Although the complete picture for alopecia areata (AA) pathogenesis has yet to be determined, recent research has made much progress in our understanding of the disease mechanism. Numerous circumstantial evidence supports the notion that AA is fundamentally a disease mediated by inflammatory cells and may be autoimmune in nature. Recent research has shown the hair-loss phenotype is precipitated predominantly by CD8+ lymphocytes, but the disease mechanism is driven by CD4+ lymphocytes. Although genetic susceptibility is a key contributor to disease development, disease onset and phenotypic presentation are probably modified by complex environmental interplay. On the basis of our current understanding of AA disease pathogenesis, several experimental and theoretical therapeutic approaches might be possible. However, the pathogenetic disease mechanism is particularly robust and the development of a cure for AA will be a significant challenge.

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Background African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine reproductive and respiratory syndrome virus (PRRSV) are still prevalent in many regions of China. Co-infections make it difficult to distinguish their clinical symptoms and pathological changes. Therefore, a rapid and specific method is needed for the differential detection of these pathogens. Objectives The aim of this study was to develop a multiplex real-time quantitative reverse transcription polymerase chain reaction (multiplex qRT-PCR) for the simultaneous differential detection of ASFV, CSFV, and PRRSV. Methods Three pairs of primers and TaqMan probes targeting the ASFV p72 gene, CSFV 5′ untranslated region, and PRRSV ORF7 gene were designed. After optimizing the reaction conditions, including the annealing temperature, primer concentration, and probe concentration, multiplex qRT-PCR for simultaneous and differential detection of ASFV, CSFV, and PRRSV was developed. Subsequently, 1,143 clinical samples were detected to verify the practicality of the assay. Results The multiplex qRT-PCR assay could specifically and simultaneously detect the ASFV, CSFV, and PRRSV with a detection limit of 1.78 × 10 0 copies for the ASFV, CSFV, and PRRSV, but could not amplify the other major porcine viruses, such as pseudorabies virus, porcine circovirus type 1 (PCV1), PCV2, PCV3, foot-and-mouth disease virus, porcine parvovirus, atypical porcine pestivirus, and Senecavirus A. The assay had good repeatability with coefficients of variation of intra- and inter-assay of less than 1.2%. Finally, the assay was used to detect 1,143 clinical samples to evaluate its practicality in the field. The positive rates of ASFV, CSFV, and PRRSV were 25.63%, 9.36%, and 17.50%, respectively. The co-infection rates of ASFV+CSFV, ASFV+PRRSV, CSFV+PRRSV, and ASFV+CSFV+PRRSV were 2.45%, 2.36%, 1.57%, and 0.17%, respectively. Conclusions The multiplex qRT-PCR developed in this study could provide a rapid, sensitive, specific diagnostic tool for the simultaneous and differential detection of ASFV, CSFV, and PRRSV.

Background African swine fever virus (ASFV), classical swine fever virus (CSFV) and atypical porcine pestivirus (APPV) have caused great economic losses to the swine industry in China. Since coinfections of ASFV, CSFV and APPV occur in certain pig herds, it is necessary to accurately and differentially detect these pathogens in field-collected samples. In this study, a one-step multiplex real-time quantitative reverse transcription-polymerase chain reaction (multiplex qRT–PCR) was developed for the simultaneous and differential detection of ASFV, CSFV and APPV. Results The one-step multiplex qRT–PCR presented here was able to simultaneously detect ASFV, CSFV and APPV but could not amplify other viruses, including porcine circovirus type 2 (PCV2), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), foot-and-mouth disease virus (FMDV), porcine parvovirus (PPV), porcine epidemic diarrhoea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PRoV), porcine deltacoronavirus (PDCoV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV-1), BVDV-2, etc. The limit of detection (LOD) of the assay was 2.52 × 101 copies/μL for ASFV, CSFV and APPV. A repeatability test using standard recombinant plasmids showed that the intra- and interassay coefficients of variation (CVs) were less than 2%. An assay of 509 clinical samples collected in Guangxi Province, southern China, from October 2018 to December 2020 showed that the positive rates of ASFV, CSFV and APPV were 45.58, 12.57 and 3.54%, respectively, while the coinfection rates of ASFV and CSFV, ASFV and APPV, CSFV and APPV were 4.91, 1.38, 0.98%, respectively. Phylogenetic analysis based on the nucleotide sequences of the partial ASFV p72 gene showed that all ASFV strains from Guangxi Province belonged to genotypes I and II. Conclusion A one-step multiplex qRT–PCR with high specificity, sensitivity and repeatability was successfully developed for the simultaneous and differential detection of ASFV, CSFV and APPV.

Topical immunotherapy with diphenylcyclopropenone (DPCP) is considered to be the most effective treatment of severe AA. However, the mechanism is unclear and an early predictor for the efficacy needs to be explored. The TSLP/OX40L/IL‐13 pathway is an important pathway to initiate and maintain Th2 immune responses. Our previous work suggests this pathway may play a role in severe AA treated with DPCP. Thus, to further investigate the mechanism of TSLP/OX40L/IL‐13 pathway in severe AA treated with DPCP and explore the predictor for the efficacy of DPCP therapy, we conducted a prospective study to compare expression levels of TSLP, OX40L, Th2 cytokines IL‐4, IL‐5 and IL13, and Th1 cytokine IFN‐γ in severe AA patients before and after the treatment. Results showed that 21 AA patients were responsive (responders) to the DPCP therapy and 12 were not responsive (non‐responders). Responders had lower levels of TSLP, OX40L and IL‐13 than non‐responders before the treatment. After the DPCP treatment, TSLP, IL‐5 and IL‐13 increased and IFN‐γ decreased in responders while there were no changes of TSLP, IL‐4, IL‐13 and IFN‐γ in non‐responders. Our data suggest that the TSLP/OX40L/IL‐13 pathway is down‐regulated in some severe AA patients and DPCP might play a therapeutic role by up‐regulating the pathway in these severe AA patients. The TSLP/OX40L/IL‐13 pathway could be a predictor of response to the DPCP therapy for severe AA patients.

Self-organised nanoporous anodic films were fabricated on 904L superaustenitic stainless steel by the electrochemical treatment in ethylene glycol electrolyte containing perchloric acid. The effects of experimental conditions, such as electrolyte temperature and applied voltage on nanoporous morphology, were studied by the way of scanning electron microscopy. The results show that lower solution temperature promotes formation of orderly nanopores. Nanoporous anodic films cannot be formed, and pitting will appear on the stainless steel when temperature is above 40uC. The sizes of pores gradually increase with increasing applied voltage when the applied voltage is lower than 30 V. Porous morphology will change from roughly circular to polygonal, and the size will change from uniform to uneven when the applied voltage is higher than 30 V. It is difficult to form orderly nanoporous anodic films when the applied voltage is too high. Difference of element content in stainless steel is also an important factor for the porous morphology. When the applied voltage is 50 V, orderly pores are formed on 316 austenitic stainless steel surface. However, under the same conditions, this phenomenon does not occur on 904L superaustenitic stainless steel surface. From the analysis of X-ray photoelectron spectroscopy, the films are composed of various metal oxides.

Objective: The aim of this study was to analyze T-lymphocyte subsets and Th1/Th2 cytokines in convalescent patients with Epstein-Barr virus (EBV)-associated aplastic anemia (AA). Methods: Sixty AA patients were enrolled, who were in remission following immunosuppressive therapy, including 34 EBV-negative cases and 26 EBV-positive cases. Their complete blood count (CBC), T-lymphocyte subsets, Th1/Th2 cytokines were analyzed. The correlation between EBV-DNA and T-lymphocyte subsets was evaluated, as well as the relationship between EBV-DNA and Th1/Th2 cytokines. The presence of EBV-DNA in peripheral blood mononuclear cells (PBMCs) was also assessed in 60 normal controls. Results: EBV-DNA was detected in 26/60 (43.33%) patients and 21/60 (35.00%) controls. EBV-DNA copy number in AA patients was higher than in controls (Z = −2.138, P = 0.033). The percentage of CD3 + CD4 + T-lymphocytes and the ratio of CD4 + /CD8 + T-lymphocytes in the EBV-negative group were higher than in the EBV-positive group (P = 0.001 and 0.001, respectively). EBV was positively correlated with CD3 + CD8 + T-lymphocyte percentages (Pearson R: 0.496, P = 0.009). Moreover, EBV was positively correlated with IL-10 and IFN-γ levels (Pearson R: 0.559, P = 0.002 and Pearson R: 0.621, P = 0.001, respectively). Conclusions: EBV-DNA copy number in AA patients was higher than in normal controls. Both AA and EBV infection may cause changes in the levels of T-lymphocyte subsets. We recommend monitoring the changes in the immune function and EBV infection simultaneously in AA patients, especially following immunosuppressive therapy.