Exploring the role of growth factors as potential regulators in psoriatic plaque formation.

Psoriasis is a chronic inflammatory skin disease in which growth activity is more prominent than inflammatory activity at the centre of lesional skin (CE skin). This growth activity is partly influenced by growth factors (GFs) that play an important role in cell growth and inflammation during the plaque development. In this study, we identified potential GFs in CE skin and predicted their regulatory functions and biological activity in mediating transcripts in the plaques. Samples of uninvolved skin (UN skin) and CE skin were biopsied from patients with psoriasis vulgaris for RNA-sequencing analysis in order to identify differentially expressed genes (DEGs). Our finding revealed that epidermal growth factor (EGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) and hepatocyte growth factor (HGF) signalling were enriched by CE/UN skin-derived DEGs. Additionally, several EGFR ligands, namely EGF, heparin-binding EGF like growth factor (HB-EGF), amphiregulin (AREG) and transforming growth factor (TGF)-α, as well as TGF-ß1, TGF-ß2, vascular endothelial growth factor-A, FGFs, PDGF-B and HGF, were predicted to be GF regulators. The regulatory pattern and biological activity of these GF regulators on mediating the CE/UN skin-derived DEGs was demonstrated. This study provides a novel hypothesis regarding the overall regulatory function of GFs, which appear to modulate the expression of the transcripts involved in inflammation and growth in the CE skin. In addition, some GFs may exert anti-inflammatory effects. Further investigations on the mechanisms underlying this regulation may contribute to a deeper understanding of psoriasis and the identification of potential therapeutic targets for patients with psoriasis.

Exosomal microRNAs from PBMCs stimulated with culprit drugs enhanced keratinocyte cell death in Stevens-Johnson syndrome/toxic epidermal necrolysis.

BACKGROUND: Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reactions with eosinophilia and systemic symptoms (DRESS) are both severe cutaneous adverse reactions. Keratinocyte death is much more prominent in SJS/TEN compared to DRESS. OBJECTIVE: This study aimed to investigate the role of exosomal miRNAs on keratinocyte death in SJS/TEN. METHODS: Peripheral blood mononuclear cells (PBMCs) from SJS/TEN and DRESS patients were stimulated with the culprit drugs. The exosomes released in cell supernatants were co-incubated with HaCaT cells to study the cytotoxic effects on keratinocytes. Exosomal miRNA sequencing analysis was performed to compare the expression patterns between SJS/TEN and DRESS subjects. HaCaT cells were then transfected with miRNA mimics and inhibitors to explore the functions of miRNAs on keratinocyte cell death. RESULTS: Cytotoxic effects of PBMC-derived exosomes on keratinocytes were demonstrated in SJS/TEN and could be neutralized with exosome inhibitors. Cytotoxic effects of PBMC-derived exosomes from SJS/TEN subjects were higher after incubating PBMCs with the culprit drugs than those incubating with irrelevant drugs and unstimulated controls. The sequencing data revealed differential expressions of 61 exosomal miRNAs between SJS/TEN and DRESS. Exosomal miR-4488 was upregulated while miR-486-5p, miR-96-5p and miR-132-3p were downregulated in SJS/TEN compared to DRESS as determined by quantitative real-time PCR. The increased percentage of apoptotic cells upon transfection of HaCat cells was 36.3% and 34.9% with miR-4488 mimic and miR-96-5p inhibitor, respectively. CONCLUSION: This study illustrated the regulatory functions of exosomal miRNAs in controlling keratinocyte death in SJS/TEN. Exosome inhibitors might have a therapeutic role in SJS/TEN.

Determination of specific autoantibodies in patients with systemic lupus erythematosus by Line immunoassay, ELISA and CLIF assay.

BACKGROUND: Detection of specific antinuclear-antibodies is very importance in term of diagnosis, prognosis and management of patients with systemic lupus erythematosus (SLE). To date, Line immunoassay (LIA), enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae indirect immunofluorescence (CLIF) assay are commonly used for detection of specific antinuclear-antibodies. OBJECTIVE: To determine the performance of LIA, ELISA and CLIF for the detection of anti-double-stranded DNA (dsDNA), anti-nucleosome, and anti-extractable nuclear antigens (ENA) antibodies in patients with SLE. METHODS: A total 100 sera from 50 patients with SLE, 25 patients with disease control and 25 healthy control subjects were tested for anti-dsDNA, anti-nucleosome, and anti-ENA antibodies by LIA, ELISA, and CLIF assay. Agreement and diagnostic performance of each assay were analyzed using Cohen's kappa coefficient and receiver operating characteristic curve analysis. RESULTS: For the detection of anti-dsDNA antibody, ELISA had a substantial agreement with CLIF assay (? = 0.74) but LIA had a fair agreement with ELISA and CLIF assay (? = 0.37, and 0.35 respectively). For the detection of anti-nucleosome, anti-nRNP/Sm, anti-Sm, anti-SSA, and anti-SSB antibodies, LIA had a substantial to perfect agreement with ELISA (? = 0.64, 0.78, 0.68, 0.91, and 0.74, respectively). Anti-dsDNA-NcX ELISA and anti-dsDNA CLIF assay had equally diagnostic performance (sensitivity, 66% vs. 68%, and specificity, 96% vs. 94%, respectively) whereas, anti-dsDNA LIA has low sensitivity (22%) but high specificity (100%). CONCLUSIONS: LIA, ELISA, and CLIF demonstrated comparable performance for the detection of specific antinuclear-antibodies. However, there were some discrepancy between assays particularly in the detection of anti-dsDNA antibody.

Comparison of Malassezia spp. colonization between human skin exposed to high- and low-ambient air pollution.

The skin microbiota is essential for human health; altered skin microbiome colonization and homeostasis may be associated with several inflammatory skin conditions and other inflammatory diseases. Malassezia spp. are commensal fungi commonly found on the human skin, and they also play a pathogenic role in various skin diseases. It is hypothesized that the exposure of human skin to air pollution might be associated with Malassezia spp. colonization. The aim of this study was to compare Malassezia spp. colonization on healthy human skin between people living in two major cities in Thailand with different air qualities: one city with highly polluted ambient air and the other with less polluted air. Skin microbiome samples from 66 participants were collected using swabbing and scraping techniques. The skin fungal composition was analysed using high-throughput sequencing based on internal transcribed spacer 2 (ITS2) rDNA. A significant difference was found in alpha and beta diversities and the relative abundance of fungal profiles between the groups. The relative abundance of Malassezia spp. was found to be significantly higher in the highly polluted area than in the less polluted area. This study demonstrates that high-ambient air pollution may alter Malassezia spp. colonization on healthy human skin, which could lead to dysbiosis of the cutaneous ecosystem and eventually result in some skin disorders.

Alteration of gut microbiota during narrowband ultraviolet B therapy in psoriasis: A preliminary study.

Gut microbiome dysbiosis is associated with psoriasis development. A relationship between gut microbiota and psoriasis treatment response has been reported. No study has reported the effect of narrowband ultraviolet B (NBUVB) therapy, a standard treatment of psoriasis, on gut microbiota. This study aimed to evaluate gut microbiota change during NBUVB therapy. Stool samples from 22 participants, including 13 patients with chronic plaque psoriasis and nine healthy controls, were recruited. Faecal microbiota composition was analysed using 16S rRNA sequencing before and after NBUVB therapy. Serum 25-OH vitamin D of patients with psoriasis was evaluated simultaneously. The most abundant phyla of gut microbiota in patients with psoriasis were Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria in all participants. Bilophila, Paraprevotella, Alistipes, Sutterella, Romboutsia, Clostridium sensu stricto and Agathobacter are significantly more enriched in healthy controls. Lactobacillales and Ruminococus torques appeared more enriched after NBUVB treatment in responders but not non-responders. Serum vitamin D levels significantly increased after NBUVB treatment. The present study revealed that gut microbiota altered after NBUVB treatment. The change might be treatment-specific and influence the treatment response.

Analysis of cutaneous bacterial microbiota of Thai patients with seborrheic dermatitis.

Seborrheic dermatitis (SD) is a chronic inflammatory skin condition that occurs in body areas that contain profuse sebaceous glands. Skin microbiota are diverse across ethnic groups and its dysbiosis has been implicated in the pathogenesis of SD. Here, we reported the contribution of cutaneous bacterial microbiota to SD in the Thai population. Healthy individuals and patients with scalp SD were recruited into the study. Normal skin, scalp skin lesion (SL) and non-lesion sites (SNL) samples were collected using a tape stripping method and next-generation sequencing of 16S rRNA for microbiome analysis. Although bacterial diversity in all sample groups was not statistically different, a population of bacteria commonly found on skin of scalp showed signs of dysbiosis. Apart from the reduction of Corynebacterium spp., SD-specific microbiota was dominated by Firmicutes at taxa level and Pseudomonas spp., Staphylococcus spp. and Micrococcus spp. at genus level. The dysbiosis of the skin microbiota in SD was specifically described as an alteration of bacteria populations commonly found on scalp skin, implying that managing and controlling the cutaneous bacterial microbiome can alleviate and prevent SD and pave the way for the development of new SD treatments.

The immediate patch test reaction to fragrance in patients with allergic contact dermatitis to fragrance: A prospective study.

BACKGROUND: Fragrance is one of the common causes of immediate contact reaction. Knowing the prevalence of a reaction in a given population enables prioritization of allergy screening. OBJECTIVE: The purpose of this study was to determine the prevalence of an immediate patch test reaction to fragrance in patients with fragrance allergic contact dermatitis. METHODS: This prospective study enrolled 291 patients who were given standard patch tests for allergic contact dermatitis. Those with positive reactions were then asked to undergo additional patch tests to assess both immediate and delayed reactions to 28 different fragrance substances. RESULTS: Cinnamic aldehyde and cinnamic alcohol were the most frequently encountered substances in positive immediate reactions and standard (delayed) patch test reactions. Immediate patch reactions to benzyl alcohol, sorbic acid, and coumarin were more frequently observed than standard patch test reactions. LIMITATIONS: Because of the small sample size of patients who agreed to continue further patch testing evaluation, a statistical association between patient characteristics and fragrance-positive patch test reactions was difficult to establish. CONCLUSIONS: In this population, cinnamic aldehyde and cinnamic alcohol were the most common fragrance allergens causing both immediate and delayed reactions, whereas reactions to benzyl alcohol, sorbic acid, and coumarin were frequently observed in immediate patch tests.

Transcriptomic Profiling of Peripheral Edge of Lesions to Elucidate the Pathogenesis of Psoriasis Vulgaris.

Elucidating transcriptome in the peripheral edge of the lesional (PE) skin could provide a better understanding of the molecules or signalings that intensify inflammation in the PE skin. Full-thickness biopsies of PE skin and uninvolved (UN) skin were obtained from psoriasis patients for RNA-seq. Several potential differentially expressed genes (DEGs) in the PE skin compared to those in the UN skin were identified. These DEGs enhanced functions such as angiogenesis, growth of epithelial tissue, chemotaxis and homing of cells, growth of connective tissues, and degranulation of myeloid cells beneath the PE skin. Moreover, the canonical pathways of IL-17A, IL-6, and IL-22 signaling were enriched by the DEGs. Finally, we proposed that inflammation in the PE skin might be driven by the IL-36/TLR9 axis or IL-6/Th17 axis and potentiated by IL-36α, IL-36γ, IL-17C, IL-8, S100A7, S100A8, S100A9, S100A15, SERPINB4, and hBD-2. Along with IL-36α, IL-17C, and IκBζ, ROCK2 could be an equally important factor in the pathogenesis of psoriasis, which may involve self-sustaining circuits between innate and adaptive immune responses via regulation of IL-36α and IL-36γ expression. Our finding provides new insight into signaling pathways in PE skin, which could lead to the discovery of new psoriasis targets.

Liver fibrosis improvement assessed by magnetic resonance elastography and Mac-2-binding protein glycosylation isomer in patients with hepatitis C virus infection receiving direct-acting antivirals.

AIM: Fibrosis regression has been observed in patients with chronic hepatitis C virus (HCV) infection treated with direct-acting antivirals. This study was aimed at evaluating dynamic changes of serum Mac-2-binding protein glycosylation isomer (M2BPGi) in patients with HCV genotype 1 receiving elbasvir/grazoprevir. METHODS: M2BPGi were serially measured at baseline, during and after therapy. Its diagnostic performance at baseline and sustained virological response at 24 weeks after treatment (SVR24) were compared with transient elastography (TE) and the aspartate aminotransferase/platelet ratio index (APRI) using magnetic resonance elastography (MRE) as a reference. RESULTS: Overall, 60 HCV mono-infected and 36 HCV/HIV co-infected patients were included with SVR24 rates of 93.3% and 97.2%, respectively. At baseline, TE, M2BPGi and APRI were correlated with MRE (r = 0.788, r = 0.703 and r = 0.564, respectively, p < 0.001). The area under the receiver operator characteristics curves for TE, M2BPGi and APRI in differentiating significant fibrosis were 0.88 (95% confidence interval; 0.81-0.95, p < 0.001), 0.86 (0.79-0.94, p < 0.001) and 0.74 (0.64-0.83, p < 0.001), respectively. The corresponding figures for cirrhosis were 0.95 (0.90-1.00, p < 0.001), 0.96 (0.92-1.00, p < 0.001) and 0.88 (0.79-0.97, p < 0.001), respectively. Compared with baseline, all fibrosis markers significantly declined after achieving SVR24. The correlations of TE, M2BPGi and APRI with MRE at time of SVR24 were r = 0.587 (p < 0.001), r = 0.457 (p < 0.001) and r = 0.293 (p = 0.004), respectively. In multivariate analysis, high baseline alanine aminotransferase level, HCV mono-infection and advanced fibrosis were factors associated with M2BPGi reduction. CONCLUSIONS: HCV eradication is associated with liver fibrosis improvement. M2BPGi has a better performance than APRI in monitoring liver fibrosis in patients treated with direct-acting antivirals. This marker is applicable in resource-limited settings where imaging-based modalities are not widely accessible.

A randomized controlled trial of adding intravenous corticosteroids to H1 antihistamines in patients with acute urticaria.

BACKGROUND: Acute urticaria is a common dermatological condition in emergency departments (EDs). The main therapy involves controlling pruritus with antihistamines. Although guidelines have promoted the use of corticosteroids in addition to H1 antihistamines, well-designed clinical trials evaluating this approach are scarce. METHODS: Adult ED patients with acute urticaria and a pruritus score > 5 on a visual analog scale (VAS) were randomized into three groups: (i) IV chlorpheniramine (CPM) treatment, (ii) IV CPM and IV dexamethasone (CPM/Dex) and (iii) IV CPM and IV dexamethasone with oral prednisolone as discharge medication for 5 days (CPM/Dex/Pred). The primary outcomes were self-reported pruritus VAS scores at 60 min after treatment. We also evaluated 1-week and 1-month urticaria activity scores for 7 days and adverse events. RESULTS: Seventy-five patients (25 per group) were enrolled. The VAS scores of all groups decreased, but no significant difference was found in the VAS scores at 60 min after treatment between patients in the CPM group (n = 25) and those who received both CPM and dexamethasone (n = 50). At the 1-week and 1-month follow-ups, active urticaria (indicated by the urticaria activity score at 7 days) was more prevalent in the CPM/Dex/Pred group (n = 25) than in the control group. CONCLUSIONS: The present study did not find evidence that adding IV dexamethasone improves the treatment of severe pruritus from uncomplicated acute urticaria. Oral corticosteroid therapy may be associated with persistent urticaria activity. Due to the lack of clinical benefits and the potential for side effects, using corticosteroids as an adjunctive treatment is discouraged.

Down-regulation of miR-155 after treatment with narrow-band UVB and methotrexate associates with apoptosis of keratinocytes in psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease arising from a complex interaction between genetics, epigenetics, the host's immune system and the environment. Recent accumulated data revealed the dysregulation of various microRNAs (miRNAs) in several diseases including psoriasis. OBJECTIVE: We explored the functional role and regulation of hsa-miR-155-5p (miR-155) in an immortalized keratinocyte cell line (HaCaT), in relation to the pathogenesis and treatment of psoriasis. METHODS: miR-155 expression in normal skin and psoriatic skin lesion before and after treatment with methotrexate (MTX) and narrow-band ultraviolet B phototherapy (NB-UVB) were analyzed using quantitative reverse transcription PCR (qRT-PCR). Apoptotic activity, cell cycle and viable cells of miR-155 transfected HaCaT were measured using flow cytometry and MTS assay. Since, caspase-3 (CASP3) gene was predicted as a target gene of miR-155, the expression of CASP3 was detected in transfected HaCaT using western blot. RESULTS: We discovered that both MTX and NB-UVB significantly down-regulated miR-155 expression in psoriatic skin lesions. We also found that overexpression of miR-155 in HaCaT led to suppression of cell apoptosis and induced cell arrest at G0/G1 phase. Moreover, CASP3 expression was down-regulated in miR-155 transfected HaCaT. CONCLUSIONS: This study demonstrates down-regulation of miR155 after treatment with MTX and NB-UVB in psoriatic skin lesion. miR155 plays significant role in apoptosis on HaCaT via CASP3. This finding provides a better understanding of the pathogenesis of psoriasis and might aid on developing the new monitoring tool or therapy for psoriasis in the future.

Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan/Alginate Nanoparticles on Tumor Necrosis Factor-Alpha-Induced Psoriasis-Like Proliferation of Keratinocytes.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation of the epidermal cells and is clinically presented as thick, bright red to pink plaques with a silvery scale. Photodynamic therapy (PDT) using visible light has become of increasing interest in the treatment of inflammatory skin diseases. In this study, we demonstrate that a combination of curcumin-loaded chitosan/alginate nanoparticles (Cur-CS/Alg NPs) and blue light emitting diodes (LED) light irradiation effectively suppressed the hyperproliferation of tumor necrosis factor-alpha (TNF-α)-induced cultured human kerlatinocyte (HaCaT) cells. The Cur-CS/Alg NPs were fabricated by emulsification of curcumin in aqueous sodium alginate solution and ionotropic gelation with calcium chloride and chitosan using an optimized formulation derived from a Box-Behnken design. The fabricated Cur-CS/Alg NPs were characterized for their particle size, zeta potential, encapsulation efficiency, and loading capacity. The surrogate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, to measure the relative number of viable cells, showed that the CS/Alg NPs were nontoxic to normal HaCaT cells, while 0.05 µg/mL and 0.1 µg/mL of free curcumin and Cur-CS/Alg NPs inhibited the hyperproliferation of HaCaT cells induced by TNF-α. However, the Cur-CS/Alg NPs demonstrated a stronger effect than the free curcumin, especially when combined with blue light irradiation (10 J/cm²) from an LED-based illumination device. Therefore, the Cur-CS/Alg NPs with blue LED light could be potentially developed into an effective PDT system for the treatment of psoriasis.

Comparison of specific IgE detection by immunoblotting and fluorescence enzyme assay with in vivo skin prick test.

BACKGROUND: Diagnostic tools to identify allergens that cause allergic symptom is important part in the care of allergic patients. Detection of causative allergen can be performed by in vivo skin prick test (SPT) or in vitro tests for detection serum specific immunoglobulin E (sIgE). The common methods used are fluorescent enzyme assay and immunoblotting assay. OBJECTIVE: We aim to evaluate performance of the two sIgE determination systems, immunoblotting assay (Euroline) and fluorescent enzyme assay (ImmunoCAP) in comparison with SPT. METHODS: Two hundred and two participants with allergic diseases were enrolled. Sensitization to common allergens was identified using skin prick test and serum specific IgE assays with Euroline and ImmunoCAP. Both systems provide the result in the same unit and the same cut-off value (0.35 kUA/L). The specific IgE levels of 4 aeroallergens, 6 food allergens and 3 food allergen components were analyzed to evaluate the performance of both sIgE assays with SPT. RESULTS: When compared with the result of SPT, ImmunoCAP has 63.9-93.2% agreement and Euroline has 68.4-86.2% agreement for allergen detection. Both sIgE assays have significant correlation in measuring sIgE of almost all allergens (r=0.626-0.901, p< 0.001) except for dog. For food allergen components, both sIgE tests have outstanding correlation and agreement (r=0.816-0.952, p< 0.001; agreement =87.0-92.9%, respectively). The receiver-operating characteristic curve analysis indicated slight discrepancy of both sIgE assays. CONCLUSIONS: Both sIgE determination systems demonstrate fair to good performance when compared to SPT depending on type of allergens. The two sIgE determination systems had favorable correlation and agreement.

Patterns and risk factors of causative contact allergens in Thai adult patients with contact dermatitis

BACKGROUND: Surveillance on common allergens identified by patch testing plays an important role in emerging allergen detection, which leads to both individual and societal level prevention. OBJECTIVE: To study the changes in the pattern of contact sensitization and to identify risk factors associated with allergens. METHOD: The data of 206 patients who underwent patch testing at King Chulalongkorn Memorial Hospital during 2012 to 2015 were assessed. The associations between patient risk factors and positive reactions to each allergen were evaluated. The results were compared with data from 2003-2004. RESULTS: The top five most common allergens during 2012-2015 were nickel sulfate (19.4%), methylchloroisothiazolinone/ methylisothiazolinone (MCI/MI) (13.6%), fragrance mix I (FM I) (10.7%), carba mix (9.2%) and cobalt chloride (6.3%) whereas, during 2003-2004, these were nickel sulfate, cobalt chloride, FM I, potassium dichromate and Myroxylon pereirae. A positive patch test to nickel was strongly associated with a history of metal and seafood allergy (p<0.001; OR, 4.94; 95% CI = 2.33-10.47 and p=0.028; OR, 2.55; 95% CI, 1.11-5.85, respectively). MCI/MI was correlated with a history of personal care products allergy, and fragrance was correlated with a history of urticaria (p=0.005; OR, 4.05; 95% CI = 1.54-10.66 and p=0.031; OR, 2.71; 95% CI, 1.10-6.68, respectively). CONCLUSIONS: There was an alteration in the pattern of contact sensitization detected by our standard series. MCI/MI has become the most common preservative causing contact allergy.

BCAP 31 expression and promoter demethylation in psoriasis.

BACKGROUND: Psoriasis is the disease of abnormal keratinocyte differentiation and apoptosis. Alterations in DNA methylation leading to keratinocyte hyperproliferation is one of the proposed pathogenic mechanisms of psoriasis. B-cell receptor associated protein (BCAP31) has been reported to be involved in the proliferation and apoptosis of keratinocytes. Up-regulation and changing in BCAP31 promoter methylation has been reported to be associated with some cancers. To date, there has been no study of psoriasis. OBJECTIVE: We investigated BCAP31 protein expression and the status of BCAP31 promoter methylation in psoriasis. METHODS: Ten patients with psoriasis and 10 healthy subjects were enrolled. The immunohistochemistry was performed on paraffin-embedded tissue section to detect BCAP31 protein expression and compared between psoriasis and normal skin. The laser capture micro-dissected keratinocyte were analyzed using bisulfite PCR method and cloning and sequencing. RESULTS: Increased BCAP31 protein expression was observed in psoriatic epidermis compared with normal epidermis. Interestingly the methylation level of the BCAP31 promoter was significantly lower in patients with psoriasis compared with healthy subjects (p < 0.001, % psoriasis vs. normal skin methylation = 14.94 vs. 60.61). CONCLUSION: The present study demonstrated increase expression of BCAP31 protein related to BCAP31 DNA demethylation in psoriasis. Future study is needed to indicate the mechanism of BCAP31 promoter demethylation and its potential use as a novel treatment for psoriasis in the future.

An Outbreak of Acute Hepatitis Caused by Genotype IB Hepatitis A Viruses Contaminating the Water Supply in Thailand.

BACKGROUND: In 2000, an outbreak of acute hepatitis A was reported in a province adjacent to Bangkok, Thailand. AIMS: To investigate the cause of the 2000 hepatitis A outbreaks in Thailand using molecular epidemiological analysis. METHODS: Serum and stool specimens were collected from patients who were clinically diagnosed with acute viral hepatitis. Water samples from drinking water and deep-drilled wells were also collected. These specimens were subjected to polymerase chain reaction (PCR) amplification and sequencing of the VP1/2A region of the hepatitis A virus (HAV) genome. The entire genome sequence of one of the fecal specimens was determined and phylogenetically analyzed with those of known HAV sequences. RESULTS AND CONCLUSIONS: Eleven of 24 fecal specimens collected from acute viral hepatitis patients were positive as determined by semi- nested reverse transcription PCR targeting the VP1/2A region of HAV. The nucleotide sequence of these samples had an identical genotype IB sequence, suggesting that the same causative agent was present. The complete nucleotide sequence derived from one of the samples indicated that the Thai genotype IB strain should be classified in a unique phylogenetic cluster. The analysis using an adjusted odds ratio showed that the consumption of groundwater was the most likely risk factor associated with the disease.

Patterns and functional roles of LINE-1 and Alu methylation in the keratinocyte from patients with psoriasis vulgaris.

Alterations in LINE-1 methylation are related to many diseases. The levels and patterns of LINE-1 hypomethylation were associated with a higher risk in developing several cancers, having a poorer prognosis and more aggressiveness. To evaluate the LINE-methylated status in psoriasis, LINE-1 methylation in various cells from patients with psoriasis, squamous cell carcinoma and normal controls were assessed by combined bisulfite restriction analysis of LINE-1. The results of the epigenetic changes for intragenic LINE-1 gene expression were also tested on two known expression microarrays. In patients with psoriasis, hypomethylation of LINE-1 and increase in %(u)C(u)C were prominent in the keratinocytes when compared with normal controls (P=0.014 and P=0.020, respectively). Alternatively, %(u)C(m)C was significantly lower in patients with severe psoriasis compared with mild psoriasis (P=0.022). The receiver-operating characteristic curve analysis indicated the high specificity and sensitivity of (u)C(u)C and (u)C(m)C in detecting psoriasis and severity of psoriasis. From expression array analysis, genes with LINE-1 were downregulated more than those genes without LINE-1 (P=3.84 × 10(-27) and P=2.14 × 10(-21), respectively). Modification in LINE-1 methylation may alter the gene expression resulting in a phenotypic change of the psoriatic skin. %(u)C(u)C and %(u)C(m)C may be used as biomarkers for psoriasis.

Characterization of the house dust mite allergen Der p 21 produced in Pichia pastoris.

BACKGROUND: The development of recombinant house dust mite (HDM) allergens opened the way for the in-depth characterization of these molecules but also provided new opportunities to refine the diagnostic procedures of HDM allergy as well as the allergen-specific immunotherapy through tailor-made treatments. OBJECTIVE: In the present study, the HDM allergen Der p 21 was expressed in Pichia pastoris under a secreted form. The physico-chemical as well as the allergenic characterizations of recombinant Der p 21 (rDer p 21) were performed. METHODS: Purified rDer p 21, secreted from recombinant P. pastoris was characterized by CD and MS analysis and the frequency of IgE reactivity was determined by ELISA using 96 sera of HDM-allergic patients from Bangkok. The direct airway epithelial cell activation by rDer p 21 was also evaluated. RESULTS: rDer p 21 was highly expressed under a secreted form in P. pastoris. The physico-chemical characterization of purified rDer p 21 showed that the allergen displayed appropriate α-helix secondary structure content although a two amino acids truncation at the N-terminus of the protein was evidenced by MS. The prevalence of IgE reactivity to rDer p 21 reached 25% in the cohort of the HDM-allergic patients. rDer p 21 could trigger IL-8 production in airway epithelial cells through TLR2-dependent signaling. CONCLUSION: Properly folded rDer p 21 produced in P. pastoris is appropriate for HDM allergy diagnosis as well for future recombinant allergen-based specific immunotherapy.

Component-resolved diagnostics for the evaluation of peanut allergy in a low-prevalence area.

BACKGROUND: Major allergenic components of peanut from distinct geographical regions are widely dispersed. Most of the diagnostic studies are from countries with a high prevalence. There have been only few reports of allergen component sensitizations from countries with a low prevalence of peanut allergy. We aimed to investigate roles of component-resolved diagnostic (CRD) to differentiate peanut allergy and peanut tolerance in the Asian population from a country with low prevalence of peanut allergy. METHODS: Participants with peanut sensitization were enrolled. Clinical reactions were determined. Skin prick test (SPT) and specific IgE (sIgE) to peanut and related allergen components were performed. RESULTS: Forty subjects with peanut sensitization were included. The mean wheal sizes of SPT and peanut sIgE were not good predictors for differentiating peanut reactions. SIgE to rAra h 2 was more often found in patients with peanut allergy and anaphylaxis. sIgE to rAra h 9 was also more frequent in the peanut-allergic group but not related to severe reactions. In the peanut-tolerant group, despite positive SPT and/or sIgE to peanut, 90% had negative sIgE to rAha h 2 and rAra h 9. Combining rAra h 2 and rAra h 9 resulted in high performance of the test with sensitivity, specificity, positive predictive value, and negative predictive value of 84%, 90%, 0.89, and 0.86, respectively. The ratio between rAra h 2 sIgE to peanut sIgE of 0.6 can be helpful in predicting patients who will develop severe reaction. SIgE to cross-reactive carbohydrate determinants (CCD) was exclusively found in the peanut-tolerant group (33.3% vs. 0%, p = 0.012). CONCLUSIONS: Our study identifies three allergen components: rAra h 2, rAra h 9, and CCD as important components in the diagnosis of peanut allergy in an Asian country with low prevalence. The ratio between rArah h 2 sIgE to peanut sIgE can be used for predicting patients who will develop anaphylaxis.