The Role of Caspase-12 in Retinal Bystander Cell Death and Innate Immune Responses against MCMV Retinitis.

(1) Background: caspase-12 is activated during cytomegalovirus retinitis, although its role is presently unclear. (2) Methods: caspase-12-/- (KO) or caspase-12+/+ (WT) mice were immunosup eyes were analyzed by plaque assay, TUNEL assay, immunohistochemical staining, western blotting, and real-time PCR. (3) Results: increased retinitis and a more extensive virus spread were detected in the retina of infected eyes of KO mice compared to WT mice at day 14 p.i. Compared to MCMV injected WT eyes, mRNA levels of interferons α, ß and γ were significantly reduced in the neural retina of MCMV-infected KO eyes at day 14 p.i. Although similar numbers of MCMV infected cells, similar virus titers and similar numbers of TUNEL-staining cells were detected in injected eyes of both KO and WT mice at days 7 and 10 p.i., significantly lower amounts of cleaved caspase-3 and p53 protein were detected in infected eyes of KO mice at both time points. (4) Conclusions: caspase-12 contributes to caspase-3-dependent and independent retinal bystander cell death during MCMV retinitis and may also play an important role in innate immunity against virus infection of the retina.

Cytomegalovirus DNA polymerase activity and an 80 kDa-associated polypeptide: a potential diagnostic tool for CMV disease.

CMV has been reported to be associated with a DNA polymerase activity (DPA). In this communication its purification, characterization and potential diagnostic value were examined. CMV DNA polymerase was prepared from cell free supernatants of CMV (AD 169) infected cultures. Separation and purification of the enzyme was accomplished by column chromatography of the purified, lysed virus. CMV DPA was measured on an oligo (dT)-poly (dA) template primer. SDS-PAGE and western blot analysis under reducing conditions using an anti-CMV early antibody showed an 80 kDa protein band that was associated with the peak of polymerase activity. However, CMV isolates and CMV from urines from CMV retinitis patients immunoblotted by the same Ab revealed 140 kDa and 80 kDa bands under non-reducing and reducing conditions respectively, the latter was also associated with a 58 kDa band. The diagnostic value of the CMV associated DAP was tested using CMV positive urines. The latter demonstrated high PAA-sensitive DPA activity, compared to normal, HSV positive urines and urines from HBSAg positive patients. Taken collectively, these findings indicate the potential usefulness of CMV-associated DNA polymerase activity in the diagnosis and follow-up of patients with CMV-related illnesses.

Expression of inducible nitric oxide synthase in cytomegalovirus-infected glial cells of retinas from AIDS patients.

The inducible isoform of nitric oxide synthase has been detected in cytomegalovirus (CMV)-infected retinas from acquired immunodeficiency syndrome (AIDS) patients by immunohistochemistry and NADPH-diaphorase staining. Subsequent immunohistochemistry using antibodies against CMV antigens and glial fibrillary acidic protein (GFAP) demonstrated that inducible NOS was localized in CMV-infected glial cells, particularly Müller cells. These findings indicate that inducible NOS is expressed in vivo in the human retina as a result of viral infection, and suggest that high levels of NO production might be involved in CMV-induced retinitis.