Real-time quantitative polymerase chain reaction diagnosis of infectious posterior uveitis.

OBJECTIVE: To validate a real-time polymerase chain reaction (PCR) assay allowing rapid and sensitive detection and quantitation of 4 common infectious posterior uveitis pathogens. METHODS: A real-time PCR assay using previously validated primer sets for cytomegalovirus, herpes simplex virus, varicella-zoster virus, and Toxoplasma gondii was developed. A standard curve for quantitation of pathogen load was generated for each pathogen using SYBR Green I fluorescence detection. Ocular samples from patients with posterior uveitis and from negative control samples were assayed and compared with standards to identify pathogens and quantify infectious load. RESULTS: Sensitivity for detection of purified pathogen DNA by PCR was not reduced by application of the real-time method. Standard curves for the quantitation of pathogen loads showed sensitivity to fewer than 10 organisms for all pathogens. The technique was applied to 2 clinical problems. First, sensitivities of existing monoplex and multiplex PCR were compared by real-time PCR. No significant difference in sensitivity was observed between multiplex and monoplex techniques. Second, pathogen loads of vitreous specimens from patients previously diagnosed as having infectious posterior uveitis were calculated. Pathogen loads were found to be generally higher for patients with disease caused by varicella-zoster virus than those caused by cytomegalovirus or herpes simplex virus. CONCLUSIONS: Real-time PCR may be applied to infectious agents responsible for posterior uveitis. This technique will likely prove useful for the diagnosis of posterior uveitis as well as the linkage of pathogen to disease. CLINICAL RELEVANCE: Real-time PCR provides a rapid technique for quantitatively evaluating ocular samples for the presence of infectious pathogens.

Pleiotropic effects of cryptochromes 1 and 2 on free-running and light-entrained murine circadian rhythms.

Cryptochromes function in both light entrainment of circadian rhythms and in a peripheral circadian clock mechanism in Drosophila. Mice have two closely related cryptochrome genes (mCry1 and mCry2). To further understand the roles of mammalian cryptochromes, we bred mice to carry all possible combinations of wild-type and cryptochrome knockout alleles, and tested these mice for free-running and entrained circadian rhythmicity. We find that a single wild-type copy of mCry1, but not mCry2, is sufficient for free running circadian rhythmicity; however, these mice show markedly variable free-running periods. Two wild-type copies of either mCry1 or mCry2 are sufficient to establish a stable free-running clock. A subset of mCry1-/mCry-; mCry2-/mCry2- mice have a diurnal activity preference, suggesting that cryptochromes function in light-dependent behavioral masking. We also analyzed mice lacking both cryptochromes and carrying the homozygous rd retinal degeneration mutation. These mice have markedly depressed behavioral photoresponses in light-dark conditions, despite having an anatomically intact retinohypothalamic tract and normal expression of melanopsin. These results suggest that, similar to insect cryptochromes, mammalian cryptochromes function pleiotropically in both circadian rhythm generation and in photic entrainment and behavioral responses.

Influence of the period-dependent circadian clock on diurnal, circadian, and aperiodic gene expression in Drosophila melanogaster.

We measured daily gene expression in heads of control and period mutant Drosophila by using oligonucleotide microarrays. In control flies, 72 genes showed diurnal rhythms in light-dark cycles; 22 of these also oscillated in free-running conditions. The period gene significantly influenced the expression levels of over 600 nonoscillating transcripts. Expression levels of several hundred genes also differed significantly between control flies kept in light-dark versus constant darkness but differed minimally between per(01) flies kept in the same two conditions. Thus, the period-dependent circadian clock regulates only a limited set of rhythmically expressed transcripts. Unexpectedly, period regulates basal and light-regulated gene expression to a very broad extent.