Fabry disease exacerbates renal interstitial fibrosis after unilateral ureteral obstruction via impaired autophagy and enhanced apoptosis

Background@#Fabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the GLA gene encoding alpha-galactosidase A. Despite some data showing that profibrotic and proinflammatory cytokines and oxidative stress could be involved in Fabry disease-related renal injury, the pathogenic link between metabolic derangement within cells and renal injury remains unclear. @*Methods@#Renal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys. @*Results@#Compared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice. @*Conclusion@#These findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.

Fabry disease exacerbates renal interstitial fibrosis after unilateral ureteral obstruction via impaired autophagy and enhanced apoptosis

Background@#Fabry disease is a rare X-linked genetic lysosomal disorder caused by mutations in the GLA gene encoding alpha-galactosidase A. Despite some data showing that profibrotic and proinflammatory cytokines and oxidative stress could be involved in Fabry disease-related renal injury, the pathogenic link between metabolic derangement within cells and renal injury remains unclear. @*Methods@#Renal fibrosis was triggered by unilateral ureteral obstruction (UUO) in mice with Fabry disease to investigate the pathogenic mechanism leading to fibrosis in diseased kidneys. @*Results@#Compared to kidneys of wild-type mice, lamellar inclusion bodies were recognized in proximal tubules of mice with Fabry disease. Sirius red and trichrome staining revealed significantly increased fibrosis in all UUO kidneys, though it was more prominent in obstructed Fabry kidneys. Renal messenger RNA levels of inflammatory cytokines and profibrotic factors were increased in all UUO kidneys compared to sham-operated kidneys but were not significantly different between UUO control and UUO Fabry mice. Protein levels of Nox2, Nox4, NQO1, catalase, SOD1, SOD2, and Nrf2 were not significantly different between UUO control and UUO Fabry kidneys, while the protein contents of LC3-II and LC3-I and expression of Beclin1 were significantly decreased in UUO kidneys of Fabry disease mouse models compared with wild-type mice. Notably, TUNEL-positive cells were elevated in obstructed kidneys of Fabry disease mice compared to wild-type control and UUO mice. @*Conclusion@#These findings suggest that impaired autophagy and enhanced apoptosis are probable mechanisms involved in enhanced renal fibrosis under the stimulus of UUO in Fabry disease.

Erratum: In Vitro Evaluation of Allergen Potencies of Commercial House Dust Mite Sublingual Immunotherapy Reagents

Corrections for Table. 1 in page 125 are needed. We apologize for any inconvenience that this may have caused.

Allergenic Characterization of 27-kDa Glycoprotein, a Novel Heat Stable Allergen, from the Pupa of Silkworm, Bombyx mori

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.

Cross-Reactivity between Oak and Birch Pollens in Korean Tree Pollinosis

Oak and birch trees belong to Fagales order. Specific IgE to pollen allergens of both trees are frequently found in Korea pollinosis patients. Oak trees which comprise 40% of forest area are common in Korea. However, birch trees are sparse. We compared the allergenicity of pollen extracts of white oak, sawtooth and Mongolian oaks which are prevalent species in Korea, with the pollen extract of birch. The cross-reactivity of four pollen extracts was examined with pooled sera of 12 patients by ELISA, immunoblotting and CAP inhibitions. A protein of 17 kDa, putatively homologous to a major birch allergen Bet v 1, displayed strong IgE reactivity from white oak and sawtooth oak pollen extract but not from Mongolian oak pollen. Notably, a 23-kDa protein from sawtooth and white oaks showed strong IgE reactivity and inhibited by Bet v 1. IgE binding to white oak was inhibited a maximum of 94.6% by white oak, 93.4% by sawtooth oak, 83.2% by Mongolian oak, and 68.8% by birch. Furthermore, sawtooth oak, white oak, and Mongolian oak extracts were able to inhibit up to 78.5%, 76.6% and 67.3% of IgE binding to birch extract, while birch extract itself inhibited up to 94.3%. Specific IgE to Bet v 1 was inhibited a maximum of 79.1% by sawtooth oak, 77.4% by white oak, and 72.7% by Mongolian oak, while 81.5% inhibition was shown by birch. Bet v 1 was able to partially inhibit its homologous molecules from sawtooth oak and white oak in immunoblotting. Birch pollen extract was found to be cross-reactive primarily with Bet v 1-homologous allergen from oak pollens in Korea pollinosis patients. Considering the sparseness of birch tree in Korea, oak, especially sawtooth oak may be the main cause of tree pollinosis in Korea, rather than birch.

Standardization of Weed Pollen Extracts, Japanese Hop and Mugwort, in Korea

PURPOSE: Japanese hop (Humulus spp.) and mugwort (Artemisia spp.) are notable causes of autumn pollinosis in East Asia. However, Japanese hop and mugwort pollen extracts, which are widely used for the diagnosis, have not been standardized. This study was performed to standardize Japanese hop and mugwort pollen extracts. MATERIALS AND METHODS: Allergen extracts were prepared in a standardized way using locally collected Humulus japonicus and purchased Artemisia vulgaris pollens. The immunoglobulin E (IgE) reactivities of prepared extracts were compared with commercial extracts via IgE immunoblotting and inhibition analyses. Intradermal skin tests were performed to determine the bioequivalent allergy unit (BAU). RESULTS: The IgE reactive components of the extracts via IgE immunoblotting were similar to those of commercial extracts. A 11-kDa allergen showed the strongest IgE reactivity in Japanese hop, as did a 28-kDa allergen in mugwort pollen extracts. Allergenic potencies of the investigatory Japanese hop and mugwort extracts were essentially indistinguishable from the commercial ones. Sums of erythema of 50 mm by the intradermal skin test (SigmaED50) were calculated to be 14.4th and 13.6th three-fold dilutions for Japanese hop and mugwort extracts, respectively. Therefore, the allergenic activity of the prepared extracts was 90827.4 BAU/mg for Japanese hop and 34412 BAU/mg for mugwort. CONCLUSION: We produced Japanese hop and mugwort pollen extracts using a standardized method. Standardized Japanese hop and mugwort pollen extracts will facilitate the production of improved diagnostic and immunotherapeutic reagents.

Allergenic Characterization of a Novel Allergen, Homologous to Chymotrypsin, from German Cockroach

PURPOSE: Cockroach feces are known to be rich in IgE-reactive components. Various protease allergens were identified by proteomic analysis of German cockroach fecal extract in a previous study. In this study, we characterized a novel allergen, a chymotrypsin-like serine protease. METHODS: A cDNA sequence homologous to chymotrypsin was obtained by analysis of German cockroach expressed sequence tag (EST) clones. The recombinant chymotrypsins from the German cockroach and house dust mite (Der f 6) were expressed in Escherichia coli using the pEXP5NT/TOPO vector system, and their allergenicity was investigated by ELISA. RESULTS: The deduced amino acid sequence of German cockroach chymotrypsin showed 32.7 to 43.1% identity with mite group 3 (trypsin) and group 6 (chymotrypsin) allergens. Sera from 8 of 28 German cockroach allergy subjects (28.6%) showed IgE binding to the recombinant protein. IgE binding to the recombinant cockroach chymotrypsin was inhibited by house dust mite chymotrypsin Der f 6, while it minimally inhibited the German cockroach whole body extract. CONCLUSIONS: A novel allergen homologous to chymotrypsin was identified from the German cockroach and was cross-reactive with Der f 6.

In Vitro Evaluation of Allergen Potencies of Commercial House Dust Mite Sublingual Immunotherapy Reagents

PURPOSE: The clinical efficacy of allergen-immunotherapy is known to be dose dependent. However, optimal maintenance dosage has not yet been determined for sublingual immunotherapy (SLIT). Furthermore, since companies adopt their own units for expression of allergenicity, the allergen concentrations of individual reagents cannot be compared easily. We sought to measure and compare the allergenicities of 3 commercially available house dust mite (HDM) SLIT regents and a subcutaneous immunotherapy reagent. METHODS: We measured the HDM allergenic potency of the maintenance dosages of three SLIT reagents: Staloral(R) (300 index of reactivity [IR] /mL, recommended maintenance dosage [MD]: 120 IR), SLITone(R) (1,000 standard therapeutic unit [STU]/mL, recommended MD: 200 STU), Wolwopharma(R) (100 microg/mL, recommended MD: 20 microg), and subcutaneous immunotherapy regents of Hollister-Stier (10,000 allergy unit [AU] /mL). The allergenic potency was assessed by measuring the total protein concentrations, mite group 1 and 2 allergens using 2-site ELISA, and an inhibition test against IgE specific to Dermatophagoides farinae and Dermatophagoides pteronyssinus. RESULTS: The protein content of the Wolwopharma(R) reagent was 1.5-261.4 times higher than that of the other 2 SLIT reagents. The concentration of group 1 major allergens in Staloral(R) (132.03 microg/mL) was 33- to 44.5-fold higher than in SLITone(R) (4.00 microg/mL) and Wolwopharma(R) (2.97 microg/mL). The concentration of group 2 major allergen was also 8.9- to 10.5-fold higher in Staloral(R) (15.7 microg/mL) than in SLITone(R) (1.8 microg/mL) or Wolwopharma(R) (1.5 microg/mL). An ELISA inhibition study against HDM-specific IgE showed that the allergen potency of Staloral(R) reagent is 8.5-fold and 21-fold higher than that of SLITone(R) or Wolwopharma(R), respectively. The differences between the maintenance dosages are further exaggerated by the differences in the recommended volumes of SLIT reagents. CONCLUSIONS: The allergen potencies of commercially available HDM SLIT reagents are markedly different. Consensus regarding the optimal allergen concentration for SLIT reagents used to treat HDM respiratory allergies is needed.

Profiles of IgE Sensitization to Der f 1, Der f 2, Der f 6, Der f 8, Der f 10, and Der f 20 in Korean House Dust Mite Allergy Patients

PURPOSE: Measurement of IgE specific to purified house dust mite (HDM) allergens may improve allergy diagnosis. This study aimed to investigate the sensitization profiles of Korean HDM allergic subjects suffering from respiratory allergy and atopic dermatitis (AD) to Der f 1, Der f 2, Der f 6, Der f 8, Der f 10, and Der f 20. METHODS: Recombinant HDM allergens were produced in Pichia pastoris (Der f 1) or Escherichia coli (5 allergens). IgE reactivity to the individual recombinant allergens and total extract of mite was assessed by ELISA. RESULTS: Der f 1 was recognized by 79.1%, Der f 2 by 79.1%, Der f 6 by 9.3%, Der f 8 by 6.2%, Der f 10 by 6.2%, and Der f 20 by 6.6% of the patients' sera tested, while the prevalence of IgE reactivity to total mite extract was 94.7%. Combination of Der f 1 and Der f 2 had a sensitivity of 87.6%. Specific IgE to Der f 2 alone was detected from 89.4% of HDM-sensitized respiratory allergy subjects and 92.3% to the combination of the 2 major allergens Der f 1 and Der f 2. However, sera from fewer patients with AD, namely 72.4% and 71.0%, recognized Der f 1 and Der f 2, respectively. The combination of 2 major allergens allowed diagnosis of 84.5% of the AD patients. No correlation between sensitization to specific allergens and HDM allergy entity was found. CONCLUSIONS: Der f 2 was the most frequently sensitized allergen among the HDM-sensitized respiratory and AD patients in Korea, and the combination of the group 1 and 2 major allergens increased the diagnostic sensitivity. Minor allergens did not significantly improve diagnostic sensitivity. However, further studies are needed to analyze the relationship between sensitization to other HDM allergens and the disease entity of the HDM allergy.