also demonstrated that picrylated cells from NDV infected mice fail to trigger the release of non-specific inhibitor (nsINH) in the T-suppressor circuit. The effect of the adherent suppressor T-cell in that circuit was determined and the results indicate that the virus induced T-cell is able to suppress the release of nsINH by blocking the function of the APC.