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.     

Monocyte retention and migration in pulmonary inflammation. Requirement for neutrophils

The acute inflammatory process is characterized by an orderly progression of events; an initial phase of early neutrophil accumulation and a later phase of mononuclear cell (including monocyte) accumulation. The mechanisms which control the transition from one phase to the other are largely unknown. We present a rabbit model of C5 fragment (C5f)-induced lung inflammation in which purified radiolabeled peripheral blood neutrophils and monocytes were used as probes to monitor the retention and emigration of these leukocytes into well localized areas of inflammation. Neutrophil preparations (greater than 95% pure) were isolated by discontinuous plasma-Percoll density gradients, and monocyte preparations (greater than 91% pure) were isolated by counterflow cell elutriation, labeled with 111Indium-tropolonate, and intravenously infused into separate recipient animals. The monocytes circulated with a half-life of approximately 30 hours. The retention of labeled monocytes or neutrophils within the lung was monitored scintigraphically. C5f-induced monocyte lung retention was delayed 2 to 4 hours compared with neutrophil lung retention. Radiolabeled neutrophils were selectively retained in the area of C5f-induced inflammation (right cranial lung lobe, RCL) as early as 20 minutes after the induction of the inflammatory response, reached a maximum by 2 hours, and were not retained by 48 hours after C5f instillation. The signal inducing C5f-induced monocyte lung retention was shown to be transient. Monocytes were selectively retained in the RCL if the area of inflammation was induced 2 to 4 hours but not 15 minutes or 16 hours before their infusion. The time course of C5f-induced monocyte migration into the alveolar space determined by lavage analysis was delayed 2 to 3 hours compared with neutrophil migration. Neutrophils selectively migrated into the RCL 1 to 2 hours after the induction of the inflammatory response, reached a maximum by 4 hours, and had disappeared by 48 hours. Radiolabeled monocytes selectively migrated into the RCL 3 to 4 hours after the induction of the inflammatory response, reached a maximum by 4 hours, and remained present through 48 hours. The total number of labeled and unlabeled mononuclear cells present in the C5f-treated RCL lavage at 48 hours was significantly increased above controls. The signal for this monocyte migration (as for lung retention) was shown to be transient in that radiolabeled monocytes did not migrate when infused 16 hours after the induction of the inflammatory response. C5f did not induce monocyte lung retention nor monocyte migration into the alveolar space of animals rendered neutropenic.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Human cytomegalovirus. DNA synthesis and migration in infected cells studied autoradiographically

Autoradiographic studies were made of the synthesis and migration of DNA fofnd in the intranuclear and intracytoplasmic inclusions of human cytomegalovirus-infected cells. The DNA of the intranuclear inclusion, presumably viral DNA, forms de novo, beginning aboft 24 hofrs after infection and continuing until' at least 96 hofrs after infection. This DNA migrates to the intracytoplasmic inclusion and is the only sofrce of the DNA fofnd there.     

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.     

Anti-poliovirus IgG subclass antibodies in cytomegalovirus infected mice

A considerable strain difference was noted in BALB/c and C57BL/6 mice with regard to the impairment of antibody responses to poliovirus antigens in the course of infection with murine cytomegalovirus (MCMV): a long lasting reduction in antibody formation in BALB/c mice contrasted with an only moderate depression observed in C57BL/6 animals. Analysis of antibody classes and IgG subclasses revealed that anti-poliovirus VP1 antibodies in BALB/c mice were predominantly of the IgG3 subclass, a subclass most drastically affected by MCMV infection, while C57BL/6 mice produced antibodies of the IgM class and of IgG1 and IgG2 subclasses which were reduced to a lesser extent by the infection with MCMV. It is concluded that the strain difference observed may be explained on the basis of differences in the handling of poliovirus antigenic determinants by BALB/c and C57BL/6 mice.     

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.     

Monocyte activation in horses persistently infected with equine infectious anemia virus.

The monocytes of horses infected with equine infectious anemia virus were shown by their failure to migrate from capillary tubes and their increased adherence to erythrocytes to be activated.     

氟伐他汀对巨细胞病毒感染后载脂蛋白E基因敲除小鼠动脉硬化的影响

目的 探讨巨细胞病毒（Cytomegalovirus）与动脉粥样硬化（Atherosclerosis,AS）形成的相关性及氟伐他汀对其的影响.方法 以载脂蛋白E基因缺陷（Apolipoprotein-E Knockout,apoE-/-）小鼠为研究对象,给予低剂量小鼠巨细胞病毒（Murine Cytomegalovirus,MCMV）,观察动脉粥样硬化病变的变化.并在氟伐他汀的干预下,观察病毒感染对AS的影响,以探讨MCMV在动脉粥样硬化形成及发展过程中所起的作用.结果 研究显示在慢性潜伏感染阶段,MCMV感染明显加重apoE-/-小鼠动脉粥样硬化病变面积;小鼠动脉壁中没有MCMV mRNA的表达;血浆MCMV抗体水平、唾液腺MCMV DNA含量和动脉粥样硬化病变程度没有相关性.氟伐他汀干预后,MCMV感染组与未感染组在AS病变的面积、数目、内膜/中膜比值方面的显著性差异消失.结论 在慢性潜伏感染阶段,MCMV感染可以明显加重apoE-/-小鼠主动脉AS病变,但在血管壁局部并无MCMV活动性感染的证据.氟伐他汀可以改善MCMV感染后apoE-/-小鼠的AS病变进程.但这种作用并不是通过减少病毒量来实现的.     

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.     

Flow cytometric analysis of pulmonary lymphocytes from mice infected with respiratory syncytial virus.

BALB/c mice were infected intra-nasally with respiratory syncytial virus (RSV) and cells recovered from the lungs by single or repeated bronchoalveolar lavage (BAL). Single BAL gave enough cells (1 - 2 x 10(5) cells/mouse) for morphological study of Giemsa stained cytospin preparations, and allowed the same mouse lungs to be assayed for virus titre or processed for histology. In normal or sham-infected mice the majority of recovered cells were macrophages, with less than 5% lymphocytes. Following infection the proportion of lymphocytes increased to about 20% between days 10 and 16 and decreased thereafter. By contrast, histological changes in the lung peaked at day 7 and resolved by day 9 or 10. Repeated BAL yielded a higher proportion of lymphocytes and provided enough cells for flow cytometric study of cell surface markers. The results of two-colour stains with antibodies to L3T4 (CD4), Lyt2 (CD8), mouse CD3, Thy 1.2 and surface immunoglobulin (SIg) were studied by flow cytometry. Analysis of the small non-granular cells (lymphocytes) showed that fewer than 5% of recovered lymphocytes were SIg+ (B cells), while most bore T cell surface markers. Early during infection (day 3-6), 30-60% of lymphocytes were Thy 1.2-, T3-, CD4-/CD8- and SIg- CD8+ cells outnumbered CD4+ cells 2:1 or 4:1 from day 6 of infection. In conclusion, CD8+ T cells constitute the major subpopulation of lymphocytes recovered from the lungs of mice recovering from RSV infection. BAL provides a useful quantitative method of assessing the cellular immune response in the lung following RSV infection.     

Thymidine-kinase in cytomegalovirus infected cells

Summary In human diploid fibroblast LEP cells infected with AD 169 strain of human cytomegalovirus (CMV) a sharp increase of cytosol thymidine kinase activity was observed. The properties of the cytosol enzymes from infected and non-infected cells were compared. No significant differences between the enzymes from infected and control cells were observed in substrate specificity, pH dependence, thermostability and relative electrophoretic mobility. Human sera containing high titres of CMV complement-fixing antibodies did not neutralize the enzyme from infected cells. It is concluded from these results that the increase of cytosol thymidine-kinase activity in CMV-infected cells was due to an enhancement of cellular thymidine kinase.With 4 Figures     

DNA synthesis in mouse embryo fibroblast cells infected with murine cytomegalovirus.

The overall rate of DNA synthesis in mouse embryo fibroblast (MEF) cells infected with murine cytomegalovirus (MCMV) decreased immediately after infection, reaching its minimum at 10–12 hr postinfection (PI), and then increased gradually. CsCl equilibrium centrifugation analysis of synthesis of both host and viral DNA in MEF cells infected with MCMV revealed the following: (1) Host cell DNA synthesis was inhibited by more than 95% by 10–12 hr PI. (2) Viral DNA synthesis began at 10–12 hr PI, and by 22–24 hr PI the rate was slightly higher than the rate of host cell DNA synthesis observed at 0–2 hr PI. (3) Viral DNA synthesis occurred without the concurrent synthesis of any significant amount of host cell DNA, indicating that there was preferential viral DNA synthesis. The inhibition of host DNA synthesis occurred when cells were infected with either uv- or heat-inactivated MCMV. This occurred in the absence of both cytopathic effects (CPE) and the synthesis of viral-induced proteins. In these instances, the suppression of host DNA synthesis was observed only during the first 12 hr PI. At 12–14 hr PI, host DNA synthesis began to increase, and finally reached or surpassed the level seen in mock-infected cells. Since the suppression of the host DNA synthesis occurred in the absence of viral-induced protein synthesis, the possibility that the suppression is caused by one or more of the structural proteins of the infecting viral particles is discussed.     

Human cytomegalovirus. Assay by counting infected cells

An assay method for human cytomegalovirus is described that is based on counts of infected embryonic human skin and muscle fibroblastic cells on coverslips inoculated with 0.1 ml of diluted virus suspension. Counts made 48 hours after inoculation, when the preparations are fixed and stained, include all initially infected cells. Counts of infected cells are linearly proportional to the inoculum dilution. There is no significant loss of infected cells from the coverslips during preparation, and infected cells are randomly distributed on the coverslips.     

Polyamine biosynthesis in cells infected with different clinical isolates of human cytomegalovirus.

Previous work in this laboratory showed that polyamine biosynthesis was stimulated in fibroblasts following infection with the AD169 strain of human cytomegalovirus (HCMV) or with murine cytomegalovirus (MCMV) (Tyms et al: Biophysics Research Communications 86:312-318, 1979; Advances in Polyamine Research 4:507-517, 1983). Here we compare the affect of AD169 on polyamine production in infected fibroblasts with that of the unusual Colburn strain of HCMV. The Colburn virus is unusual in that it was isolated from a 7 year old boy with encephalitis and molecular studies indicated the virus was simian like (Huang et al: Journal of Virology 26:718-723, 1978). As a consequence of CMV infection a two to ten fold increase in the spermine content of fibroblast cells is observed. Radiolabel transfer experiments show that spermine is synthesized throughout virus infection. Indeed, spermidine and spermine are specifically incorporated into the purified virions of the AD169 and Colburn strains of HCMV. Furthermore, polyamine biosynthesis is stimulated in fibroblast cells infected with a number of low passage clinical isolates of HCMV. Inhibition of polyamine biosynthesis in HCMV infection may provide a specific and novel target for antiviral chemotherapy.     

Acute cytomegalovirus infections in leukemic mice.

Mice infected with 2 x 10(3) plaque-forming units of mouse cytomegalovirus (MCMV) 3 days after receiving 300 to 400 spleen focus-forming units of Friend leukemia virus developed a more severe MCMV infection than did normal animals. Increased severity was demonstrated by the increased amounts of MCMV recoverable from the salivary glands of leukemic mice 1 to 5 weeks postinfection. In addition, the difference in the number of virus isolations from the kidneys, spleens, livers, and lungs of animals (74 to 120) coinfected with MCMV and Friend leukemia virus compared with animals (49 of 120) infected with MCMV alone was significant (P less than 0.01). Both the 50% lethal dose and 50% infectious dose of MCMV in leukemic mice were lower than in normal animals. MCMV and Friend leukemia virus appear to interact by suppressing the ability of infected spleen cells to respond to mitogen-induced stimulation. The observations of increased severity of MCMV infections in leukemic mice closely parallel the situation observed in human leukemia patients who are at an increased risk of disease due to human cytomegalovirus infections. This mouse model may be useful in assessing the effect of antiviral (cytomegalovirus) therapy.     

Chemokine response in mice infected with Mycobacterium tuberculosis.

We show here that infection of murine macrophages with various strains of Mycobacterium tuberculosis induces the rapid in vitro expression of genes encoding chemokines macrophage inflammatory protein 1 alpha and macrophage inflammatory protein 2, which recruit neutrophils to sites of infection, and macrophage-recruiting chemokines 10-kDa, interferon-inducible protein (IP-10) and macrophage chemotactic protein 1. Three strains of M. tuberculosis, Erdman and the clinical isolates CSU 22 and CSU 46, induced similar levels of secretion of macrophage chemotactic protein 1 from infected macrophage monolayers; however, the Erdman strain failed to induce levels of secretion of tumor necrosis factor alpha similar to those induced by either CSU 22 or CSU 46. Using a low-dose aerosol infection model, we also found that while the Erdman strain induced negligible increases in chemokine mRNA levels in the lungs, infection with either CSU 22 or CSU 46 resulted in greater levels of mRNA production for all four chemokines tested. The growth of these strains in the lungs was, however, equally well contained by acquired host immunity. These data allow us to hypothesize that the chemokine response in the lungs probably does not control the protective granulomatous response and that perhaps other T-cell- or macrophage-associated cytokines such as tumor necrosis factor alpha or interleukin 12 may be involved in this process.     

人巨细胞病毒感染胎鼠大脑皮质神经元显微和超微结构观察

目的：研究人巨细胞病毒（HCMV）经小鼠胎盘垂直传播致胎鼠大脑皮质神经元病理改变的显微和超微结构特征。方法：在8－12周龄Balb/c雌雄小鼠腹腔内接种HCMV后,交配,特孕鼠临产时剖腹取出10份胎鼠双侧大脑皮质,用光镜和透射电射观察了神经元的显微和超微结构,同时进行了病毒分离。结果：光镜下可见胎脑微血管扩张充血,神经元数目明显减少,残存的神经元缺血性改变,间质水肿;可见到软化灶及受染神经元核内及胞质内HCMV特征性哮碱性和哮酸性包涵体,电镜下可见受染神经元核仁消失,胞质内线粒体,内质网和高尔基复合体等重要细胞器结构严重破坏呈空泡状或溶解;并找到人类疱疹病毒样颗粒。病毒分离阳性,在同时设置的正常对照组则无上述阳性发现。结论：HCMV能通过小鼠胎盘感染子代胎鼠大脑皮质神经元,并导致神经元结构破坏死亡。