Cytomegalovirus replication within the lung allograft is associated with bronchiolitis obliterans syndrome.

Early studies reported cytomegalovirus (CMV) pneumonitis as a risk factor for development of bronchiolitis obliterans syndrome (BOS) following lung transplantation. While improvements in antiviral prophylaxis have resulted in a decreased incidence of CMV pneumonitis, molecular diagnostic techniques allow diagnosis of subclinical CMV replication in the allograft. We hypothesized that this subclinical CMV replication was associated with development of BOS. We retrospectively evaluated 192 lung transplant recipients (LTR) from a single center between 2001 and 2009. Quantitative (PCR) analysis of CMV viral load and histological evidence of CMV pneumonitis and acute cellular rejection was determined on 1749 bronchoalveolar lavage (BAL) specimens and 1536 transbronchial biopsies. CMV was detected in the BAL of 41% of LTR and was significantly associated with the development of BOS (HR 1.8 [1.1-2.8], p = 0.02). This association persisted when CMV was considered more accurately as a time-dependent variable (HR 2.1 [1.3-3.3], p = 0.003) and after adjustment for significant covariates in a multivariate model. CMV replication in the lung allograft is common following lung transplantation and is associated with increased risk of BOS. As antiviral prophylaxis adequately suppresses CMV longer prophylactic strategies may improve long-term outcome in lung transplantation.

Intracellular topography of rhodopsin bleaching.

In a vertebrate eye, the photoreceptor cells are aligned so that most of the light passes through them lengthwise. At the light-transducing outer segment region of the photoreceptor, photons are absorbed in a time-varying, spatially dependent fashion. Because the transduction event is spatially localized around the site of photon absorption, the spatiotemporal patterns of light absorption in outer segments are an important receiver input characteristic. This aspect of receptor biophysics has now been measured; the results were consistent with a theoretical model proposed for bleaching of a pigment in an unstirred layer.

The Rpe65 Leu450Met variation increases retinal resistance against light-induced degeneration by slowing rhodopsin regeneration.

Excessive light can cause retinal degeneration and may be an environmental cofactor accelerating retinal dystrophies and age-related diseases. In rodent models, the light damage susceptibility (LDS) of the retina is determined genetically. In two mouse strains, with different degrees of LDS, a Leu450Met variation in the pigment epithelial protein RPE65 was shown recently to cosegregate with low LDS. Because light damage is rhodopsin-mediated, and RPE65 is essential for the regeneration of rhodopsin in the visual cycle, we analyzed this variation regarding rhodopsin metabolism and LDS in four mouse strains. We found that, in contrast to previous assertions, LDS does not correlate with the maximal retinal content of rhodopsin present after dark adaptation. Instead, LDS correlated positively with the kinetics of rhodopsin regeneration, which determine rhodopsin availability during light exposure. Light damage occurred after absorption of a threshold dose of photons and thus fast regeneration, as observed in those two strains having Leu at position 450 of RPE65, was correlated with the occurrence of photoreceptor apoptosis after short exposure. In contrast, mice with the Leu450Met variation of Rpe65 regenerated rhodopsin with slow kinetics and showed an increased resistance to light-induced retinal degeneration. In these mice, RPE65 protein levels were reduced by a post-transcriptional mechanism. F(1) hybrid mice, carrying one normal and one variant Rpe65 gene, had intermediate levels of the corresponding protein and showed intermediate rhodopsin regeneration kinetics and an intermediate LDS. Thus, none of the two variants of Rpe65 had a dominant effect.

The effect of phospholipid structure on the thermal stability of rhodopsin.

The effect of the major headgroup classes of phospholipids on the conformational stability of rhodopsin is investigated. This is accomplished by measuring the effect of L-alpha-dimyristylphosphatidic acid (DMPA), L-alpha-dimyristylphosphatidylcholine (DMPC), L-alpha-dimyristylphosphatidylethanolamine (DMPE) and L-alpha-dimyristylphosphatidylserine (DMPS) on the thermal decay rate of rhodopsin in dodecyltrimethylammonium bromide (DTAB) and octylglucoside detergent systems. In the DTAB system the relative stabilization by these phospholipids is DMPA less than DMPS greater than DMPC greater than DMPE. In the octylglucoside system, the relative stabilization ability is DMPA greater than DMPS congruent to DMPC greater than DMPE. The relative stabilization ability in a series of lecithin derivatives that differ in fatty acid chain structure is also reported. This series of experiments demonstrate that the structure of the fatty acid chains is as important as the headgroup structure in determining the stabilization ability of a phospholipid.

Some properties of old and new rhodopsin in single Bufo rods.

Rod photoreceptors renew the membranous disks of the outer segments (ROS). New disks are assembled at the proximal base and old disks are shed at the distal tip. Rhodopsin, the major protein of the disk, remains with the disk into which it was inserted. Thus, it is true that the oldest rhodopsin is at the tip and the newest at the base. A microspectrophotometer is used to examine the properties of rhodopsin in the two ends of the toad ROS. No differences between the two are found in absorption spectrum, concentration, dichroism, photoconversion rates, or lateral diffusion rates. Regeneration of rhodopsin from the bleached state is also studied but cannot be used to discriminate old from new rhodopsin because the point of entry of regeneration retinoids and/or their concentrations cannot be controlled. However, a new insight into pigment regeneration in the living toad eye is gained: regeneration is faster in the basal disks than in the distal.

Intracellular topography of rhodopsin regeneration in vertebrate rods.

The vertebrate visual pigment of rods, rhodopsin, bleaches in light and regenerates in darkness. When the bleaching and regeneration are carried out in vivo, it is found that the regeneration takes place at nonuniform rates along the rod outer segment (ROS): toads and frogs regenerate rhodopsin faster in the proximal ends of the ROS than in the distal ends. Rats do the reverse. These patterns of regeneration persist whether the bleaching is done with flashes or with steady light. They are also independent of the extent to which the retinal pigment epithelium contains melanin. Furthermore, the dichotomy of patterns (proximal faster vs. distal faster) does not seem to depend upon the presence of an excess of stored retinoid in the eye. Instead, it is suggested that the villous processes of the epithelial cells may play an important role in the regeneration patterns. These processes in amphibia extend nearly to the rod inner segment but in the rat they surround only the apical end of the outer segment. If they "funnel" the retinoids back to the ROS, their location and morphology could explain the two different kinds of patterns seen.

Axial gradients of rhodopsin in light-exposed retinal rods of the toad.

Exposure of an intact vertebrate eye to light bleaches the rhodopsin in the photoreceptor outer segments in spatially nonuniform patterns. Some axial bleaching patterns produced in toad rods were determined using microspectrophotometric techniques. More rhodopsin was bleached at the base of the outer segment than at the distal tip. The shape of the bleaching gradient varied with the extent of bleach and with the spectral content of the illuminant. Monochromatic light at the lambda max of the rhodopsin gave rise to the steepest bleaching gradients and induced the greatest changes in the form of the gradient with increasing extent of bleach. These results were consistent with a mathematical model for pigment bleaching in an unstirred sample. The model did not fit bleaching patterns resulting from special lighting conditions that promoted the photoregeneration of rhodopsin from the intermediates of bleaching. Prolonged light adaptation of toads could also produce axial rhodopsin gradients that were not fit by the bleaching model. Under certain conditions the axial gradient of rhodopsin in a rod outer segment reversed with time in the light: the rhodopsin content became highest at the base. This result could be explained by an interaction between the pattern of bleaching and the intracellular topography of regeneration.

Action spectrum of retinal light-damage in albino rats.

The right eyes of anesthetized, 10-week-old albino rats were exposed to constant photon fluxes at 6 wavelengths for 6 hrs. The left eye of each animal was patched during the exposure and was used as control. Histologic examination of retinal sections disclosed a region in the superior retina that was more damaged than were other areas. Attempting to ascertain an action spectrum by measuring ONL lost in this "sensitive" region failed. However, it was shown that, when ONL thickness was integrated over the entire retinal sections, a rhodopsin action-spectrum emerged. It was concluded that (1) retinal light damage in the albino rat under these conditions was rhodopsin mediated, and (2) proper assessment of the extent of damage could only be made by some method that integrates over the entire retinal section.

Effect of eye closures and openings on photostasis in albino rats.

PURPOSE: To determine the effects of eye closure and opening on photostasis, the regulation of light absorption by retinal rods in the albino rat. METHODS: The approach was to measure the effect of eye closure and opening on rhodopsin bleaching in situ and to use those results to simulate what happens to rhodopsin when a living rat opens or closes its eyes during daylight exposure. Completely dark-adapted, dead albino rats, each with one eye closed or open, were exposed to a standard lighting situation. The rhodopsin bleaching rate in closed versus open eyes was measured. Rhodopsin bleached at a more reduced rate in closed eyes than in open eyes. This measured reduction of rate in closed eyes was applied to a simulation of rhodopsin bleaching in open and closed eyes. The simulation used idealized conditions to verify the simulation itself, and then it was applied to previously published photostasis results. RESULTS: Rhodopsin in closed eyes bleaches at half the rate found in open eyes. The absorption spectrum of rat red blood cells was compared with the rate rhodopsin absorption spectrum, and the comparison showed that blood does not absorb the main-band wavelengths of rhodopsin. Simulating rhodopsin bleaching with eyes closed (half intensity) and open (full intensity) during daylight hours showed a slight effect on the total number of photons absorbed in an entire day. The simulation set limits to the maximal effect of eyes open all day versus eyes closed all day. At a habitat intensity of 200 lux, for example this maximal effect (eyes always open versus always closed) was calculated to be +/- 9%. At the lowest intensity, 3 lux, this maximal effect was +/- 28%, but it is only 1% at the highest intensity, 400 lux. CONCLUSIONS: Eye closures and openings have a slight effect on photostasis in albino rats. There are two reasons for this: The eyelids reduce the effective bleaching intensity by half. Moreover, during the "dim-out" of closure, rhodopsin continues to regenerate and approaches a new, higher value. This accumulation of rhodopsin enhances the rate of photon absorption because the rate is proportional to the product (rhodopsin x intensity). Thus, the increased rhodopsin concentration in the rods partially compensates for the reduced intensity of lid closure, and the photon absorption rates, with eyes closed, do not decrease by the full factor of 2 implied by the intensity reduction. In addition, when the eyes are subsequently opened after such a dim-out, the retina is suddenly exposed again to the full intensity of the environment. At this time, photon absorption rate, rhodopsin x intensity, is transiently higher than just before eye opening. Thus, the compensatory interplay between bleaching and regeneration in closed and open eyes results in the near compensation of light absorption and maintenance of the stasis close to 10(16) photons per eye per day.

The effect of light history on the aspartate-isolated fast-PIII responses of the albino rat retina.

PURPOSE: To assess the effect of light-rearing history on the photon-capturing ability, amplitude, and kinetics of the fast-PIII response of the retina. METHODS: Albino rats were raised on 12-hour light-12-hour dark cycles, with illumination at 3 lux or 200 lux, and killed at approximately 12 weeks. Retinal rhodopsin content was measured spectrophometrically. The morphology of the rod outer segments (ROS) and the thickness of the outer nuclear layer were determined histologically. Electroretinograms of isolated retinas to 3-microsecond flashes were recorded. The kinetics of fast PIII responses were assessed with a model of the phototransduction cascade. RESULTS: Total rhodopsin of 200 lux animals was reduced to 60% that of 3 lux animals: 2.3 +/- 0.2 versus 1.4 +/- 0.1 nmol/eye (mean +/- SD). Length of ROS of 200 lux animals was reduced to 68% of the length of that of 3 lux animals: 20.1 +/- 1.2 versus 13.7 +/- 0.5 microns. The saturated amplitude of fast PIII of 200 lux animals was reduced to 56% that of the 3 lux group: 134 +/- 27 versus 239 +/- 37 microV (T = 22 degrees C). Fast PIII responses of both groups are well described by the kinetic model before slow PIII intrusion (up to 100 ms). Estimated kinetic parameters of the transduction cascade did not differ reliably between the two groups. CONCLUSIONS: Diminished saturated amplitude of fast PIII in 200 lux animals is accounted for by the hypothesis that fast PIII is directly proportional to the rod photocurrent and by the finding that the ROS of 200 lux animals are short compared to those of 3 lux animals. Similarity in estimated kinetic parameters of phototransduction suggests that the rods of the two groups differ little in the biochemistry underlying the activation phase of phototransduction.

Dietary deficiency of N-3 fatty acids alters rhodopsin content and function in the rat retina.

PURPOSE: To investigate the possibility that previously demonstrated reductions in photoreceptor sensitivity to light in n-3 fatty-acid-deficient rats can be explained by alterations in rhodopsin content and/or function. METHODS: Sprague-Dawley rats were reared throughout gestation, lactation, and up to 24 weeks of age on a diet containing safflower oil (n-3 fatty-acid-deficient) or soybean oil as the sole source of lipids. Dark-adapted content and in vivo regeneration of rhodopsin after bleaching were measured by detergent extraction. The regeneration rate constants and number of photons absorbed by rhodopsin under steady-state bleach conditions were calculated from these values. The rate of metarhodopsin II (MII) formation in vitro was determined by flash bleaching extracted pigment and native rod outer segment membranes. Rod outer segment length and photoreceptor cell density were determined in histologic sections through the inferior central retina. RESULTS: Dark-adapted rhodopsin content of retinas from rats reared on safflower oil was 12% to 15% higher than that of rats raised on soybean oil at every age measured. The rate of rhodopsin regeneration was significantly slower in rats reared on safflower oil while the level of steady-state bleach was the same. This meant that the rats reared on safflower oil absorbed about one half as many photons during light exposure. The rate of metarhodopsin II formation in vitro was unaffected by n-3 fatty acid deficiency. No difference in either rod outer segment length or cell number was detected. CONCLUSION: A reduced capacity for photon absorption by rhodopsin could play a role in lowering retinal sensitivity to light in n-3 fatty-acid-deficient rats.

The rhodopsin content of human eyes.

PURPOSE: To measure the total amount of rhodopsin in human eyes across the life span and to test the hypothesis that the rhodopsin content of infants' and the elderly's eyes is lower than at other ages. METHODS: Rhodopsin was extracted from retinal and pigment epithelial fractions of 196 eyes of 102 donors, ages 27 weeks' gestation through 94 years, using quantitative procedures. To recover photopigment bleached by unavoidable light exposure, the fractions from 78 eyes were incubated with 9-cis retinal. The total photopigment (retinal plus pigment epithelial fractions) per eye was examined for significant changes with age, using the higher value from pairs of eyes. RESULTS: The median rhodopsin content of the higher eye of adults is 6.45 nmoles (range, 3.33-10.84 nmoles) with 8 nmoles or more recovered from 28% of all adult eyes. The rhodopsin content of infants' eyes (< 12 months post-term) is significantly lower than that of older individuals and increases with age. After infancy, no change with age is found. For both infants and adults, 9-cis retinal significantly increases the amount of photopigment recovered without reducing the variance in the amount of photopigment recovered. The rhodopsin content is estimated to be 50% of the median adult amount early in infancy, approximately 5 weeks postterm (95% confidence interval, 0-10 weeks postterm). CONCLUSIONS: A developmental increase in rhodopsin content occurs during infancy. Thereafter rhodopsin content remains constant. The amount of rhodopsin recovered from human eyes is quite variable. Bleaching alone cannot explain the variability.

Distribution of melanosomes across the retinal pigment epithelium of a hooded rat: implications for light damage.

Distribution of melanosomes across the retinal pigment epithelium of hooded rats (Long-Evans) is studied at the light microscopic and electron microscopic levels. This distribution is shown to be nonuniform: more melanosomes exist in the periphery than elsewhere and, importantly, there are very few melanosomes in a restricted area of the central portion of the superior hemisphere compared with the corresponding part of the inferior hemisphere. The region with fewest melanosomes is precisely the one that is highly susceptible to light damage. Because this region is the same in both pigmented and albino eyes, the paucity of melanin in this region is not the cause of its great sensitivity to light damage. Nor does light cause the nonuniform distribution of melanin. A possible explanation, involving a proposed vestigial tapetum, is given in order to explain the correlation of melanosome counts and sensitivity to light damage.

Blue light's effects on rhodopsin: photoreversal of bleaching in living rat eyes.

PURPOSE: To determine whether blue light induces photoreversal of rhodopsin bleaching in vivo. METHODS: Eyes of anesthetized albino rats were exposed to either green (550 nm) or deep blue (403 nm) light, and the time course of rhodopsin bleaching was determined. Rhodopsin was isolated from whole retinas by detergent extraction and measured photometrically. To inhibit photoreversal of bleaching, rats were perfused with 70 mM hydroxylamine (NH(2)OH), a known inhibitor of photoreversal. To determine whether blue-absorbing, photoreversible photoproducts were formed, rhodopsin was bleached to near completion with green light and then exposed to blue light. Finally, experimental results were simulated on a computer by means of a simple, three-component model involving a long-lived photoreversible photoproduct. RESULTS: Photoreversal of bleaching in blue light occurs in vivo as evidenced by the following: In the absence of NH(2)OH, bleaching of rhodopsin by blue light was slow and complex. In the presence of NH(2)OH, however, blue light bleached rhodopsin very fast with a simple, pseudo-first-order kinetic. A long-lived bleaching intermediate produced by green light exposure was photoreversed to rhodopsin by exposure to blue light. The three-component computer model, invoking a blue-absorbing, photoreversible, long-lived intermediate accurately described the data. CONCLUSIONS: Because of the instantaneous, nonmetabolic regeneration of rhodopsin by the process of photoreversal of bleaching, blue light exposure permits the absorption of large numbers of photons by rhodopsin and by a photoreversible intermediate of bleaching in vivo. These data may have an important impact on resolving mechanisms of blue light-mediated damage to the retina.

Photoreceptor autophagy: effects of light history on number and opsin content of degradative vacuoles.

PURPOSE: To investigate whether regulation of rhodopsin levels as a response to changed lighting environment is performed by autophagic degradation of opsin in rod inner segments (RISs). METHODS: Groups of albino rats were kept in 3 lux or 200 lux. At 10 weeks of age, one group was transferred from 3 lux to 200 lux, another group was switched from 200 lux to 3 lux, and two groups remained in their native lighting (baselines). Rats were killed at days 1, 2, and 3 after switching. Another group was switched from 3 lux to 200 lux, and rats were killed at short intervals after the switch. Numbers of autophagic vacuoles (AVs) in RISs were counted, and immunogold labeling was performed for opsin and ubiquitin in electron microscopic sections. RESULTS: The number of AVs increased significantly after switching from 3 lux to 200 lux at days 1 and 2 and declined at day 3, whereas the reverse intensity change did not cause any increase. Early time points after change from 3 lux to 200 lux showed a significant increase of AVs 2 and 3 hours after switching. Distinct opsin label was observed in AVs of rats switched to 200 lux. Ubiquitin label was present in all investigated specimens and was also seen in AVs especially in 200-lux immigrants. CONCLUSIONS: Earlier studies had shown that an adjustment to new lighting environment is performed by changes in rhodopsin levels in ROSs. Autophagic degradation of opsin or rhodopsin may subserve, at least in part, the adaptation to abruptly increased habitat illuminance by removing surplus visual pigment.

Rod photoreceptors in infant rats with a history of oxygen exposure.

PURPOSE: To study in an infant rat model of retinopathy of prematurity, the rod photoreceptors, which are known to have attenuated photoresponses. METHODS: Rhodopsin was extracted from whole retinas, the thickness of the rod outer segment (ROS) layer was measured, large phagosomes were counted, and the ROS ultrastructure was examined in the retinas of oxygen-exposed and control rats, ages 13 and 18 days. Rhodopsin absorbances in the ROS were measured by microspectrophotometry at age 20 days. RESULTS: The rhodopsin content did not differ significantly between the oxygen-exposed and control rats at either 13 or 18 days. The thickness of the ROS layer was equal in 13-day-old oxygen-exposed and control rats; however, at 18 days, the ROS layer was significantly thinner in the oxygen-exposed rats than in the control rats. The number of phagosomes did not vary significantly among the oxygen-exposed and control groups. Opsin immunoreactivity was seen only in the ROS layer in oxygen-exposed and control rats. The ROS were disorganized in oxygen-exposed rats. The rhodopsin absorbances of the oxygen-exposed ROS were significantly more variable and higher than in the control rats. CONCLUSIONS: Attenuation of the rod photoresponse parameters does not result simply from shortening of the outer segments and consequent low rhodopsin content. Rather, the structure of the outer segments is altered. A fault in the synthesis of the outer segments, rather than disposal of outer segment discs, is suspected.

Rhodopsin in immature rod outer segments.

PURPOSE: To test the hypothesis that rhodopsin concentration is low in immature rat rod outer segments (ROS). METHODS: Microspectrophotometry (MSP) was used to assess rhodopsin absorbances in localized regions of isolated ROS from dark-adapted 13-, 19-, and 34-day-old and adult rats. Photopigment was extracted from the retinas of paired eyes in dark-adapted and light-adapted rats. One retina of each pair was treated with 9-cis retinal before extraction of photopigment. Rhodopsin with native 11-cis retinal was extracted from the fellow retina. RESULTS: By MSP, rhodopsin absorbance was low in the short ROS of 13-day-old rats. In 19-day-old rats with ROS lengths approximately equal to those of adults, absorbance was low at the tip, but at the base, it was equal to the high absorbance at both the tip and the base in adults. The 9-cis retinal did not add absorbance to the photopigment extracts of dark-adapted retinas at any age, but it did add absorbance to extracts of the light-adapted retinas at every age. CONCLUSIONS: The MSP results show that the accumulation of rhodopsin in developing rat rods depends on increasing concentrations in localized regions. No evidence of apo-opsin is found in immature rat rods. Thus, in immature ROS regions, the low rhodopsin absorbances suggest that the amount of opsin is also low. Greater disk-to-disk spacing in immature ROS regions than in mature regions could account for these findings.

A new microspectrophotometric method for measuring absorbance of rat photoreceptors.

A method has been developed for obtaining visual pigment absorbance spectra from fixed, frozen sections of the albino rat retina. The retinas are transversely sectioned at various thicknesses on a cryo-microtome and the resulting slices are examined with a photon-counting microspectrophotometer. Problems caused by the limited pigment content of small receptors are reduced and measurements can be made at well-specified locations along the retinal section. This method is both precise and accurate and permits comparisons across sections and across animals.

Flicker detection in the albino rat following light-induced retinal damage.

The effect of light-induced retinal damage on the behaving rat's critical flicker-fusion frequency (CFF) was studied by determining the CFF at scotopic and photopic luminances both before and after exposure to damaging light. The CFF was reduced but not abolished following damaging light exposure. The shapes of the functions relating CFF to luminance before and after exposure suggested that scotopic visual function may have survived the light damage better than did photopic function. Anatomical and biochemical measures of retinal damage indicated that 91-93% of the outer nuclear layer (ONL) and 99% of the rodopsin had been lost.

Retinal damage in pigmented and albino rats exposed to low levels of cyclic light following a single mydriatic treatment.

The present study demonstrated that less than three days of exposure to low levels of normally cycled ambient illumination are sufficient to cause death to photoreceptor cells in adult pigmented and albino rat. Cyclic light levels as low as 133 and 320 lux were found to destroy photoreceptor cells. A single mydriatic treatment with atropine immediately preceding the three-day exposure was sufficient to permit the effect in pigmented rats. No mydriasis was required for albino rats. When pigmented rats were reared in either 3 lux or 100 lux, it was found that these different light histories did not significantly affect the rats' subsequent susceptibility, during mydriasis, to retinal damage by cyclic illumination.