Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells

AIM:To investigate cytokeratin 8(CK8)overexpression during hepatitis C virus(HCV)infection and its pathogenesis,and the effect of ectopic CK8 expression on hepatoma cell lines.METHODS:We successfully established an in vitro HCV cell culture system(HCVcc)to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells.The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction(RT-PCR).The expression of CK8 at the protein level was evaluated by Western blotting.We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene.CK8cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFPCK8 was obtained.After confirming the sequence,the recombinant plasmid was transfected into SMMC7721cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy,RTPCR and Western blotting.Besides,we identified biological function of CK8 on SMMC7721 cells,including cell proliferation,cell cycle and apoptosis detection.RESULTS:RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88and 2.95 times higher than in control cells.Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells,respectively.We found that CK8at mRNA and protein levels were both significantly increased in HCVcc.CK8 was up-regulated in SMMC7721cells.CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells.CK8expression in SMMC7721/pEGFP-CK8 cells was 2.69times higher than in SMMC7721 cells,and was 2.64times higher than in SMMC7721/pEGFP-C1 cells.CK8expression at the protein level in SMMC7721/pEGFPCK8 cells was 2.46 times higher than in SMMC7721cells,and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells.Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells.CONCLUSION:CK8 up-regulation might have a functional role in HCV infection and pathogenesis,and could be a promising target for the treatment of HCV infection.

Sociodemographic characteristics and risk factor analysis of Demodex infestation(Acari:Demodicidae)

To identify sociodemographic characteristics and risk factor of Demodex infestation,756 students aged 13-22 years in Xi'an,China were sampled for the school-based cross-sectional study.Demodex was examined using the cellophane tape method(CTP).The results showed that the total detection rate of Demodex was 67.6%.Logistic regression analysis revealed that five variables(gender,residence,sharing sanitary ware,frequency of face-wash per day,and use of facial cleanser) were found to be uncorrelated with Demodex infestation,whereas three variables(age,skin type,and skin disease) were found to be independent correlates.Students aged over 18 years had 22.1 times higher odds of Demodex infestation compared to those under 16 years and students aged 16-18 years also had 2.1 times higher odds compared to those aged 13-15 years.Odds of having a Demodex infestation for oily or mixed skin were 2.1 times those for dry or neutral skin.Students with a facial skin disease had 3.0 times higher odds of being infested with Demodex compared to those without.The inception rate of students with facial dermatoses increased in parallel with increasing mite count.The inception rates were 21.3%,40.7%,59.2%,and 67.7% in the negative,mild,moderate,and severe infestation groups,respectively(χ2=60.6,P<0.001).Specifically,the amount of infested mites and inception rate of acne vulgaris were positively correlated(R2=0.57,moderate infestation odds ratio(OR)=7.1,severe infestation OR=10.3).It was concluded that Demodex prevalence increases with age,and Demodex presents in nearly all adult human.Sebaceous hyperplasia with oily or mixed skin seems to favour Demodex proliferation.Demodex infestation could be associated with acne vulgaris.The CTP is a good sampling method for studies of Demodex prevalence.

A meta-analysis of association between acne vulgaris and Demodex infestation

Until now, etiology of acne vulgaris is still uncertain. Although clinicians usually deny the association between Demodex infestation and acne vulgaris, it has been proved in some clinical practices. To confirm the association between Demodex infestation and acne vulgaris, a meta-analysis was conducted. Predefined selection criteria were applied to search all published papers that analyzed the association between Demodex infestation and acne vulgaris (January 1950 to August 2011) in ISI Web of Knowledge, MEDLINE, and China National Knowledge Infrastructure (CNKI) databases. A meta-analysis was performed to calculate odds ratios (ORs) and 95% confidence intervals (CIs) based on fixed effects models or random effects models. We enrolled the 60 Chinese and 3 English papers in this meta-analysis, which covered Turkey and 25 different provinces/municipalities in China and 42 130 participants in-cluding students and residents, aged from 1 to 78 years. The pooled OR in random effects models is 2.80 (95% CI, 2.34-3.36). Stability is robust according to sensitivity analysis. The fail-safe number is 18 477, suggesting that at least 18 477 articles with negative conclusions would be needed to reverse the conclusion that acne vulgaris was related to Demodex infestation. So the effect of publication bias was insignificant and could be ignored. It was concluded that acne vulgaris is associated with Demodex infestation. This indicates that when regular treatments for acne vulgaris are ineffective, examination of Demodex mites and necessary acaricidal therapies should be considered.

Cloning and sequence analysis of chitin synthase gene fragments of Demodex mites

To our knowledge,few reports on Demodex studied at the molecular level are available at present.In this study our group,for the first time,cloned,sequenced and analyzed the chitin synthase(CHS) gene fragments of Demodex folliculorum,Demodex brevis,and Demodex canis(three isolates from each species) from Xi'an China,by designing specific primers based on the only partial sequence of the CHS gene of D.canis from Japan,retrieved from GenBank.Results show that amplification was successful only in three D.canis isolates and one D.brevis isolate out of the nine Demodex isolates.The obtained fragments were sequenced to be 339 bp for D.canis and 338 bp for D.brevis.The CHS gene sequence similarities between the three Xi'an D.canis isolates and one Japanese D.canis isolate ranged from 99.7% to 100.0%,and those between four D.canis isolates and one D.brevis isolate were 99.1%-99.4%.Phylogenetic trees based on maximum parsimony(MP) and maximum likelihood(ML) methods shared the same clusters,according with the traditional classification.Two open reading frames(ORFs) were identified in each CHS gene sequenced,and their corresponding amino acid sequences were located at the catalytic domain.The relatively conserved sequences could be deduced to be a CHS class A gene,which is associated with chitin synthesis in the integument of Demodex mites.

Discrimination between Demodex folliculorum(Acari:Demodicidae) isolates from China and Spain based on mitochondrial cox1 sequences

For a long time,classification of Demodex mites has been based mainly on their hosts and phenotypic characteristics.A new subspecies of Demodex folliculorum has been proposed,but not confirmed.Here,cox1 partial sequences of nine isolates of three Demodex species from two geographical sources(China and Spain) were studied to conduct molecular identification of D.folliculorum.Sequencing showed that the mitochondrial cox1 fragments of five D.folliculorum isolates from the facial skin of Chinese individuals were 429 bp long and that their sequence identity was 97.4%.The average sequence divergence was 1.24% among the five Chinese isolates,0.94% between the two geographical isolate groups(China(5) and Spain(1)),and 2.15% between the two facial tissue sources(facial skin(6) and eyelids(1)).The genetic distance and rate of third-position nucleotide transition/transversion were 0.0125,2.7(3/1) among the five Chinese isolates,0.0094,3.1(3/1) between the two geographical isolate groups,and 0.0217,4.4(3/1) between the two facial tissue sources.Phylogenetic trees showed that D.folliculorum from the two geographical isolate groups did not form sister clades,while those from different facial tissue sources did.According to the molecular characteristics,it appears that subspecies differentiation might not have occurred and that D.folliculorum isolates from the two geographical sources are of the same population.However,population differentiation might be occurring between isolates from facial skin and eyelids.

Facial dermatosis associated with Demodex:a case-control study

Demodex has been considered to be related with multiple skin disorders,but controversy persists.In this case-control study,a survey was conducted with 860 dermatosis patients aged 12 to 84 years in Xi'an,China to identify the association between facial dermatosis and Demodex.Amongst the patients,539 suffered from facial dermatosis and 321 suffered from non-facial dermatosis.Demodex mites were sampled and examined using the skin pressurization method.Multivariate regression analysis was applied to analyze the association between facial dermatosis and Demodex infestation,and to identify the risk factors of Demodex infestation.The results showed that total detection rate of Demodex was 43.0%.Patients aged above 30 years had higher odds of Demodex infestation than those under 30 years.Compared to patients with neutral skin,patients with mixed,oily,or dry skin were more likely to be infested with Demodex(odds ratios(ORs) were 2.5,2.4,and 1.6,respectively).Moreover,Demodex infestation was found to be statistically associated with rosacea(OR=8.1),steroid-induced dermatitis(OR=2.7),seborrheic dermatitis(OR=2.2),and primary irritation dermatitis(OR=2.1).In particular,ORs calculated from the severe infestation(≥5 mites/cm2) rate were significantly higher than those of the total rate.Therefore,we concluded that Demodex is associated with rosacea,steroid-induced dermatitis,seborrheic dermatitis,and primary irritation dermatitis.The rate of severe infestation is found to be more correlated with various dermatosis than the total infestation rate.The risk factors of Demodex infestation,age,and skin types were identified.Our study also suggested that good hygiene practice might reduce the chances of demodicosis and Demodex infestation.