Wiparat Manuyakorn

Nanomechanical assessment of human and murine collagen fibrils via atomic force microscopy cantilever-based nanoindentation

The nanomechanical assessment of collagen fibrils via atomic force microscopy (AFM) is of increasing interest within the biomedical research community. In contrast to conventional nanoindentation there exists no common standard for conducting experiments and analysis of data. Currently used analysis approaches vary between studies and validation of quantitative results is usually not performed, which makes comparison of data from different studies difficult. Also there are no recommendations with regards to the maximum indentation depth that should not be exceeded to avoid substrate effects. Here we present a methodology and analysis approach for AFM cantilever-based nanoindentation experiments that allows efficient use of captured data and relying on a reference sample for determination of tip shape. Further we show experimental evidence that maximum indentation depth on collagen fibrils should be lower than 10-15% of the height of the fibril to avoid substrate effects and we show comparisons between our and other approaches used in previous works. While our analysis approach yields similar values for indentation modulus compared to the Oliver-Pharr method we found that Hertzian analysis yielded significantly lower values. Applying our approach we successfully and efficiently indented collagen fibrils from human bronchi, which were about 30 nm in size, considerably smaller compared to collagen fibrils obtained from murine tail-tendon. In addition, derived mechanical parameters of collagen fibrils are in agreement with data previously published. To establish a quantitative validation we compared indentation results from conventional and AFM cantilever-based nanoindentation on polymeric samples with known mechanical properties. Importantly we can show that our approach yields similar results when compared to conventional nanoindentation on polymer samples. Introducing an approach that is reliable, efficient and taking into account the AFM tip shape, we anticipate that the present work may act as a guideline for conducting AFM cantilever-based nanoindentation of collagen fibrils. This may aid understanding of collagen-related diseases such as asthma, lung fibrosis or bone disease with potential alterations of collagen fibril mechanics.

Phenobarbital‐induced severe cutaneous adverse drug reactions are associated with CYP2C19*2 in Thai children

Aromatic anticonvulsant–induced severe cutaneous adverse drug reactions (SCARs), including Stevens–Johnson syndrome (SJS), toxic epidermal necrosis (TEN), and drug rash with eosinophilia and systemic symptoms (DRESS), are fatal immune‐mediated adverse drug reactions. CYP2C19, a cytochrome P450 isoform, plays a role in metabolic rate of aromatic anticonvulsant. HLA‐B*1502 has also been demonstrated to be associated with carbamazepine‐induced SJS‐TEN.

Regulation of A Disintegrin And Metalloprotease-33 Expression by Transforming Growth Factor-β

The asthma susceptibility gene, a disintegrin and metalloprotease-33 (ADAM33), is selectively expressed in mesenchymal cells, and the activity of soluble ADAM33 has been linked to angiogenesis and airway remodeling. Transforming growth factor (TGF)-β is a profibrogenic growth factor, the expression of which is increased in asthma, and recent studies show that it enhances shedding of soluble ADAM33. In this study, we hypothesized that TGF-β also affects ADAM33 expression in bronchial fibroblasts in asthma. Primary fibroblasts were grown from bronchial biopsies from donors with and those without asthma, and treated with TGF-β(2) to induce myofibroblast differentiation. ADAM33 expression was assessed using quantitative RT-PCR and Western blotting. To examine the mechanisms whereby TGF-β(2) affected ADAM33 expression, quantitative methylation-sensitive PCR, chromatin immunoprecipitation, and nuclear accessibility assays were conducted on the ADAM33 promoter. We found that TGF-β(2) caused a time- and concentration-dependent reduction in ADAM33 mRNA expression in normal and asthmatic fibroblasts, affecting levels of splice variants similarly. TGF-β(2) also induced ADAM33 protein turnover and appearance of a cell-associated C-terminal fragment. TGF-β(2) down-regulated ADAM33 mRNA expression by causing chromatin condensation around the ADAM33 promoter with deacetylation of histone H3, demethylation of H3 on lysine-4, and hypermethylation of H3 on lysine-9. However, the methylation status of the ADAM33 promoter did not change. Together, these data suggest that TGF-β(2) suppresses expression of ADAM33 mRNA in normal or asthmatic fibroblasts. This occurs by altering chromatin structure, rather than by gene silencing through DNA methylation as in epithelial cells. This may provide a mechanism for fine regulation of levels of ADAM33 expression in fibroblasts, and may self-limit TGF-β(2)-induced ectodomain shedding of ADAM33.

Airway remodelling in asthma: role for mechanical forces

Asthma is a chronic airway inflammatory disease with functional and structural changes, leading to bronchial hyperresponsiveness and airflow obstruction. Airway structural changes or airway remodelling consist of epithelial injury, goblet cell hyperplasia, subepithelial layer thickening, airway smooth muscle hyperplasia and angiogenesis. These changes were previously considered as a consequence of chronic airway inflammation. Even though inhaled corticosteroids can suppress airway inflammation, the natural history of asthma is still unaltered after inhaled corticosteroid treatment. As such there is increasing evidence for the role of mechanical forces within the asthmatic airway contributing to airway structural changes.

Association analysis of CYP2C9*3 and phenytoin-induced severe cutaneous adverse reactions (SCARs) in Thai epilepsy children

CYP2C9 is the key enzyme in aromatic antiepileptic drugs (AEDs) metabolism. CYP2C9*3 is a loss of function polymorphism. This study was designed to investigate genetic association between CYP2C9*3 and aromatic AED-induced severe cutaneous adverse reactions (SCARs) in Thai children. The 37 aromatic AED-induced SCARs patients (20 phenobarbital and 17 phenytoin) and 35 tolerances (19 phenobarbital and 16 phenytoin) were enrolled. CYP2C9*3 was genotyped by allele-specific PCRs. The association between CYP2C9*3 with phenytoin-induced SCARs and phenobarbital-induced SCARs were analyzed in comparison with tolerances and healthy samples. Significant association between phenytoin-induced SCARs and CYP2C9*3 was discovered (odds ratio=14.52; 95% confidence interval (CI)=1.18–∞, P-value=0.044). CYP2C9*3 was not associated with phenobarbital-induced SCARs. This study is the first report of CYP2C9*3 association to phenytoin-induced SCARs in Thai epileptic children. The CYP2C9*3 is a reasonable predictive genetic marker to anticipate SCARs from phenytoin.

Pediatric anaphylaxis: triggers, clinical features, and treatment in a tertiary-care hospital

BACKGROUND Anaphylaxis is a life-threatening condition. There are limited data about its etiology and clinical characteristics in Asian children with anaphylaxis. OBJECTIVE To investigate triggers, presenting symptoms, treatment and clinical course of anaphylaxis in Thai children. METHOD Medical record of children who were diagnosed with anaphylaxis between 2004 and 2013 at Ramathibodi Hospital, Bangkok, Thailand were reviewed. RESULTS One hundred-seventy two episodes of anaphylaxis occurred in 160 children (91 boys, 69 girls) aged 3 months to 18 years. Anaphylaxis increased from 2.7 cases/1000 pediatric admission to 4.51 cases/1000 pediatric admission between 2004-2008 and 2009-2013. The main causes were food (34.92%), drug (33.1%), blood components (23.8%), insect sting (9%), and unidentified causes (2.8%). Allergy to the triggers was known prior to anaphylaxis in 42 episodes (24.6%). Treatment consisted of epinephrine intramuscularly (93.8%), corticosteroids (92.5%), H₁antihistamines (96%), H₂antihistamines (50%), and β₂agonists nebulization (35.1%). Biphasic anaphylaxis occurred in 8.7% of the documented episodes and severe anaphylaxis in 34.3% of the documented episodes. Biphasic anaphylaxis and severe anaphylaxis were associated with fewer administrations of intramuscular epinephrine (OR 0.08 [95% CI 0.014-0.43]; p =0.01 and OR 9.36 [95% CI 2.5-34.7]; p <0.001 respectively). There were no fatality cases. There were associations between triggers of anaphylaxis and atopic histories, patients with severe anaphylaxis and cardiovascular involvement (p <0.01). CONCLUSIONS The incidence of anaphylaxis in Thai children is increasing. Anaphylaxis in children commonly occurred without the histories of prior reaction to the causative agent. Less frequent treatment with intramuscular epinephrine was associated with biphasic and severe anaphylaxis. A better knowledge of patterns and causes of anaphylaxis might contribute to a better management.

Association of HLA genotypes with phenobarbital hypersensitivity in children

Phenobarbital hypersensitivity is one of the common drug hypersensitivity syndromes in children. Clinical symptoms of phenobarbital hypersensitivity vary from maculopapular rashes (MPs) to severe cutaneous adverse drug reactions (SCARs) including drug reactions with eosinophilia and systemic symptoms (DRESS), Stevens‐Johnson syndrome (SJS), and toxic epidermal necrolysis (TEN). Drug hypersensitivity has been demonstrated to be associated with variations in the HLA genotypes. This study was to investigate the association between the variations of HLA genotypes and phenobarbital hypersensitivity in Thai children.

Mechanical strain causes adaptive change in bronchial fibroblasts enhancing profibrotic and inflammatory responses

Asthma is characterized by periodic episodes of bronchoconstriction and reversible airway obstruction; these symptoms are attributable to a number of factors including increased mass and reactivity of bronchial smooth muscle and extracellular matrix (ECM) in asthmatic airways. Literature has suggested changes in cell responses and signaling can be elicited via modulation of mechanical stress acting upon them, potentially affecting the microenvironment of the cell. In this study, we hypothesized that mechanical strain directly affects the (myo)fibroblast phenotype in asthma. Therefore, we characterized responses of bronchial fibroblasts, from 6 normal and 11 asthmatic non-smoking volunteers, exposed to cyclical mechanical strain using flexible silastic membranes. Samples were analyzed for proteoglycans, α-smooth muscle actin (αSMA), collagens I and III, matrix metalloproteinase (MMP) 2 & 9 and interleukin-8 (IL-8) by qRT-PCR, Western blot, zymography and ELISA. Mechanical strain caused a decrease in αSMA mRNA but no change in either αSMA protein or proteoglycan expression. In contrast the inflammatory mediator IL-8, MMPs and interstitial collagens were increased at both the transcriptional and protein level. The results demonstrate an adaptive response of bronchial fibroblasts to mechanical strain, irrespective of donor. The adaptation involves cytoskeletal rearrangement, matrix remodelling and inflammatory cytokine release. These results suggest that mechanical strain could contribute to disease progression in asthma by promoting inflammation and remodelling responses.

Stevens-Johnson Syndrome and Toxic Epidermal Necrolysis Standard Reporting and Evaluation Guidelines: Results of a National Institutes of Health Working Group

Importance Toxic epidermal necrolysis (TEN) and Stevens-Johnson Syndrome (SJS) are rare, acute, life-threatening dermatologic disorders involving the skin and mucous membranes. Research into these conditions is hampered by a lack of standardization of case reporting and data collection. Objective To establish a standardized case report form to facilitate comparisons and maintain data quality based on an international panel of SJS/TEN experts who performed a Delphi consensus-building exercise. Evidence Review The elements presented for committee scrutiny were adapted from previous case report forms and from PubMed literature searches of highly cited manuscripts pertaining to SJS/TEN. The expert opinions and experience of the members of the consensus group were included in the discussion. Findings Overall, 21 out of 29 experts who were invited to participate in the online Delphi exercise agreed to participate. Surveys at each stage were administered via an online survery software tool. For the first 2 Delphi rounds, results were analyzed using the Interpercentile Range Adjusted for Symmetry method and statements that passed consensus formulated a new case report form. For the third Delphi round, the case report form was presented to the committee, who agreed that it was “appropriate and useful” for documenting cases of SJS/TEN, making it more reliable and valuable for future research endeavors. Conclusions and Relevance With the consensus of international experts, a case report form for SJS/TEN has been created to help standardize the collection of patient information in future studies and the documentation of individual cases.

Efficacy of Nasal Cellulose Powder in the Symptomatic Treatment of Allergic Rhinitis: A Randomized, Double-Blind, Placebo-Controlled Trial

Purpose Nasal Cellulose Powder (NCP), which can prevent from binding an allergen to nasal mucosa, may reduce allergic rhinitis (AR) symptoms in dust mite-sensitized children. This study was conducted to assess the efficacy of NCP in improving clinical symptoms of a nasal airflow limitation and the response of nasal inflammatory cells. Methods Children with dust mite-sensitized AR aged 6-18 years were recruited. After a 4-week run-in period, NCP or a placebo was administered, 1 puff per nostril 3 times daily for 4 weeks. The nasal provocation test (NPT) with Dermatophagoides pteronyssinus (Der p) was performed before and after treatment. The daily symptom scores (DSS), daily medication scores (DMS), the peak nasal inspiratory flows (PNIF), nasal airway resistance (NAR), as well as the maximum tolerated dose of NPT and eosinophil counts in nasal scraping, were evaluated. Results Sixty children (30 NCP and 30 placebos) were enrolled. Before treatment, there were no significant differences in age, dust mite control measures, DSS, DMS, PNIF, NAR, the maximum tolerated dose of NPT, or nasal eosinophil scores between children receiving NCP and placebos. After treatment, there were no significant differences between the NCP and placebo groups in the median (range) of the outcomes—DSS: 2.06 (0.18-3.77) vs 1.79 (0.08-7.79), P=0.756; DMS: 1.60 (0-5.13) vs 0.56 (0-4.84), P=0.239; PNIF (L/min): 110 (60-160) vs 100 (50-180), P=0.870; NAR (Pa/cm3/s): 0.40 (0.20-0.97) vs 0.39 (0.24-1.32), P=0.690; the maximum tolerated dose of NPT and the nasal eosinophil scores: 1 (0-4) vs 1 (0-4), P=0.861. Conclusions NCP treatment may not be more effective than placebo treatment in dust mite-sensitized AR children.