Applications of the polymerase chain reaction to diagnosis of ophthalmic disease.

The polymerase chain reaction (PCR) is a powerful molecular biologic technique for the analysis of very small amounts of DNA. This technique has found increasing use in the past 10 years for the detection of pathogenic organisms associated with many forms of ocular inflammatory and infectious disease. PCR has shown utility in the diagnosis of viral uveitis, infectious endophthalmitis, and parasitic eye disease. The strengths and weaknesses of this diagnostic technique are discussed. Additionally, uses of PCR in linking known pathogens to disease, and to discovering novel pathogens, are addressed.

Validation of a diagnostic multiplex polymerase chain reaction assay for infectious posterior uveitis.

OBJECTIVE: To validate a multiplex polymerase chain reaction (PCR) assay capable of simultaneously screening vitreous biopsy specimens for a panel of common pathogens in posterior uveitis. METHODS: A multiplex PCR assay using novel primer sets for cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Toxoplasma gondii was developed. The sensitivity of the assay was determined for purified pathogen DNA. Twenty-one vitreous specimens from patients with posterior uveitis were tested by both multiplex and monoplex PCR. RESULTS: Fewer than 10 genomes of VZV and fewer than 100 genomes of HSV, CMV, and T gondii could be detected using the new primer sets. When used in multiplex, the assay lost less than 1 log of sensitivity. Monoplex PCR detected pathogen DNA in 18 of 21 patient samples; multiplex PCR detected pathogen DNA in 15 of the 18 samples positive by monoplex PCR. None of 10 negative control samples were positive for pathogen DNA. CONCLUSIONS: Multiplex PCR has adequate sensitivity to simultaneously screen a substantial differential diagnosis for posterior uveitis in a single reaction, without loss of specificity. This assay may reduce the time and cost involved in PCR-based molecular diagnostics of infectious pathogens. CLINICAL RELEVANCE: Mutiplex PCR may allow rapid diagnosis of infectious posterior uveitis.

Preservation of light signaling to the suprachiasmatic nucleus in vitamin A-deficient mice.

To investigate the role of retinal-based pigments (opsins) in circadian photoreception in mice, animals mutated in plasma retinol binding protein were placed on a vitamin A-free diet and tested for photic induction of gene expression in the suprachiasmatic nucleus. After 10 months on the vitamin A-free diet, the majority of mice contained no detectable retinal in their eyes. These mice demonstrated fully intact photic signaling to the suprachiasmatic nucleus as measured by acute mPer mRNA induction in the suprachiasmatic nucleus in response to bright or dim light. The data suggest that a non-opsin pigment is the primary circadian photoreceptor in the mouse.

Frontiers of polymerase chain reaction diagnostics for uveitis.

The polymerase chain reaction (PCR) is a powerful molecular biologic technique for the detection of pathogen DNA. Recent technical advances in the technique have broadened its scope considerably. Emerging applications of PCR in pathogen strain typing, rapid screening of differential diagnoses, and pathogen discovery are discussed.

Herpes simplex virus type 2 as a cause of acute retinal necrosis syndrome in young patients.

PURPOSE: To determine the causative virus in acute retinal necrosis (ARN) syndrome in a series of patients by calculation of modified Witmer coefficients. DESIGN: Noncomparative case series. PARTICIPANTS: Ten patients with ARN syndrome from four medical centers. METHODS: Aqueous samples, vitreous samples, or both were collected prospectively during surgery from patients with a clinical diagnosis of ARN syndrome. Serologic measures of intraocular and serum antibodies to potentially causative viruses were measured by enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES: Modified Witmer coefficients (immunoglobulin G and immunoglobulin A) for herpes simplex virus types 1 (HSV-1) and 2 (HSV-2), varicella zoster virus (VZV), and cytomegalovirus (CMV), as well as adenovirus type 2, were calculated from aqueous or vitreous samples, or both. RESULTS: Intraocular antibody measurements were strongly suggestive of a single diagnosis in 9 of 10 patients tested. Modified Witmer coefficients demonstrated intraocular antibody production to HSV in five patients and antibodies to VZV in four patients, and the measurement was inconclusive in one patient. No patients were positive for adenovirus or CMV. Strain-specific antibody titers demonstrated that all HSV-positive patients were reactive only to HSV-2. Herpes simplex virus type 2 was found predominantly in younger patients with ARN syndrome (mean age, 21.2 +/- 10 years; range, 17-39 years), whereas VZV was more commonly seen in older patients (mean age, 40.8 +/- 12.2 years; range, 29-58 years; P = 0.033). Immunoglobulin A testing confirmed immunoglobulin G testing in all patients examined. CONCLUSIONS: Although VZV is thought to be the most common cause of ARN syndrome, HSV-2 is an important cause of ARN syndrome, particularly in younger patients. Because infection with HSV-2 has important medical ramifications, these results suggest that determination of a causal agent should be considered in some cases of ARN syndrome.

Non-visual ocular photoreception.

At least six light-regulated phenomena are preserved in the eyes of retinally degenerate mice, including the entrainment of circadian rhythms, the gating of ocular immune response, and pupillary reactivity. Some of these phenomena have also been observed in blind human patients. These findings have prompted the search for a non-visual ocular phototransduction mechanism. Molecular genetic studies have identified several candidate genes for these effects. These include genes encoding novel ocular opsins, such as melanopsin, as well as potential flavin-based photopigments. Data linking these potential photoreceptors to these phenomena are discussed, and the clinical implications of these findings are explored.

Functional redundancy of cryptochromes and classical photoreceptors for nonvisual ocular photoreception in mice.

The daily light-dark (LD) cycle exerts a powerful influence on the temporal organization of behavior and physiology. Much of this influence is preserved in behaviorally blind retinally degenerate mice; the photoreceptors underlying this nonvisual phototransduction are unknown. The mammalian eye contains at least two classes of photoactive pigments, the vitamin A-based opsins and the vitamin B(2)-based cryptochromes. To genetically define the roles of these pigments in light modulation of behavior, we generated rd/rd;mCry1(-)/mCry1(-);mCry2(-)/mCry2(-) mutant mice lacking rods and most cones as well as both cryptochrome proteins. The response of the mutant mouse to photic input was analyzed at both behavioral and molecular levels. Behaviorally, mice lacking either classical photoreceptors or cryptochromes exhibited strongly rhythmic locomotor responses to 10 and 100 lux daily LD 12 h/12-h cycles; however, triple mutant mice carrying both cryptochrome and retinal degenerate mutations were nearly arrhythmic under both LD cycles and in constant darkness. At the molecular level, the light induction of c-fos transcription in the suprachiasmatic nucleus was markedly reduced in the triple mutant mouse compared with either rd/rd or cryptochrome mutant mice. These data indicate that classical opsins and cryptochromes serve functionally redundant roles in the transduction of light information to behavioral modulation and suggest a pleomorphic role for cryptochromes in both photoreception and central clock mechanism.

Guidelines for the use of immunosuppressive drugs in patients with ocular inflammatory disorders: recommendations of an expert panel.

PURPOSE: To provide recommendations for the use of immunosuppressive drugs in the treatment of patients with ocular inflammatory disorders. PARTICIPANTS: A 12-person panel of physicians with expertise in ophthalmologic, pediatric, and rheumatologic disease, in research, and in the use of immunosuppressive drugs in patient care. EVIDENCE: Published clinical study results. Recommendations were rated according to the quality and strength of available evidence. PROCESS: The panel was convened in September of 1999 and met regularly through May 2000. Subgroups of the panel summarized and presented available information on specific topics to the full panel; recommendations and ratings were determined by group consensus. CONCLUSIONS: Although corticosteroids represent one of the mainstays in the management of patients with ocular inflammation, in many patients, the severity of the disease, the presence of corticosteroid side effects, or the requirement for doses of systemic corticosteroids highly likely to result in corticosteroid complications supports the rationale for immunosuppressive drugs (for example, antimetabolites, T-cell inhibitors, and alkylating agents) being used in the management of these patients. Because of the potential for side effects, treatment must be individualized and regular monitoring performed. With careful use of immunosuppressive drugs for treatment of ocular inflammatory disorders, many patients will benefit from them either with better control of the ocular inflammation or with a decrease in corticosteroid side effects.

Circadian rhythms: eyes of the clock.

The daily light-dark cycle synchronizes the internal circadian clock with the outside world. Blind organisms maintain this light-induced entrainment, suggesting the existence of a non-visual phototransduction pathway. The photoreceptor is unknown, but several intriguing candidates have recently come to light.

Neural network computer program to determine photorefractive keratectomy nomograms.

PURPOSE: To evaluate a commercially available neural network program for calculation of photorefractive keratectomy treatment nomograms. SETTING: University referral refractive surgery clinic. METHODS: PRK/LASIK Brain, a commercial neural network computer program, was trained using the demographics, preoperative clinical data, surgical parameters, and 1 year postoperative clinical data of 44 patients treated with a Summit Technology excimer laser using a 5.0 mm optical zone. The neural-network derived nomogram was compared with the standard treatment nomogram for each patient. The relative contribution of age, sex, keratometry, and intraocular pressure (IOP) to the predicted nomograms was also assessed. RESULTS: Nomograms produced by the neural network were qualitatively similar to the standard nomogram. The sequence of data entry during training affected the network's predictions. Entry ordered by outcome (as opposed to entry by chronological order) yielded a nomogram that was more consistent with the standard nomogram. However, both outcome- and chronologically ordered network-derived nomograms diverged from the standard nomogram in individual patients, including a subset for whom use of the standard nomogram yielded desired refractive results (within 0.25 diopter of emmetropia). Further analysis of the neural-network-derived nomograms revealed marked sensitivity to sex, age, keratometry readings, and IOP. CONCLUSIONS: Neural networks offer a potential means of individualizing treatment nomograms, to account for patient demographics, preoperative examination, surgeon style, and equipment bias. However, a data set of 44 patients was not sufficient to train the PRK/LASIK Brain network to accurately predict treatment parameters in individual cases in the training set. A larger training set or a different learning algorithm may be required to improve the neural network's performance.

A novel circadianly expressed Drosophila melanogaster gene dependent on the period gene for its rhythmic expression.

The Drosophila melanogaster period (per) gene is required for expression of endogenous circadian rhythms of locomotion and eclosion. per mRNA is expressed with a circadian rhythm that is dependent on Per protein; this feedback loop has been proposed to be essential to the central circadian pacemaker. This model would suggest the Per protein also controls the circadian expression of other genetic loci to generate circadian behavior and physiology. In this paper we describe Dreg-5, a gene whose mRNA is expressed in fly heads with a circadian rhythm nearly identical to that of the per gene. Dreg-5 mRNA continues to cycle in phase with that of per mRNA in conditions of total darkness and also when the daily feeding time is altered. Like per mRNA, Dreg-5 mRNA is not expressed rhythmically in per null mutant flies. Dreg-5 encodes a novel 298 residue protein and Dreg-5 protein isoforms also oscillate in abundance with a circadian rhythm. The phase of Dreg-5 protein oscillation, however, is different from that of Per protein expression, suggesting that Dreg-5 and per have common translational but different post-translational control mechanisms. These results demonstrate that the per gene is capable of modulating the rhythmic expression of other genes; this activity may form the basis of the output of circadian rhythmicity in Drosophila.

Partners in time. Circadian rhythms.

The timeless gene is a second essential component of the circadian clock in Drosophila; its product interacts physically with the only other known clock component, the period gene product. Together they control the daily cycle of expression of their own and other loci.

Extent and character of circadian gene expression in Drosophila melanogaster: identification of twenty oscillating mRNAs in the fly head.

BACKGROUND: Although mRNAs expressed with a circadian rhythm have been isolated from many species, the extent and character of circadianly regulated gene expression is unknown for any animal. In Drosophila melanogaster, only the period (per) gene, an essential component of the circadian pacemaker, is known to show rhythmic mRNA expression. Recent work suggests that the encoded Per protein controls its own transcription by an autoregulatory feedback loop. Per might also control the rhythmic expression of other genes to generate circadian behavior and physiology. The goals of this work were to evaluate the extent and character of circadian control of gene expression in Drosophila, and to identify genes dependent on per for circadian expression. RESULTS: A large collection of anonymous, independent cDNA clones was used to screen for transcripts that are rhythmically expressed in the fly head. 20 of the 261 clones tested detected mRNAs with a greater than two-fold daily change in abundance. Three mRNAs were maximally expressed in the morning, whereas 17 mRNAs were most abundant in the evening--when per mRNA is also maximally expressed (but when the flies are inactive). Further analysis of the three 'morning' cDNAs showed that each has a unique dependence on the presence of a light-dark cycle, on timed feeding, and on the function of the per gene for its oscillation. These dependencies were different from those determined for per and for a novel 'evening' gene. Sequence analysis indicated that all but one of the 20 cDNAs identified previously uncloned genes. CONCLUSIONS: Diurnal control of gene expression is a significant but limited phenomenon in the fly head, which involves many uncharacterized genes. Diurnal control is mediated by multiple endogenous and exogenous mechanisms, even at the level of individual genes. A subset of circadianly expressed genes are predominantly or exclusively dependent on per for their rhythmic expression. The per gene can therefore influence the expression of genes other than itself, but for many rhythmically expressed genes, per functions in conjunction with external inputs to control their daily expression patterns.

Real-time automated sleep scoring: validation of a microcomputer-based system for mice.

Long-term circadian studies of sleep and wakefulness in rodents have been hindered by the labor required to analyze long polygraph records. To expedite such studies, we have designed and implemented SCORE, a microcomputer-based real-time sleep scoring system for rodents. The electroencephalograph is digitized in 10-s epochs at 100 Hz. Frequency and amplitude information from the waveform are extracted into a 48-dimension vector that is then compared to previously taught vectors representing the canonical features of four arousal states: wakefulness, theta-dominated wakefulness, rapid eye movement (REM) sleep, and nonREM (NREM) sleep. Match values are assigned for each state to each epoch; after excluding states based on wheel-running or drinking activity data, the nonexcluded state with the best match value for the epoch is scored. Analysis of over 23,000 epochs for four mice yielded an overall agreement of 94.0% between two human scorers and the program, compared with a 94.5% agreement between the two human scorers. The SCORE algorithm matched the human concensus best for wakefulness (97.8%) and NREM sleep (94.7%), but was lower for REM sleep (75.2%) and theta-dominated wakefulness (83.3%). Most errors in scoring of REM sleep were in close temporal proximity to human-scored REM epochs. SCORE is capable of scoring arousal states for eight animals simultaneously in real time on a standard IBM PC equipped with a commercially available analog-to-digital conversion board, and should considerably facilitate the performance of long-term studies of sleep and wakefulness in the rodent.

Amplified RNA synthesized from limited quantities of heterogeneous cDNA.

The heterogeneity of neural gene expression and the spatially limited expression of many low-abundance messenger RNAs in the brain has made cloning and analysis of such messages difficult. To generate amounts of nucleic acids sufficient for use in standard cloning strategies, we have devised a method for producing amplified heterogeneous populations of RNA from limited quantities of cDNA. Whole cerebellar RNA was primed with a synthetic oligonucleotide containing the T7 RNA polymerase promoter sequence 5' to a polythymidylate region. After second-strand cDNA synthesis, T7 RNA polymerase was used to generate amplified antisense RNA (aRNA). Up to 80-fold molar amplification has been achieved from nanogram quantities of cDNA. The amplified material is similar in size distribution to the parent cDNA and shows sequence heterogeneity as assessed by Southern and Northern blot analysis. Specific messages for moderate-abundance mRNAs for actin and guanine nucleotide-binding protein (G-protein) alpha subunits have been detected in the amplified material. By using in situ transcription to generate cDNA, sequences for cyclophilin have been detected in aRNA derived from single cerebellar tissue sections. cDNA derived from a single cerebellar Purkinje cell also has been amplified and yields material that hybridizes to cognate whole RNA and mRNA but not to Escherichia coli RNA.

Factors affecting plasma benzo[a]pyrene levels in environmental studies.

Benzo[a]pyrene (BaP) is a useful indicator of exposure to polycyclic aromatic hydrocarbons (PAHs), airborne carcinogenic compounds. Radioimmunoassay was used to test for plasma BaP differences in 61 subjects divided into three groups based on geographic-demographic locale: urban-industrial, urban-residential, and outer suburban. The results showed that the urban-industrial area participants had a significantly higher mean plasma BaP level than did the outer suburban subjects. The urban-residential subjects did not have a significantly different mean plasma benzo[a]pyrene level from either of the other two groups. Obesity, as measured by Quetelet's index, was found to have a significant correlation with BaP levels. These results indicate that radioimmunoassay of plasma for BaP may be used successfully to judge environmental exposure to PAHs, provided physiological considerations such as obesity are taken into account.