Enhancing the efficacy of AREDS antioxidants in light-induced retinal degeneration.

PURPOSE: Light-induced photoreceptor cell degeneration and disease progression in age-related macular degeneration (AMD) involve oxidative stress and visual cell loss, which can be prevented, or slowed, by antioxidants. Our goal was to test the protective efficacy of a traditional Age-related Eye Disease Study antioxidant formulation (AREDS) and AREDS combined with non-traditional antioxidants in a preclinical animal model of photooxidative retinal damage. METHODS: Male Sprague-Dawley rats were reared in a low-intensity (20 lux) or high-intensity (200 lux) cyclic light environment for 6 weeks. Some animals received a daily dietary supplement consisting of a small cracker infused with an AREDS antioxidant mineral mixture, AREDS antioxidants minus zinc, or zinc oxide alone. Other rats received AREDS combined with a detergent extract of the common herb rosemary, AREDS plus carnosic acid, zinc oxide plus rosemary, or rosemary alone. Antioxidant efficacy was determined by measuring retinal DNA levels 2 weeks after 6 h of intense exposure to white light (9,000 lux). Western blotting was used to determine visual cell opsin and arrestin levels following intense light treatment. Rhodopsin regeneration was determined after 1 h of exposure to light. Gene array analysis was used to determine changes in the expression of retinal genes resulting from light rearing environment or from antioxidant supplementation. RESULTS: Chronic high-intensity cyclic light rearing resulted in lower levels of rod and cone opsins, retinal S-antigen (S-ag), and medium wavelength cone arrestin (mCAR) than found for rats maintained in low cyclic light. However, as determined by retinal DNA, and by residual opsin and arrestin levels, 2 weeks after acute photooxidative damage, visual cell loss was greater in rats reared in low cyclic light. Retinal damage decreased with AREDS plus rosemary, or with zinc oxide plus rosemary whereas AREDS alone and zinc oxide alone (at their daily recommended levels) were both ineffective. One week of supplemental AREDS plus carnosic acid resulted in higher levels of rod and cone cell proteins, and higher levels of retinal DNA than for AREDS alone. Rhodopsin regeneration was unaffected by the rosemary treatment. Retinal gene array analysis showed reduced expression of medium- wavelength opsin 1 and arrestin C in the high-light reared rats versus the low-light rats. The transition of rats from low cyclic light to a high cyclic light environment resulted in the differential expression of 280 gene markers, enriched for genes related to inflammation, apoptosis, cytokine, innate immune response, and receptors. Rosemary, zinc oxide plus rosemary, and AREDS plus rosemary suppressed 131, 241, and 266 of these genes (respectively) in high-light versus low-light animals and induced a small subset of changes in gene expression that were independent of light rearing conditions. CONCLUSIONS: Long-term environmental light intensity is a major determinant of retinal gene and protein expression, and of visual cell survival following acute photooxidative insult. Rats preconditioned by high-light rearing exhibit lower levels of cone opsin mRNA and protein, and lower mCAR protein, than low-light reared animals, but greater retention of retinal DNA and proteins following photooxidative damage. Rosemary enhanced the protective efficacy of AREDS and led to the greatest effect on the retinal genome in animals reared in high environmental light. Chronic administration of rosemary antioxidants may be a useful adjunct to the therapeutic benefit of AREDS in slowing disease progression in AMD.

Prevention of retinal light damage by zinc oxide combined with rosemary extract.

PURPOSE: Zinc oxide effectively reduces visual cell loss in rats exposed to intense visible light and is known to slow the rate of disease progression in advanced stages of age-related macular degeneration. Our goal was to determine the efficacy of zinc oxide in combination with novel and well-established antioxidants in an animal model of light-induced oxidative retinal damage. METHODS: One group of male Sprague-Dawley rats was pretreated with zinc oxide with or without a detergent extract of rosemary powder and then exposed to intense visible light for 4-24 h. Another group of animals received zinc oxide combined with rosemary oil diluted with a mixture of polyunsaturated fatty acids (ROPUFA) and a third group was given an antioxidant mineral mix containing zinc oxide, as recommended by the Age Related Eye Disease Study group's first clinical trial (AREDS1). Visual cell survival was determined 2 weeks after intense light treatment by measuring rhodopsin and photoreceptor cell DNA levels and confirmed by retinal histology and agarose gel electrophoresis of DNA. Western analysis was used to determine the effects of zinc and antioxidants on the oxidative stress markers, glial fibrillary acidic protein (GFAP), heme-oxygenase-1 (HO-1), and carboxyethylpyrrole (CEP). Rod and cone opsin and arrestin levels were used as markers of photoreceptor cell function. RESULTS: Dark-reared rats treated with 1.3 mg/kg zinc oxide and 17 mg/kg rosemary extract, or with one-half those doses, and exposed to moderate intensity green light retained 75%-85% of the rhodopsin and retinal DNA measured in unexposed rats. These levels were significantly higher than found for zinc oxide or rosemary treatment alone. Rosemary oil was also effective when combined with zinc oxide, but ROPUFA alone was no more effective than the detergent vehicle. Prolonged intense green light led to increases in retinal GFAP and HO-1 levels and to decreases in cone cell opsin and rod and cone arrestins. Rosemary plus zinc treatment reduced the expression of oxidative stress protein markers and enhanced visual cell survival, as shown by improved photoreceptor cell morphology and by decreased retinal DNA degradation. Using higher intensity white light for exposures in cyclic light-reared rats, treatment with an AREDS antioxidant/mineral mixture was found to be ineffective, whereas rosemary extract plus an equivalent dose of zinc oxide was significantly more effective in preserving visual cells. CEP protein adduct formation was reduced by all antioxidant treatments, but rosemary plus zinc oxide also prevented the loss of cone cell opsin and arrestin more effectively than AREDS. CONCLUSIONS: In the rat model of acute retinal light damage, zinc oxide combined with a detergent extract of rosemary powder or rosemary oil is more effective than treatment with either component alone and significantly more effective than an AREDS mixture containing a comparable dose of zinc oxide. Light-induced oxidative stress in animal models of retinal degeneration can be a useful preclinical paradigm for screening novel antioxidants and for testing potential therapeutics designed to slow the progression of age-related ocular disease.

Distribution and removal of cat, dog and mite allergens on smooth surfaces in homes with and without pets.

BACKGROUND: Removing allergen from the indoor environment should be a primary strategy for the management and treatment of allergic disease. OBJECTIVE: The aims of this study were to characterize the distribution of dog, cat, and mite allergen on hard surfaces in homes with and without pets and to evaluate the efficiency of removing allergen from hard surfaces by wiping with a dry dust cloth and by vacuum cleaning using the dustbrush attachment. METHODS: The amount of allergen collected from adjacent areas of two smooth floors, a wall, and finished furniture by wiping with a Pledge Grab-it dust cloth (S. C. Johnson & Son, Inc, Racine, WI) and by brush-vacuuming were compared for 24 homes with and without pets. In addition, the areas first wiped with the dust cloth were then brush-vacuumed and the amounts of allergen collected by the first and second cleaning were compared. RESULTS: A key finding was that 23 of the 24 homes had Can f 1 allergen on one or more of the sampled areas regardless of whether a dog was present. Most homes with pets and many homes without pets had Can f 1 and Fel d 1 allergens on walls, smooth floors, and finished furniture. Carpets were the major reservoir for pet allergens in homes with pets whereas allergen was more uniformly distributed in homes without pets. Little mite allergen was found on hard surfaces even when it was present in carpets. CONCLUSIONS: Dog and cat allergens are prevalent on walls, smooth floors, and finished furniture in homes with and without pets. Dry dusting with a Grab-it dust cloth was an effective cleaning method for removing allergen from hard smooth surfaces.

Reducing relative humidity is a practical way to control dust mites and their allergens in homes in temperate climates.

BACKGROUND: Maintaining a relative humidity (RH) of less than 50% is one recommendation for reducing numbers of house dust mites and their allergens in homes. OBJECTIVE: The purpose of this study was to determine whether, in a humid temperate climate, indoor RH could be sufficiently lowered to control dust mites and their allergens. METHODS: During a period spanning 2 humid summers (May 1998 to October 1999), dust mite and allergen densities were determined in 3 groups of homes. One group (low RH group, n = 23) maintained an RH of less than 51%. Most of these homes used a high-efficiency dehumidifier and air conditioning. A second group of homes (group A) used air conditioning only (n = 19) or air conditioning and dehumidification (n = 5) but did not maintain an RH of less than 51%. A third group of homes (group C, n = 24) controlled climate by opening windows and had an RH of greater than 51%. Normal housecleaning was maintained in all homes during the study. RESULTS: The low RH group homes started in June with a mean +/- SE of 401 +/- 124 live mites and 17 +/- 3 microg of total Der 1 allergen per gram of dust. After 17 months of maintaining an RH of less than 51%, these declined significantly to 8 +/- 3 live mites per gram (P =. 004) and 4 +/- 1 microg of Der 1 per gram of dust (P <.001). In contrast, group A and C homes exhibited seasonal peaks of 500 to 1000 mites and 40 to 70 microg of Der 1 per gram of dust. At all time points after the baseline sample, the low RH group homes had significantly less (P <.001) allergen than the group A and C homes. After 17 months, allergen levels were more than 10 times lower in low RH homes compared with humid homes. CONCLUSION: This study showed that it is practical to maintain an indoor RH of less than 51% during the humid summer season in a temperate climate, and this resulted in significant reductions in mite and allergen levels.

Population dynamics of the house dust mites Dermatophagoides farinae, D. pteronyssinus, and Euroglyphus maynei (Acari: Pyroglyphidae) at specific relative humidities.

Experiments were conducted to determine the effects of relative humidity on the population dynamics of single and mixed species of Dermatophagoides farinae Hughes, D. pteronyssinus (Trouessart), and Euroglyphus maynei (Cooreman) at specific relative humidities maintained at 20 degrees C, with unlimited food. The population density of single and mixed species (D. farinae and D. pteronyssinus) increased exponentially when cultured at 65, 70, and 75% RH. The mean population growth rates were 17.3 +/- 4.4 SD and 32.5% +/- 4.7/wk for D. farinae and D. pteronyssinus, respectively. Mean population doubling times were 2.2 +/- 0.3 and 4.2 +/- 1.3 wk for D. pteronyssinus and D. farinae, respectively. Mixed species cultures, started with equal numbers of D. farinae and D. pteronyssinus, resulted in higher percentages of D. farinae than D. pteronyssinus. In cultures started with 75% of one species and 25% of the other, the more numerous species remained dominant and in similar ratios throughout the experiment. Both D. farinae and D. pteronyssinus population densities maintained at 85% RH declined over a 12-wk culture period because of mold growth. E. maynei were unable to survive at 65, 70, 75, and 85% RH, which indicated that their climatic requirements were different from those of D. farinae and D. pteronyssinus. Population densities of D. farinae and D. pteronyssinus cultures declined when held at 21-22 degrees C and relative humidities of < or = 50%; however, at 50% RH, significant proportions of the populations survived for 10 wk. Half-life for desiccation of D. farinae and D. pteronyssinus at 45% RH was 11.5 and 1.2 wk, respectively, but at 50% RH was 86.3 and 4.0 wk, respectively. The data indicated that a < or = 50% RH would have to be maintained for long periods to reduce both D. farinae and D. pteronyssinus by desiccation procedures. The results of this study show that D. farinae and D. pteronyssinus have high reproductive potentials and population growth rates, which indicate that mite reduction procedures must be thorough or mite densities will return to high levels quickly following remediation if adequate food and suitable microclimatic conditions exist.

Characterization of lymphocyte subtypes in scabietic skin lesions of naive and sensitized dogs.

We delineated the density of cells expressing CD4, CD8, CD21 and CD45RA antigens in the cellular infiltrates in the epidermis, dermis and follicular epithelium in scabietic skin lesions of naive hosts and sensitized hosts that expressed resistance to scabies infestation. No cells expressing CD21 (B-lymphocytes and follicular dendritic cells) were present in the epidermis and only a few were occasionally present in the dermis during both the first and second infestations. Naive T-cells (CD45RA+) and CD8+ cells (cytotoxic and suppressor T-lymphocytes) were present in varying densities in the infiltrates throughout the epidermis, dermis and follicular epithelium with no apparent differences in density and the rate of appearance between sensitizing and challenge infestations. CD4+ cells were abundant in fluctuating densities in the dermis, epidermis, and follicular epidermis during the sensitizing infestation and these cells became the dominant cell type early during the challenge infestation. The density of CD4+ cells in the infiltrate was much greater during the challenge than during the sensitization infestation. This population of CD4+ cells consisted of both T-helper/inducer cells and neutrophils and the large increase in their numbers during the challenge suggested they played a key role in the successful immune/inflammatory response that resulted in resistance to scabies infestation.

Characterization of antigen presenting cells and T-cells in progressing scabietic skin lesions.

Experimentally infested dogs expressed successful adaptive immunity and self-cured of scabies after previously having scabies that required treatment to cure. A biphasic increase and decrease of CD1a+ Langerhans cells (LCs) in the epidermis of hosts infested the first time (sensitized) and infested a second time (challenged) suggested that these cells were actively involved in the hosts' early immune response to scabies. In contrast, in the dermis CD1a+ cell densities during both infestations increased to a single peak that followed the first peak of these cells in the epidermis. In addition, there was an influx of T-lymphocytes (CD3 epsilon + cells) and CD11c+ cells into the dermis following the first peak of LCs in the epidermis. The influx of T-lymphocytes in the dermis coincided with the peak density of CD1a+ cells in the dermis and epidermis during the second infestation. In both the epidermis and dermis, MHC Class II+ cell density profiles were similar to that of CD1a during the first infestation and then exhibited single peaks during the second infestation. The increases in CD1a+, CD3 epsilon + (T-lymphocytes), CD11c+, and MHC Class II+ cell responses in the dermis occurred earlier and were more intense in the challenge infestation compared with the first infestation. These data indicate that T-lymphocytes (CD3 epsilon +), CD11c+, MHC Class II+, and CD1a+ cells in the dermis played a major role in the successful immune response to scabies mites.

Production of IL-1 alpha and IL-1 beta by human skin equivalents parasitized by Sarcoptes scabiei.

Human skin equivalents (HSEs) were used as a model to investigate interleukin (IL)-1 alpha and IL-1 beta secretions by keratinocytes stimulated by Sarcoptes scabiei (SS). SS mites burrowed into the stratum corneum when placed on the surface of cultured HSEs. Mites lived for 14 days. Mites and mite products induced cells in the HSEs to secrete IL-1 alpha and IL-1 beta within 16 hr. Scabies mites induced production of greater amounts of IL-1 alpha than IL-1 beta. Hepatocyte growth factor in the culture medium at 3 and 30 ng/ml upregulated the secretions of both IL-1 alpha and IL-1 beta by mite-infested skin equivalents, whereas 10 ng/ml of IL-6 upregulated production of only IL-1 beta. Therefore, these cytokines were important immunomodulating factors influencing keratinocyte secretion of IL-1 alpha and IL-1 beta in vitro. The results of this study provide the first evidence that keratinocytes (possibly fibroblasts) in the skin produce these cytokines in response to scabies mites or other ectoparasitic arthropods. Because IL-1 alpha and IL-1 beta are potent inducers of inflammation and keratinocytes are among the first effector cells to encounter scabies mites and their products, these cells may be key initiators of the inflammatory/immune reaction to scabies.

The development of protective immunity in canine scabies.

Seven of eight dogs that had been previously infested with Sarcoptes scabiei var. canis and then cured, expressed protective immunity when experimentally reinfested with scabies. All seven dogs that expressed resistance were spontaneously cleared of scabies by 64 days after they were experimentally reinfested. Five of the eight dogs were free of scabies by 24 days. The sequential changes in the inflammatory/immune cellular infiltrate in the scabietic lesions of each dog were determined during the sensitizing infestation, cure and the subsequent experimental reinfestation (challenge). During the initial infestation and in the subsequent challenge reinfestation, dogs developed mixed cellular infiltrates in their scabietic lesions that contained mononuclear cells, neutrophils, plasma cells and mast cells. Reinfestation induced more rapid increases in the densities of these cells than had occurred during the sensitizing infestation. Mononuclear and mast cells were the most numerous infiltrating cells during the sensitizing phase. During the challenge phase the most numerous infiltrating cells were mononuclear cells and neutrophils. The sensitizing and challenge infestations induced circulating scabies-specific antibody responses, but the response was more rapid during the reinfestation challenge. Both the cell-mediated response in the skin and the circulating antibody response waned in parallel with clearing of the mites following reinfestation.

Some effects of sarcoptic mange on dogs.

Sequential changes in pathology were examined for scabies-infested dogs to determine the effects of infestation with Sarcoptes scabiei var. canis. During 8 wk of infestation with S. scabiei, the progression of the disease was evaluated weekly by skin scrape, clinical examination, and blood analyses. At 8 wk, selected organs were microscopically examined for histopathology. All infested dogs developed an advanced level of scabies infestation by 8 wk. Of the 36 blood parameters evaluated, only values for erythrocyte sedimentation rate (ESR) deviated significantly from the normal ranges for dogs. However, infested dogs had significantly (P < 0.01) lower average hemoglobin and hematocrit concentrations after 8 wk of infestation compared to their values prior to infestation or to the values for the control dogs. Red blood cell levels for infested dogs dropped significantly (P < 0.01) from preinfestation concentrations by week 8. Conversely, by 8 wk total white blood cell and neutrophil concentrations were significantly (P < 0.01) greater than uninfested controls. Also, whereas average eosinophil concentrations were not statistically different for infested dogs compared to controls, some individual infested dogs exhibited eosinophilia at 4-8 wk of infestation. The ESRs for infested dogs were significantly (P < 0.01) greater at week 6 and 8 than for experimental dogs prior to infestation or control dogs. All parameters except neutrophils had returned to preinfestation levels by 2 wk after treatment for scabies. Neutrophil concentrations were no longer significantly different by 4 wk posttreatment. There were no significant differences in serum enzyme, biochemical and electrolyte concentrations between infested and control dogs.(ABSTRACT TRUNCATED AT 250 WORDS)

Resistance and immune response in scabies-infested hosts immunized with Dermatophagoides mites.

Seventy-one percent of rabbits immunized with a mixed (50:50) Dermatophagoides farinae and D. pteronyssinus house dust mite extract were resistant to infestation by Sarcoptes scabiei var. canis. The resistance was evidenced by a marked reduction in parasite load. All immunized hosts developed similar immunogen-specific antibody titers that were independent of the levels of scabies infestation that developed when the hosts were infested with scabies. Resistant hosts exhibited significantly lower scabies-specific immunoglobulin titers and produced antibody to fewer scabies antigens than did nonresistant hosts. All infested hosts (resistant and nonresistant) showed a cellular infiltrate in the scabietic lesions that was composed of neutrophils, plasma cells, macrophages, and mononuclear cells. Resistant hosts were characterized by fewer plasma cells in the infiltrate than were observed for non-resistant hosts. Resistant hosts exhibited a gradual increase in the number of infiltrating neutrophils, followed by a decrease that correlated with a decrease in the mite burden. Nonresistant hosts exhibited an early rapid increase, a decrease, and then a gradual increase in the concentration of neutrophils as the mite load increased. These results clearly showed that D. farinae/D. pteronyssinus antigens/epitopes can sensitize the hosts to scabies mites and induce protective immunity. The lower circulating antibody levels and generally stronger inflammatory cell-mediated response of resistant hosts compared with nonresistant hosts suggested that the mechanism by which immunization with Dermatophagoides mites induces immunity to scabies mites involved a down-regulated T helper cell type 2 (Th2) response with reduced antibody production but an up-regulated and stronger Th1 (inflammatory cell-mediated) response to scabies.

Sarcoptes scabiei: histopathological changes associated with acquisition and expression of host immunity to scabies.

The sequential changes in the histology in scabietic lesions were investigated for hosts (rabbits) during sensitization and reinfestation with Sarcoptes scabiei. Experimentally infested naive hosts developed heavy scabies infestations but 65% of the hosts showed induced immunity (resistance) to reinfestation. All hosts (naive, immune, and susceptible) exhibited an initial cell infiltrate in scabietic lesions that consisted predominantly of neutrophils and a few macrophages. This was followed by a dense plasma cell infiltrate. Upon reinfestation the progression of neutrophil infiltration was faster and the response was greater in hosts who acquired immunity than those in the susceptible hosts.

Allergenicity of Euroglyphus maynei and its cross-reactivity with Dermatophagoides species.

BACKGROUND: The house dust mite, Euroglyphus maynei (EM), is common in homes in England, Europe, the southern United States, and other parts of the world. It is often present in densities greater than 100 mites per gram of dust. EM usually occurs with Dermatophagoides farinae (DF) and D. pteronyssinus (DP) and it is frequently more abundant than Dermatophagoides species. Therefore the allergenicity of EM and the cross-reactivity between EM and Dermatophagoides species are important considerations for diagnosis of mite-induced allergy and the use of appropriate immunotherapy. METHODS: Crossed immunoelectrophoresis revealed that EM was the source of at least 33 antigens. Crossed radioimmunoelectrophoresis with 32 different sera from patients with RAST results that were positive to EM and skin test results that were positive to both DF and DP identified 15 allergens. Individual sera recognized two to eight EM antigens as allergens. RESULTS: Ninety-one percent of the patients showed serum IgE directed at three or more allergens. Eighty-four percent of the patients had moderate or strong levels of IgE directed at antigen no. 33. All 15 allergens showed moderate or strong IgE binding by one or more sera. EM shared four and six cross-reacting allergens with DF and DP, respectively. Therefore 11 and 9 allergens of EM were species-specific and not shared by DF and DP, respectively. CONCLUSIONS: The results of this study clearly indicate that sensitivity to EM should be considered in geographic areas where this mite is abundant in homes.

Development of Dermatophagoides pteronyssinus (Acari: Pyroglyphidae).

Developmental times (egg to adult) and fecundity of the European house dust mite, Dermatophagoides pteronyssinus (Trouessart), were determined for various temperatures that normally occur in the mite microhabitat in homes. Egg-to-adult developmental times were 122.8 +/- 14.5, 34.0 +/- 5.9, 19.3 +/- 2.5, and 15.0 +/- 2.0 d (means +/- SD) at 16 (61 degrees F), 23 (73 degrees F), 30 (86 degrees F), and 35 degrees C (95 degrees F), respectively. Developmental times of individual life stages were consistent proportions of the total egg-to-adult developmental times regardless of temperature. Duration of the egg and protonymphal and tritonymphal life stages were 20-26% of the total development times and for the larval stage, 28-33%. At 23 degrees C, females lived 31.2 +/- 11.1 d and produced 2.5 +/- 0.7 eggs per day during a reproductive period of 26.2 +/- 10.7 d. By comparison, at 35 degrees C, females produced 3.3 +/- 1.3 eggs per day, but female longevity and the reproductive period were reduced to 15.5 +/- 9.6 and 11.6 +/- 6.4 d, respectively. During a lifetime, females produced 68.4 +/- 30.4 and 48.0 +/- 29.6 eggs at 23 and 35 degrees C, respectively.