Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy.

OBJECTIVE: To identify stage-specific induction of molecular pathology pathways in Duchenne muscular dystrophy (DMD). METHODS: We performed mRNA profiling using muscles from fetopsies, infants (aged 8 to 10 months), and symptomatic patients (aged 5 to 12 years) with DMD, and age- and sex-matched controls. We performed immunohistochemistry to determine changes at the protein level and protein localization. RESULTS: Activated tissue dendritic cells, expression of toll-like receptor 7, and strong induction of nuclear factor-kappaB pathways occurred soon after birth in DMD muscle. Two muscle wasting pathways, atrogin-1 and myostatin, were not induced at any stage of the disease. Normal muscle showed accumulation of glycolytic and oxidative metabolism capacity with increased age, but this accumulation failed in DMD. The transforming growth factor (TGF)-beta pathway was strongly induced in symptomatic patients, with expression of TGFbeta type II receptor and apoptosis signal-regulating kinase 1 proteins on subsets of mature DMD myofibers. CONCLUSIONS: Our data show stage-specific remodeling of human dystrophin-deficient muscle, with inflammatory pathways predominating in the presymptomatic stages and acute activation of TGFbeta and failure of metabolic pathways later in the disease.

Chlamydia pneumoniae exacerbates aortic inflammatory foci caused by murine cytomegalovirus infection in normocholesterolemic mice.

Inflammatory foci induced by murine cytomegalovirus infection in normocholesterolemic mice were present temporarily in the aortic wall, but some of these foci developed into advanced lesions that persisted late after infection. The early foci induced by virus infection were significantly exacerbated following a single inoculation with Chlamydia pneumoniae.

Immunoenhancement and suppression induced by adenovirus in chicken.

Chickens injected intravenously (i.v.) with human adenovirus type 6 (Ad6) reveal a 2-17-fold increase in the number of plaque-forming cells producing antibody (Ab) against sheep red blood cells (SRBC) 2-6 days after virus infection. Further, polyclonal B-cell activation has been demonstrated by the quantitation of immunoglobulin-producing cells (IgPC) and cells producing immunoglobulin (Ig) of IgM isotype (IgPC mu) in the spleen of chicken inoculated with Ad6. Ad6 infection in chicken results in immunosuppression against SRBC when this unrelated antigen is given after virus infection. It seems that coincidence occurs between the B-cell mitogenic activation and the immunosuppression caused by Ad6, as the most pronounced change in both activities appears on the fourth day following virus infection. These findings suggest that the B-cell mitogenicity of the virus contributes to the impairment of the humoral immune response to SRBC.

Impaired interleukin 2 production by spleen cells from mice infected with human adenovirus.

Spleen cells from mice infected with human adenovirus type 6 (Ad6) showed defective interleukin 2 (IL2) production 3-5 days after the infection. The response of spleen cells to exogenous IL 2 was also deficient. The impaired capacity of concanavalin A--(Con A)-activated spleen cells from Ad6--infected mice to utilize IL 2 seemed to be related to the depressed capacity of the infected splenocytes to express IL 2 receptors. The immunologic dysfunction following infection with Ad6 may be a consequence of deficiencies in both the elaboration of and response to IL 2.

The role of interferon in the adenovirus-induced augmentation of granulocyte-mediated cytotoxicity in chicken.

The effects of human adenoviruses on the granulocyte-mediated natural cytotoxicity of chicken leukocytes were investigated. A significant, but transient augmentation of granulocyte cytotoxicity was observed 24 h after virus injection, followed by a relatively long period of its suppression. A good correlation was found between the augmented cytotoxicity and interferon induction. The interferon-inducing capacity of adenovirus type 6 and type 12 in vitro similarly ran parallel with their ability to stimulate granulocyte-mediated cytotoxicity. An adenovirus-induced elevation of cytotoxicity was not observed when IFN production was inhibited by pretreatment of the leukocytes with monoclonal antibody specific for bursal cells and monocytes. In addition, anti-IFN antibody abrogated the stimulation of cytotoxicity as well. During the in vitro experiments in which granulocyte-specific monoclonal antibody was applied, evidence was found that the effector cell activity is associated with the granulocytes. These results suggest that both the in vitro and the in vivo adenovirus-induced augmentation of granulocyte-mediated cytotoxicity is due to the IFN-inducing capacity of the virus. In chickens, the rapid augmentation of the granulocyte cytotoxicity may be important in the acute stage of infection, increasing the resistance to the virus in question and also to bacterial infections.

Involvement of prostaglandins in immunosuppression caused by adenovirus in mice.

The effect of intraperitoneally (i.p.) inoculated human adenovirus type 6 (Ad6) was tested for humoral immune response against sheep red blood cells (SRBC) in normal and indomethacin-treated mice, with the aim to elucidate the mode of the virus action in immunosuppression. The results indicate that inhibition of prostaglandin synthesis slightly influences the immunosuppressive effect of the virus. It is very likely that also other mechanisms are involved in the immunosuppression observed.

The role of macrophages in adenovirus-induced immunosuppression in mice.

A decreased humoral immune response to sheep red blood cells (SRBC) was demonstrated in CFLP mice inoculated with human adenovirus type 6 (Ad6 virus). The suppressive effect depended on the inoculation sites of the virus and SRBC. Inoculation by the same route, i.e. intraperitoneally (i.p.), resulted in a significant decrease of the humoral immune response to SRBC. When administering the virus and SRBC at different sites, no inhibitory effect could be detected. Immunosuppression was prevented by pretreatment of mice with silica depressing the macrophage function. The results indicate that peritoneal macrophages play an important role in the immunosuppression observed.

[Molecular aspects of interferon induction by adenoviruses]. / Molekuliarnye aspekty induktsii interferona adenovirusami.

The capacity of adenovirus to induce interferon in the infected cells was studied. The examined adenovirus strains of early types were grouped in 2 groups according to their capacity to induce interferon and to the sensitivity of the infecting and interferon-inducing activity to UV-irradiation. A common property of adenoviruses, potent interferon inducers, is their high sensitivity to UV-irradiation.

Effect of interferon on antibody-dependent cellular cytotoxicity (ADCC) in chickens.

The influence of chicken leukocyte interferon (IFN) on the antibody-dependent cellular cytotoxicity (ADCC) of peripheral blood mononuclear cells of chickens has been investigated in vivo and in vitro. The cytotoxic effect was studied on sheep red blood cells (SRBC) coated with chicken anti-SRBC serum. Cytotoxicity was estimated using a 51Cr release assay system. A single intravenous injection of crude IFN led to an enhanced ADCC 6 to 12 h after the IFN injection, whereas by 24 h the activity had decreased. The ability of IFN to augment the cytotoxicity was not lost after phagocytic cell depletion. In vitro, the ADCC was boosted within 1-18 h by IFN, in a dose-dependent manner. We assume that the augmenting agent in vitro is IFN itself, as purified IFN had a similar activity, but mock IFN and trypsin-treated IFN showed no enhancing effect.

Different interferon-inducing ability of human adenovirus types in chick embryo cells.

Human adenovirus (Ad) types differ in their ability to induce interferon (IFN) in chick cells. Of 12 types investigated, Ad8, Ad12, Ad 18 and Ad 31 proved to be more effective IFN inducers than Ad2, Ad3, Ad4, Ad5, Ad6, Ad7, Ad15 and Ad19. Ultraviolet (UV) irradiation decreased the IFN-inducing ability of the more effective inducers only, indicating that transcription of viral DNA might play a role in IFN induction by these types. DNAs isolated from Ad2, Ad5 and Ad12 alike induced low amounts of IFN in chick cells. The IFN-inducing capacity of phage DNA was similar to that of adenovirus DNA, but induction by non-viral DNA (prokaryotic and eukaryotic) did not result in detectable IFN production. It is assumed that viral DNA and virus particles promote IFN production in different ways. Probably the viral component responsible for IFN induction by the effective Ad types differs from those having lower IFN-inducing ability.

Effect of human adenovirus type 6 on the primary immune response in mice.

CFLP and BALB/c mice inoculated intraperitoneally with large doses of adenovirus type 6 (Ad6) showed a decreased humoral immune response to sheep red blood cells (SRBC) and circulating interferon was detected in their serum. The timing of infection was critical. Infection of mice 3--11 days before SRBC administration led to depression of the 19 S haemolytic plaque forming cell (HPFC) response in the spleen. When mice were given Ad6 and SRBC simultaneously on Ad6 14 days before or 1 day after SRBC, there was no decrease in the number of HPFC. The suppressive effect was dependent on the dose of virus and antigen. Heat and UV treatment completely abolished the immunosuppressive effect of the virus, suggesting that a great amount of infectious adenovirus is needed to induce immunosuppression in mice.

Interferon induction by adenoviruses.

All human, simian, bovine and avian adenovirus types tested so far and the canine hepatitis virus induce interferon production in chick cells. This finding indicated this property to be characteristic for viruses belonging to the adenovirus group. Trypsin treatment, which had no effect upon the infectivity, diminished or eliminated the interferon-inducing abilities of crude adenoviruses, and thus the need for a trypsin-sensitive protein in interferon induction was suggested. T antigen and interferon were formed simultaneously in chick embryo fibroblast cells infected with human adenovirus type 12, and therefore the adenovirus-specific T antigen was resistant to the action of endogenous interferon synthetized by the same cells. In chicks inoculated with human types, the appearance of interferon was biphasic: an 'early' and a 'late' interferon could be demonstrated with maximum titre 4 and 10 hr, respectively, after virus infection. In chicks infected with adenoviruses, first interferon production and then a decreased primary immune response to sheep red blood cells was observed. It was assumed that in adenovirus-infected chicks the interferon produced by viral stimulus resulted in a transient immunosuppression.

The effect of interferon on chicken spleen lymphocytes.

The inhibitory activity of chicken interferon has been investigated on phytohemagglutinin (PHA)-induced DNA synthesis of chicken spleen lymphocytes inoculated with type 12 adenovirus and was purified by selective adsorption on Na-Al-silicate. As a control, the culture fluid of chicken leukocytes purified in the same manner was used. In some instances the control preparation also showed a slight inhibitory effect. To prove that the immunosuppressive activity of our interferon preparation was indeed due to its interferon content both the interferon and the control preparation were purified further by chromatography on Sephadex G 100 superfine gel. Only the fractions with antiviral activity of interferon preparation inhibited the PHA-response of the lymphocytes. The fractions with the same molecular weight of control preparation were without effect.

Effect of human adenoviruses on the response of chickens to sheep erythrocytes.

Human adenovirus types 6, 8, and 12 were immunosuppressive in chickens. A single intravenous injection of adenoviruses markedly depressed the 19S hemolytic plaque-forming cell response in the spleen to the immunization with sheep red blood cells. Hemagglutinin production was also decreased in adenovirus type 6-infected chickens. Adenoviruses caused a transient immunosuppression in chickens which could be detected 2 to 3 days after the virus infection, and no depressive effect was found 16 to 20 days after virus injection. The possible mechanism of immunosuppression observed is discussed.