Evaluation of recombinant MGL_1304 produced by Pichia pastoris for clinical application to sweat allergy

AbstractBackgroundWe previously identified MGL_1304 secreted by Malassezia globosa as a sweat antigen for patients with atopic dermatitis (AD) and cholinergic urticaria (ChU). However, purifying native MGL_1304 from human sweat or culture supernatant of M. globosa (sup-MGL_1304) is costly and time-consuming. Moreover, recombinant MGL_1304 expressed by using Escherichia coli (TF-rMGL_1304) needs a large chaperon protein and lacks the original glycosylation of yeasts. Thus, we generated a recombinant MGL_1304 by Pichia pastoris (P-rMGL_1304) and investigated its characteristic features.MethodsRecombinant MGL_1304 proteins expressed by E. coli and P. pastoris were generated. Properties of these recombinants and native antigens were compared by western blot analysis, histamine release tests (HRT) of patients with AD and ChU, and β-hexosaminidase release tests with RBL-48 cells. P-rMGL_1304-specific IgE in sera of patients with AD were measured by sandwich ELISA.ResultsWestern blot analysis revealed that IgE of patients with AD bound to all MGL_1304 recombinants and native antigens. The histamine releasing ability of P-rMGL_1304 was 100 times higher than that of TF-rMGL_1304, and was comparable to that of sup-MGL_1304. Degranulation rates of RBL-48 cells, sensitized with sera of patients with AD in response to the stimulation of P-rMGL_1304, were comparable to those of sup-MGL_1304, whereas those of TF-rMGL_1304 were relatively weak. The levels of P-rMGL_1304-specific IgE in sera of patients with AD were correlated with their disease severities.ConclusionsP-rMGL_1304 has an antigenicity comparable to the native antigen, and is more useful than TF-rMGL_1304, especially in HRT and degranulation assay of RBL-48 cells

Pilot Validation Study of the Japanese Translation of the Brief Negative Symptoms Scale (BNSS)

Purpose: The brief negative symptoms scale (BNSS) is a concise instrument used to assess negative symptoms of subjects with schizophrenia covering five domains of negative symptoms and is suitable for use in clinical, experimental, and epidemiological settings. The original and translated version of BNSS has thus far been shown to have adequate psychometric properties. This study aimed to examine internal consistency, inter-rater and test-retest reliability, discriminant and convergent validity, and factor structure of the Japanese version of BNSS. Patients and methods: The assessment was performed by 11 raters using interview videos of nine subjects. Reliability was calculated with Cronbach's alpha for internal consistency and intra class correlation coefficient (ICC) for inter-rater reliability. Pearson's correlation coefficients were calculated to estimate the test-retest reliability. In addition to BNSS, Scale for assessment of negative symptoms (SANS) and scale for assessment of positive symptoms (SAPS) was obtained to assess the convergent and discriminant validity. Factor structure was assessed using principle factor analysis. Results: The Japanese BNSS showed excellent internal consistency (Cronbach's alpha=0.95), inter-rater reliability (intra class correlation coefficient=0.97), and test-retest reliability (r=0.94, p<0.001). The convergent validity shown by correlation with SANS total score (r=0.87, p<0.001) and discriminant validity shown by correlation with SAPS total score (r=0.17, p=-0.68) were also good. Principal factor analysis revealed a two-factor structure of BNSS, although the loading of each item differed from that in the literature. Conclusion: Our pilot study demonstrated that Japanese BNSS had good psychometric properties which were achieved with relatively brief training. Further studies with more subjects and raters with various backgrounds recruited from multiple sites are warranted