Initial updosing phase of oral immunotherapy improves quality of life and psychological burden in parents of children with food allergy.

Background: Oral immunotherapy (OIT) can impose psychological burdens on patients and their parents due to the necessary preparations and repeated adverse reactions. Objective: To investigate changes in quality of life (QoL) and psychological burden in parents of children receiving OIT for food allergy (FA). Methods: Children aged 3-13 years with FA were enrolled. Parents were asked to fill out the Korean versions of the Food Allergy Quality of Life-Parental Burden (FAQL-PB), the Korean versions of the Food Allergy Quality of Life-Parental Form (K-FAQLQ-PF), the Korean versions of the Beck Anxiety Inventory (K-BAI), and the Korean version of the Patient Health Questionnaire-9 (PHQ-9) for depression before OIT (T1), after 2 months of updosing (T2), and after the end of the updosing phase (T3). Results: A total of 111 parents were enrolled. The total FAQL-PB scores were decreased at T2 and T3 compared with those at T1 (all p < 0.001). Greater improvement in the total FAQL-PB score at T2 was noted in parents with a higher parental burden (FAQL-PB score ≥ 74 points) at baseline than in those with a lower parental burden (p = 0.001). Among the K-FAQLQ-PF domains, "food anxiety" scores were decreased at T2 and T3 compared with those at T1 (p = 0.049 and p = 0.030, respectively), whereas there was no change in "social and dietary limitation" and "emotional impact" scores between T1 and T2 and between T1 and T3. However, no differences were observed in K-BAI and PHQ-9 scores between T1 and T2 and between T1 and T3. Conclusion: Our results suggest that OIT improves parental burden and QoL in parents of children with FA.

Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion.

PURPOSE: Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein. METHODS: Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry. RESULTS: Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER. CONCLUSIONS: Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.

Analysis of glucocorticoid-induced MYOC expression in human trabecular meshwork cells.

To understand the regulatory mechanisms governing glucocorticoid-mediated MYOC induction in human trabecular meshwork (HTM) cells, the expression and degradation of MYOC mRNA were quantified in HTM cells by Northern blot analysis, and the transcriptional activity of constructs containing variable lengths of putative MYOC promoters was assessed by luciferase reporter assay. Here, we confirmed that MYOC is a delayed secondary glucocorticoid-responsive gene by demonstrating that its transcription was not initiated immediately by the addition of dexamethasone (DEX) and was completely inhibited by treatment with cycloheximide. In addition, we demonstrated that MYOC mRNA is degraded very slowly, with approximately half persisting for at least 4 days, suggesting that its mRNA is intrinsically quite stable. Promoter analysis of up to 5271 base pairs upstream of MYOC revealed that luciferase induction by DEX was increased by 280 ± 34% in HTM cells. Moreover, DEX induction required the region between base pairs -2548 and -1541. However, the putative regulatory element exhibited little activity in other cell lines, including TM-5, 293A, SH-SY5Y, and human retinal pigment epithelium (RPE) cells. To our knowledge, this study provides the first evidence for the presence of a cis-acting region for secondary glucocorticoid responsiveness in the 5'-flanking sequences of MYOC. It will be a major step towards understanding the expression pattern of MYOC in HTM cells and TM tissue.

Little evidence for association of the glaucoma gene MYOC with open-angle glaucoma.

BACKGROUND/AIM To determine if overexpression of the glaucoma gene MYOC is involved in the development of open-angle glaucoma (OAG) and if its promoter variants are associated with glaucoma in the Korean population. METHODS Human trabecular meshwork cells were cultured in the presence of ophthalmic steroids such as fluorometholone, fluorometholone acetate, dexamethasone, prednisolone acetate and rimexolone. The cells were cultured at a hydrostatic pressure of 32 mm Hg above atmospheric pressure and induction of MYOC was evaluated by northern blot analysis. Genomic DNA was extracted from blood samples obtained from 74 normal controls and 168 unrelated Korean patients with OAG, including primary OAG, normal tension glaucoma and steroid-induced glaucoma. A 461 base pair (bp) DNA fragment of the MYOC promoter region was amplified using PCR and its genotype was analysed by directly sequencing the product. RESULTS The potencies of steroid eye drops in MYOC induction in vitro was the same regardless of their potential for elevating intraocular pressure in vivo. Hydrostatic pressure had no effect on MYOC induction. A dinucleotide repeat polymorphism and three single nucleotide polymorphisms were identified, but no obvious differences in the genotype distribution and allele frequency of the variants between the control group and any type of OAG were observed. CONCLUSION Our data suggest that MYOC overexpression is not a cause or an effect of intraocular pressure elevation and that MYOC itself is not associated with OAG.

Identification of flotillin-1 as a protein interacting with myocilin: implications for the pathogenesis of primary open-angle glaucoma.

Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.

Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells.

MYOC encoding a 55kDa secretory glycoprotein named myocilin is closely linked to primary open-angle glaucoma (POAG). To understand a role played by MYOC in glaucoma, we examined the cellular fate of various mutant myocilins that were adenovirally expressed in human trabecular meshwork cells. Most myocilins with mutations such as G364V, Q368X, K423E, Y437H, and I477N were intrinsically stable, and appeared to have interactions with wild-type myocilin but not with stromelysin and thereby selectively inhibited the secretion of the former protein. The myocilins expressed were identified to be concentrated into fine punctate aggregates in endoplasmic reticulum, but never developed into the formation of aggresomes. In endoplasmic reticulum, the accumulation of the myocilins resulted in the upregulation of 78kDa glucose-regulated protein and protein disulfide isomerase. In addition, the expression of the myocilins led to deformed cellular morphology and diminished cell proliferation, an effect postulated to result in the dysfunction of trabecular cells that could be a cause of glaucoma. Therefore, our results support the statement that gain of function rather than haploinsufficiency is a critical mechanism for POAG in individuals with mutations on MYOC.