Oxyresveratrol Attenuates Inflammation in Human Keratinocyte via Regulating NF-kB Signaling and Ameliorates Eczematous Lesion in DNCB-Induced Dermatitis Mice.

Oxyresveratrol (ORV) is one of the novel antioxidants having been extensively studied in recent years. One of the main sources of ORV is Artocarpus lakoocha, which has been used in traditional medicine in Thailand for decades. However, the role of ORV in skin inflammation has not been clearly demonstrated. Therefore, we investigated the anti-inflammatory effects of ORV on dermatitis model. The effect of ORV was examined on human immortalized and primary skin cells exposed to bacterial components including peptidoglycan (PGN) and lipopolysaccharide (LPS) and 2,4-Dinitrochlorobenzene (DNCB)-induced dermatitis mouse model. PGN and LPS were used to induce inflammation on immortalized keratinocytes (HaCaT) and human epidermal keratinocytes (HEKa). We then performed MTT assay, Annexin V and PI assay, cell cycle analysis, real-time PCR, ELISA and Western blot in these in vitro models. H&E staining, immunohistochemistry (IHC) staining with CD3, CD4 and CD8 markers were used to evaluate the effects of ORV in in vivo model of skin inflammation using BALB/c mice. Pretreatment of HaCaT and HEKa cells with ORV inhibited pro-inflammatory cytokine production through inhibition of NF-κB pathway. In DNCB-induced dermatitis mouse model, ORV treatment reduced lesion severity, and skin thickness and numbers of CD3, CD4 and CD8 T cells in the sensitized skin of mice. In conclusion, it has been demonstrated that ORV treatment can ameliorate inflammation in the in vitro models of skin inflammation and in vivo models of dermatitis, suggesting a therapeutic potential of ORV for treatment of skin diseases particularly eczema.

A performance comparison between fluorescent immunoassay and immunochromatography for rapid dengue detection in clinical specimens.

Dengue virus (DENV 1-4) infection has been a global health threat where no specific treatment is currently available. Therefore, a rapid and accurate diagnosis is critical for an appropriate management as it could reduce the burden of severe clinical manifestation. Currently, dengue immunochromatography (IC) is commonly used to primarily differentiate acute febrile illnesses. Fluorescent immunoassay (FIA) utilized a highly sensitive detection system and claimed 70-100% sensitivity and 83.5-91.7% specificity for dengue infection in a preliminary report. This report recruited samples with acute febrile illnesses sent for dengue screening and tested IC and FIA in parallel. The performance of both tests was verified by a definitive diagnosis retrieved from combinatorial reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA) for IgM and IgG confirmation tests. Results showed that the viral nonstructural protein (NS1) performance of FIA was slightly higher than IC with the sensitivity, specificity, PPV, NPV, agreement, kappa, and its standard error at 79.11, 92.28, 86.81, 87.31, 352 (87.13%), 0.725 ± 0.035, respectively; whereas those of the IC were at 76.58, 92.28, 86.43, 85.98, 348 (86.14%), 0.703 ± 0.037, respectively. Moreover, the IgM and IgG performance of FIA had higher specificity, PPV, and agreement than the IgM IC performance, suggesting that the FIA was more specific but less sensitive for antibody detection. No correlation was observed in IgM and IgG levels of ELISA and FIA assays. In conclusion, the FIA and IC were highly sensitive, specific, and substantially agreed in NS1 detection but moderately agreed in IgM and IgG detection.

Quantitative Risk Assessments of Hepatitis A Virus and Hepatitis E Virus from Raw Oyster Consumption.

A quantitative risk assessment of hepatitis A virus (HAV) and hepatitis E virus (HEV) from raw oyster consumption from farm and retail was evaluated over three seasons. This risk assessment comprises four steps: hazard identification, dose-response assessment, exposure assessment, and risk characterization. We used probabilistic models for prevalence, concentration, and oyster consumption. HEV dose-response (DR) model based on HEV dosing in chimpanzees and used to perform a dose-response assessment of HEV was proposed. Both HAV and HEV were simultaneously enumerated by real-time PCR to determine viral doses. The probabilistic prevalences of HAV and HEV were in the ranges of 8-20% and 8-40%, respectively. The best-fit DR model was the beta-Poisson with alpha and N50 equal to 216.9 and 3.03 × 107 , respectively. After running the Monte Carlo simulation, the annual cases of foodborne hepatitis A and hepatitis E from raw oyster consumption from farms were 9,264-17,526 and 1-604, respectively, while those at retail were 7,694-14,591 and 1-204, respectively. This study suggested that consuming farm oysters poses a significantly higher risk of hepatitis A than hepatitis E. The best-fit DR model for HEV developed in this study could determine risks of hepatitis E from raw oyster consumption in Thailand.

Spread of genetically similar noroviruses in Bangkok, Thailand, through symptomatic and asymptomatic individuals.

Norovirus infection is a major cause of acute gastroenteritis, although some infected individuals are asymptomatic. GII.4 is the predominant genotype worldwide and, since 2000, has been the most prevalent in patients in Thailand with acute gastroenteritis. We screened stool samples for norovirus in 786 patients with acute gastroenteritis who were admitted to a hospital in Bangkok from 2017 to early 2019 and detected it in 136 specimens (17.3%). Eight and 124 specimens were positive for the GI and GII genogroups, respectively, and the remaining 4 specimens were double-positive. Nine genotypes (GI.3, GI.5, GII.2, GII.3, GII.4, GII.6, GII.8, GII.13, and GII.17) were identified from 140 strains, and 72 strains (51.4%) were GII.4. We had previously conducted a one-year survey of norovirus infection in residents of a community in Bangkok from May 2018 to April 2019 and found that a substantial portion of the residents were infected asymptomatically. The 9 genotypes identified in the patients were also commonly identified in the community residents. To investigate the relationship between noroviruses identified in the acute gastroenteritis patients and those identified in the community residents, phylogenetic tree analysis was conducted. Of the 9 genotypes, 8 showed similarities in both their genomic sequences and their deduced amino acid sequences. In addition, strain replacement of GI.3 was observed in both the patients and the community residents within the overlapping period. These results suggested that norovirus spreads efficiently to the community by simultaneously causing symptomatic and asymptomatic infections.

Reconstructing unseen transmission events to infer dengue dynamics from viral sequences.

For most pathogens, transmission is driven by interactions between the behaviours of infectious individuals, the behaviours of the wider population, the local environment, and immunity. Phylogeographic approaches are currently unable to disentangle the relative effects of these competing factors. We develop a spatiotemporally structured phylogenetic framework that addresses these limitations by considering individual transmission events, reconstructed across spatial scales. We apply it to geocoded dengue virus sequences from Thailand (N = 726 over 18 years). We find infected individuals spend 96% of their time in their home community compared to 76% for the susceptible population (mainly children) and 42% for adults. Dynamic pockets of local immunity make transmission more likely in places with high heterotypic immunity and less likely where high homotypic immunity exists. Age-dependent mixing of individuals and vector distributions are not important in determining spread. This approach provides previously unknown insights into one of the most complex disease systems known and will be applicable to other pathogens.

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.

Identification of GII.14[P7] norovirus and its genomic mutations from a case of long-term infection in a post-symptomatic individual.

Norovirus is a leading cause of acute gastroenteritis worldwide. Norovirus shedding typically lasts one week to one month after the onset of diarrhea in immunocompetent hosts. The occurrence of mutations in the genome during infection has contributed to the evolution of norovirus. It has been suggested that genomic mutations in the P2-domain of capsid protein VP1, the major antigenic site for virus clearance, are involved in the evasion of host immunity and prolonged shedding of norovirus. In our previous study, we found a case of long-term shedding of GII.14 norovirus in a post-symptomatic immunocompetent individual that lasted about three months. In this study, we characterized the genomic sequence of the GII.14 strain to gain insight into the context of long-term shedding. By sequencing a 4.8 kb region of the genome corresponding to half of ORF1 and the entire ORF2 and ORF3, which encode several non-structural proteins and the structural proteins VP1 and VP2, the GII.14 strain was found to be classified as recombinant GII.14[P7]. Six point-mutations occurred during the three-month period of infection in a time-dependent manner in the genomic regions encoding RNA-dependent RNA polymerase, VP1, and VP2. Three of the six mutations were sense mutations, but no amino acid substitution was identified in the P2-domain of VP1. These results suggest that there is a mechanism by which long-term shedding of norovirus occurs in immunocompetent individuals independent of P2-domain mutations.

Anti-Chikungunya Virus Monoclonal Antibody That Inhibits Viral Fusion and Release.

Chikungunya fever, a mosquito-borne disease manifested by fever, rash, myalgia, and arthralgia, is caused by chikungunya virus (CHIKV), which belongs to the genus Alphavirus of the family Togaviridae Anti-CHIKV IgG from convalescent patients is known to directly neutralize CHIKV, and the state of immunity lasts throughout life. Here, we examined the epitope of a neutralizing mouse monoclonal antibody against CHIKV, CHE19, which inhibits viral fusion and release. In silico docking analysis showed that the epitope of CHE19 was localized in the viral E2 envelope and consisted of two separate segments, an N-linker and a ß-ribbon connector, and that its bound Fab fragment on E2 overlapped the position that the E3 glycoprotein originally occupied. We showed that CHIKV-E2 is lost during the viral internalization and that CHE19 inhibits the elimination of CHIKV-E2. These findings suggested that CHE19 stabilizes the E2-E1 heterodimer instead of E3 and inhibits the protrusion of the E1 fusion loop and subsequent membrane fusion. In addition, the antigen-bound Fab fragment configuration showed that CHE19 connects to the CHIKV spikes existing on the two individual virions, leading us to conclude that the CHE19-CHIKV complex was responsible for the large virus aggregations. In our subsequent filtration experiments, large viral aggregations by CHE19 were trapped by a 0.45-µm filter. This virion-connecting characteristic of CHE19 could explain the inhibition of viral release from infected cells by the tethering effect of the virion itself. These findings provide clues toward the development of effective prophylactic and therapeutic monoclonal antibodies against the Alphavirus infection.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Neither a specific antiviral drug nor a commercial vaccine for CHIKV infection are available. Here, we show a detailed model of the docking between the envelope glycoprotein of CHIKV and our unique anti-CHIKV-neutralizing monoclonal antibody (CHE19), which inhibits CHIKV membrane fusion and virion release from CHIKV-infected cells. Homology modeling of the neutralizing antibody CHE19 and protein-protein docking analysis of the CHIKV envelope glycoprotein and CHE19 suggested that CHE19 inhibits the viral membrane fusion by stabilizing the E2-E1 heterodimer and inhibits virion release by facilitating the formation of virus aggregation due to the connecting virions, and these predictions were confirmed by experiments. Sequence information of CHE19 and the CHIKV envelope glycoprotein and their docking model will contribute to future development of an effective prophylactic and therapeutic agent.

Norovirus transmission mediated by asymptomatic family members in households.

The transmission of human norovirus excreted from infected persons occasionally causes sporadic infections and outbreaks. Both symptomatic patients and asymptomatic carriers have been reported to contribute to norovirus transmission, but little is known about the magnitude of the contribution of asymptomatic carriers. We carried out a 1-year survey of residents of a district of Bangkok, Thailand to determine the percentage of norovirus transmissions originating from asymptomatic individuals. We screened 38 individuals recruited from 16 families from May 2018 to April 2019 for GI and GII genotypes. Norovirus was detected every month, and 101 of 716 stool samples (14.1%) from individuals with no symptoms of acute gastroenteritis were norovirus-positive. The average infection frequency was 2.4 times per person per year. Fourteen genotypes were identified from the positive samples, with GII.4 being detected most frequently. Notably, 89.1% of the norovirus-positive samples were provided by individuals with no diarrhea episode. Similar to cases of symptomatic infections in Thailand, asymptomatic infections were observed most frequently in December. We detected 4 cases of NV infection caused by household transmission, and 3 of the 4 transmissions originated from asymptomatic individuals. We also identified a case in which norovirus derived from an asymptomatic individual caused diarrhea in a family member. These results suggest that asymptomatic individuals play a substantial role in both the maintenance and spreading of norovirus in a community through household transmission.

Long-term circulation of Zika virus in Thailand: an observational study.

BACKGROUND: Little is known about the historical and current risk of Zika virus infection in southeast Asia, where the mosquito vector is widespread and other arboviruses circulate endemically. Centralised Zika virus surveillance began in Thailand in January, 2016. We assessed the long-term circulation of Zika virus in Thailand. METHODS: In this observational study, we analysed data from individuals with suspected Zika virus infection who presented at hospitals throughout the country and had biological samples (serum, plasma, or urine) tested for confirmation with PCR at the National Institute of Health laboratories in Bangkok. We analysed the spatial and age distribution of cases, and constructed time-resolved phylogenetic trees using genomes from Thailand and elsewhere to estimate when Zika virus was first introduced. FINDINGS: Of the 3089 samples from 1717 symptomatic individuals tested between January, 2016, and December, 2017, 368 were confirmed to have Zika virus infection. Cases of Zika virus infection were reported throughout the year, and from 29 of the 76 Thai provinces. Individuals had 2·8 times (95% CI 2·3-3·6) the odds of testing positive for Zika virus infection if they came from the same district and were sick within the same year of a person with a confirmed infection relative to the odds of testing positive anywhere, consistent with focal transmission. The probability of cases being younger than 10 years was 0·99 times (0·72-1·30) the probability of being that age in the underlying population. This probability rose to 1·62 (1·33-1·92) among those aged 21-30 years and fell to 0·53 (0·40-0·66) for those older than 50 years. This age distribution is consistent with that observed in the Zika virus epidemic in Colombia. Phylogenetic reconstructions suggest persistent circulation within Thailand since at least 2002. INTERPRETATION: Our evidence shows that Zika virus has circulated at a low but sustained level for at least 16 years, suggesting that Zika virus can adapt to persistent endemic transmission. Health systems need to adapt to cope with regular occurrences of the severe complications associated with infection. FUNDING: European Research Council, National Science Foundation, and National Institutes of Health.

Characterization of a G10P[14] rotavirus strain from a diarrheic child in Thailand: Evidence for bovine-to-human zoonotic transmission.

An unusual rotavirus strain, DB2015-066 with the G10P[14] genotype (RVA/Human-wt/THA/DB2015-066/2015/G10P[14]), was detected in a stool sample from a child hospitalized with acute gastroenteritis in Thailand. Here, we sequenced and characterized the full-genome of the strain DB2015-066. On whole genomic analysis, strain DB2015-066 was shown to have a unique genotype constellation: G10-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The backbone genes of this strain (I2-R2-C2-M2-A3-N2-T6-E2-H3) are commonly found in rotavirus strains from artiodactyls such as cattle. Furthermore, phylogenetic analysis indicated that each of the 11 genes of strain DB2015-066 could be of artiodactyl (likely bovine) origin. Thus, strain DB2015-066 appeared to be derived from through zoonotic transmission of a bovine rotavirus strain. Of note, the VP7 gene of strain DB2015-066 was located in G10 lineage-6 together with ones of bovine and bovine-like rotavirus strains, away from the clusters comprising other G10P[14] strains in G10 lineage-2/4/5/9, suggesting the occurrence of independent bovine-to-human interspecies transmission events. Our observations provide important insights into the origins of rare G10P[14] strains, and into dynamic interactions between artiodactyl and human rotavirus strains.

The dynamics of norovirus genotypes and genetic analysis of a novel recombinant GII.P12-GII.3 among infants and children in Bangkok, Thailand between 2014 and 2016.

Norovirus (NoV) is the leading cause of viral acute gastroenteritis among all age groups in the world. We performed a molecular epidemiological study of the NoVs prevalent in Bangkok between November 2014 and July 2016 to investigate the emergence of new NoV variants in Thailand. A total of 332 stool specimens were collected from hospitalized pediatric patients with acute gastroenteritis in Bangkok, Thailand. NoVs were detected by real-time PCR. The genome of the N-terminal/shell domain was amplified, the nucleotide sequence was determined, and phylogenetic analyses were performed. GII NoV was detected in 58 (17.5%) of the 332 specimens. GII.17, a genotype strain prevalent from 2014 to mid-2015, was hardly detected and replaced by the GII.3 genotype strain. Entire genome sequencing followed by phylogenetic analysis of the GII.3 genotype strains indicated that they are new recombinant viruses, because the genome encoding ORF1 is derived from a GII.12 genotype strain, whereas that encoding ORF2-3 is from a GII.3 genotype strain. The putative recombination breakpoints with the highest statistical significance were located around the border of 3Dpol and ORF2. The change in the prevalent strain of NoV seems to be linked to the emergence of new forms of recombinant viruses. These findings suggested that the swapping of the structural and non-structural proteins of NoV is a common mechanism by which new epidemic variants are generated in nature.

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.

Alterations in the LINE-1 methylation pattern in patients with lichen simplex chronicus.

BACKGROUND: Long interspersed element-1 (LINE-1) and short interspersed element (Alu) retrotransposons have been identified to influence the human genome by modifications in gene expression. Variations in LINE-1 and Alu methylation have been shown to be associated with many diseases, predominantly malignancies and autoimmune diseases. Moreover, the degree and pattern of LINE-1 methylation are related to risk, prognosis and aggressiveness of several cancers. However, a similar study has not been performed in lichen simplex chronicus (LSC). OBJECTIVE: To evaluate DNA methylation status of repetitive sequences in LSC. RESULTS: The %mCmC of LINE-1 was significantly decreased in keratinocytes from patients with LSC (p=0.012). Moreover, the %mCuC was significantly lower in LSC than controls (p=0.029). Conversely, %uCmC was significantly higher LSC than controls (p=0.004). A receiver-operating characteristic (ROC) curve analysis demonstrated that % mCmC, % mCuC and % uCmC were highly sensitive and specific for LSC with an optimal cut-off value. There were no significant differences in Alu methylation in keratinocytes from LSC patients. METHODS: We determined the level and pattern of LINE-1 and Alu methylation in keratinocytes from patients with LSC (n=10) compared to normal controls (n=13), by the improved combined bisulfite restriction analysis of LINE-1 and Alu (COBRA-LINE-1 and Alu). COBRA-LINE-1 classifies LINE-1 loci according to the methylation patterns of two CpG dinucleotides in the 5'UTR into four categories: hypermethylated (mCmC), hypomethylated (uCuC), and two forms of partially methylated loci (uCmC and mCuC). CONCLUSION: Changes in the LINE-1 pattern were revealed in the epidermis from patients with LSC. A particular LINE-1 methylation pattern is indicative of LSC and might be used as a diagnostic tool.

Effect of methotrexate on serum levels of IL-22 in patients with psoriasis.

Interleukin-22 (IL-22) is the effector molecule of T-helper subset 22 (Th-22) lineage that promotes keratinocyte proliferation and dermal inflammation in psoriasis. Methotrexate is widely used as a first-line treatment in moderate to severe psoriasis. Methotrexate inhibits inflammatory and cytokinetic processes via various mechanisms, but the relevance of these to psoriasis is limited and whether methotrexate is specifically able to down-regulate Th22 cytokines is unknown. To determine if methotrexate reduces IL-22 in cases of psoriasis. Nineteen patients with moderate to severe psoriasis were given methotrexate 15 mg per week for up to 12 weeks. Serum levels of IL-22 were determined by enzyme-linked immunosorbent assay (ELISA) before and after treatment. Eleven of 19 patients (57.8%) achieved a 75% PASI score reduction. IL-22 levels were significantly higher in untreated psoriasis patients (56.63 ± 60.73 pg/mL) than in controls (12.58 ± 12.59 pg/mL). Methotrexate significantly reduced serum levels of IL-22 in psoriasis patients to 5.91 ± 7.97 pg/mL (p<0.001). Moreover, there was a significant positive correlation between IL-22 levels and PASI (r=0.63, p=0.004). Methotrexate significantly reduces serum IL-22 levels in cases of psoriasis. This is a novel mechanism by which methotrexate acts in the treatment of this disease.

Parakeratosis in skin is associated with loss of inhibitor of differentiation 4 via promoter methylation.

Parakeratosis refers to incomplete maturation of epidermal keratinocytes, resulting in abnormal retention of nuclei in the stratum corneum. It occurs in many diseases of the skin, particularly in psoriasis. Down-regulation of inhibitor of differentiation 4 messenger RNA has been demonstrated in psoriatic skin, but the specificity and mechanism for this finding are unknown. In this study, we addressed specificity by immunohistochemical staining for inhibitor of differentiation 4 protein in skin disorders showing parakeratosis, including: psoriasis (n = 9), chronic eczema (n = 6), and squamous cell carcinoma (n = 7). In these conditions, parakeratotic keratinocytes in the upper layers of the skin lacked inhibitor of differentiation 4 protein expression, whereas keratinocytes in the lower layers were densely stained, in contrast to diffuse expression in normal skin. Because promoter hypermethylation of inhibitor of differentiation 4 has been described in several cancers, we determined the methylation pattern of the inhibitor of differentiation 4 promoter in psoriasis and compared this with squamous cell carcinoma. We found a novel methylation pattern of the inhibitor of differentiation 4 promoter in both conditions. Inhibitor of differentiation 4 promoter methylation was significantly increased in psoriasis (34.8%) and squamous cell carcinoma (21.8%), compared with normal skin (0%). Moreover, cells in the upper and lower parts of psoriatic epidermis were, respectively, hypermethylated and nonmethylated, at the inhibitor of differentiation 4 promoter. Comparable studies in several cell lines confirmed that hypermethylation of the promoter was associated with loss of inhibitor of differentiation 4 messenger RNA and protein expression. Our study demonstrates a previously unreported link between gene-specific promoter hypermethylation and abnormal cellular differentiation in several skin diseases. This mechanism might provide clues for novel therapies for skin disorders characterized by parakeratosis.

A novel pathway involving melanoma differentiation associated gene-7/interleukin-24 mediates nonsteroidal anti-inflammatory drug-induced apoptosis and growth arrest of cancer cells.

Numerous studies show that nonsteroidal anti-inflammatory drugs (NSAIDs) are effective in chemoprevention or treatment of cancer. Nevertheless, the mechanisms underlying these antineoplastic effects remain poorly understood. Here, we report that induction of the cancer-specific proapoptotic cytokine melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) by several NSAIDs is an essential step for induction of apoptosis and G(2)-M growth arrest in cancer cells in vitro and inhibition of tumor growth in vivo. We also show that MDA-7/IL-24-dependent up-regulation of growth arrest and DNA damage inducible 45 alpha (GADD45alpha) and GADD45gamma gene expression is sufficient for cancer cell apoptosis via c-Jun NH(2)-terminal kinase (JNK) activation and growth arrest induction through inhibition of Cdc2-cyclin B checkpoint kinase. Knockdown of GADD45alpha and GADD45gamma transcription by small interfering RNA abrogates apoptosis and growth arrest induction by the NSAID treatment, blocks JNK activation, and restores Cdc2-cyclin B kinase activity. Our results establish MDA-7/IL-24 and GADD45alpha and GADD45gamma as critical mediators of apoptosis and growth arrest in response to NSAIDs in cancer cells.