Identification and Validation of SLC9A2 as A Potential Tumor Suppressor in Colorectal Cancer: Integrating Bioinformatics Analysis with Experimental Confirmation.

OBJECTIVE: To uncover the mechanisms underlying the development of colorectal cancer (CRC), we applied bioinformatic analyses to identify key genes and experimentally validated their possible roles in CRC onset and progression. METHODS: We performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis on differentially expressed genes (DEGs), constructed a protein-protein interaction (PPI) network to find the top 10 hub genes, and analyzed their expression in colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ). We also studied the correlation between these genes and immune cell infiltration and prognosis and validated the expression of SLC9A2 in CRC tissues and cell lines using qRT-PCR and Western blotting. Functional experiments were conducted in vitro to investigate the effects of SLC9A2 on tumor growth and metastasis. RESULTS: We found 130 DEGs, with 45 up-regulated and 85 down-regulated in CRC. GO analysis indicated that these DEGs were primarily enriched in functions related to the regulation of cellular pH, zymogen granules, and transmembrane transporter activity. KEGG pathway analysis revealed that the DEGs played pivotal roles in pancreatic secretion, rheumatoid arthritis, and the IL-17 signaling pathway. We identified 10 hub genes: CXCL1, SLC26A3, CXCL2, MMP7, MMP1, SLC9A2, SLC4A4, CLCA1, CLCA4, and ZG16. GO enrichment analysis showed that these hub genes were predominantly involved in the positive regulation of transcription. Gene expression analysis revealed that CXCL1, CXCL2, MMP1, and MMP7 were highly expressed in CRC, whereas CLCA1, CLCA4, SLC4A4, SLC9A2, SLC26A3, and ZG16 were expressed at lower levels. Survival analysis revealed that 5 key genes were significantly associated with the prognosis of CRC. Both mRNA and protein expression levels of SLC9A2 were markedly reduced in CRC tissues and cell lines. Importantly, SLC9A2 overexpression in SW480 cells led to a notable inhibition of cell proliferation, migration, and invasion. Western blotting analysis revealed that the expression levels of phosphorylated ERK (p-ERK) and phosphorylated JNK (p-JNK) proteins were significantly increased, whereas there were no significant changes in the expression levels of ERK and JNK following SLC9A2 overexpression. Correlation analysis indicated a potential link between SLC9A2 expression and the MAPK signaling pathway. CONCLUSION: Our study suggests that SLC9A2 acts as a tumor suppressor through the MAPK pathway and could be a potential target for CRC diagnosis and therapy.

Resistance of the Ginseng Gray Mold Pathogen, Botrytis cinerea, to Boscalid and Pyraclostrobin Fungicides in China.

Gray mold caused by Botrytis cinerea severely threatens the yield of ginseng (Panax ginseng). Various categories of fungicides have been utilized to control gray mold on this crop. In this study, the resistance of 102 isolates of B. cinerea from 11 commercial ginseng-growing regions in China to fungicides was examined. A total of 32.4% were resistant to boscalid, with EC50 values that ranged from 12.26 to 235.87 µg/ml, and 94.1% were resistant to pyraclostrobin, with EC50 values that ranged from 5.88 to 487.72 µg/ml. Except for sdhA and sdhD, the amino acid substitutions of P225F, P225L, N230I, H272Y, and H272R in the sdhB subunit from 24 (4 sensitive [S] and 20 resistant [R]), 5 (1 S and 4 R), 1 (S), 1 (R), and 8 (4 S and 4 R) strains, respectively, and the concurrent amino acid substitutions of G85A + I93V + M158V + V168I in the sdhC subunit from 5 (4 S and 1 R) strains were identified. A G143A substitution in cytochrome b was identified in 96 isolates that were resistant to pyraclostrobin and three that were sensitive to it. The Bcbi-143/144 intron was identified in the other three isolates sensitive to pyraclostrobin, but it was absent in the isolates that harbored the G143A mutation. The results showed that the populations of B. cinerea on ginseng have developed strong resistance to pyraclostrobin. Therefore, it is not recommended to continue using this fungicide to control gray mold on P. ginseng. Boscalid is still effective against most isolates. However, to prevent fungicide resistance, it is recommended to use a mixture of boscalid with other categories of fungicides.

First Report of Sclerotinia Rot Caused by Sclerotinia sclerotiorum on Artemisia capillaris in China.

Artemisia capillaris (Asteraceae) is an annual herb found in ˃10 provinces in China. It is cultivated on ˃670 ha, with annual production around 2,500 tons. Its shoot is used in traditional Chinese medicine (Liu et al. 2021). From April to May 2023, Sclerotinia rot symptoms were seen at the Institute of Medicinal Plant Development (40.04°N, 116.28°E), Beijing, China. Disease incidence was up to 10% in the field through investigation of 300 plants. Initial symptoms were irregular tan-brown lesions (0.5 to 5.0 mm) that expended to circumferential necrosis on the roots and basal stem, aerial mycelia and sclerotia were developed on them. The leaves and stem tips were withered and droopy in severe cases. Twelve symptomatic primary roots of 12 plants from two sites were cut into 5 × 5 mm pieces, surface sterilized with 75% ethanol for 30 s and 5% NaClO for 60 s, rinsed with distilled water for three times, dried with sterile filter paper, put on potato dextrose agar (PDA), and incubated at 25°C in the dark for 2 days. Two Sclerotinia-like isolates were obtained using the hyphaltip method. White aerial mycelia were sparse and appressed for isolate YC1-3 and dense for isolate YC1-7. After incubated at 25°C in the dark for 15 days, 10 to 25 sclerotia were developed near the colony margin. Sclerotia of isolate YC1-3 were 1.0 to 3.9 × 1.2 to 4.5 (mean 1.8 × 2.2) mm (n = 60), ovoid or arc-shaped. Sclerotia of isolate YC1-7 were 1.5 to 3.4 × 2.7 to 9.2 (mean 2.3 × 4.3) mm (n = 60), ovoid, dumbbell shaped or curved. The isolates were identified as Sclerotinia sclerotiorum based on morphology (Maas 1998). To further identify the pathogens, molecular identification was performed with isolates YC1-3 and YC1-7. DNA of the two isolates were extracted by the cetyltrimethylammonium bromide (CTAB) method. Polymerase chain reaction was performed with primers ITS1/ITS4 for the internal transcribed spacer (ITS) region (Choi et al. 2020; White et al. 1990) and primers G3PDHfor/G3PDHrev for the glyceraldehyde 3-phosphate dehydrogenase (G3PDH) gene (Garfinkel. 2021). BLAST search analysis revealed that the ITS sequence (GenBank OR229758 and OR229762) was ≥99% similar to S. sclerotiorum (MN099281, MZ379265, KX781301, etc.), and the G3PDH sequence (OR778388 and OR761975) was too (MZ493894, JQ036048, OQ790148, etc.). Phylogenetic trees were computed with ITS and G3PDH sequences using the Maximum Likelihood in MEGA 11. Nine two-month-old seedlings of A. capillaris were used to test pathogenicity. The epidermis layer of each primary root was slightly wounded (2 × 2 mm, 1 mm deep) using a sterile dissecting blade. Three plants were inoculated with mycelial plugs (5 mm in diameter) of YC1-3 and YC1-7 that cultured on PDA for 7 days. Control plants were inoculated with sterile PDA plugs. All seedlings were then incubated at 25oC and 90% relative humidity. After isolate YC1-7 inoculation 3 days and isolate YC1-3 inoculation 5 days, inoculated roots had symptoms like those in the field, controls had no symptoms. S. sclerotiorum was consistently re-isolated from diseased roots, fulfilling Koch's postulates. Diseases caused by S. sclerotiorum have been reported threatens several important economical crops (Marin and Peres 2020; Guan et al. 2022). To our knowledge, this is the first report of S. sclerotiorum causes Sclerotinia rot on A. capillaris. To avoid of significant economic losses, it is urgent to establish an effective disease-management strategy.

First Report of Corynespora cassiicola Causing Septoria Leaf Spot on Scutellaria baicalensis in China.

Scutellaria baicalensis Georgi. is a perennial herb in the Lamiaceae family, with a distribution in more than 10 provinces in China. At the current time, the cultivation area of S. baicalensis in China exceeds 58,000 hectares, with annual production approaching 28,000 tons. As a traditional Chinese herbal medicine, the root of S. baicalensis has many applications, such as anti-inflammatory, anti-neuroinflammatory and neuroprotective, anticancer, antiviral, antibacterial, and antioxidant activities, and is effective in treatment of colitis, hepatitis, pneumonia, respiratory infections, and allergic diseases. (Jang et al. 2023; Liu et al. 2023). From August to September 2022, septoria leaf spot symptoms were observed at the Institute of Medicinal Plant Development (40.04°N, 116.28°E), Beijing, China, and the incidence of this disease was up to 20% in the field through more than two weeks of continuous investigation. Initial symptoms on leaves were observed as small, dark-brown spots (0.5 to 2.0 mm), which then expanded to irregular lesions with a pale gray center surrounded by a black ring with a dark-brown edge and light brown halo (Fig. 1A1-A3). Plants were defoliated and withered in severe cases. Thirty-six symptomatic leaves of 12 diseased plants from three experimental sites were cut into 5 × 5 mm pieces, and surface sterilized with 75% ethanol for 30 s followed by 5% NaClO solution for 45 s, rinsed with sterile water three times, dried with sterile filter paper, and subsequently placed on potato dextrose agar (PDA) medium and incubated at 25°C in dark for two days. Isolates were purified by transferring hyphal tips to new PDA plates and incubated at 25°C in dark. Finally, eight isolates (A1, B3, D1, F2, E2, a4, e4 and f1) with similar colonial morphological characteristics were obtained. Colonies on PDA exhibited dense, downy, and white to grayish-green aerial mycelia and the reverse of colonies showed dark-brown in the center and grayish on the edge (Fig. 1D, E). Conidia were solitary or catenate, pale brown, obclavate to cylindrical, apex obtuse (Fig. 1B, C). The isolates were divided into two categories by examining 100 conidia (50 of each isolate), represented by isolates D1 and e4. Conidia of D1 measured 5.4 to 75.8 µm × 2.1 to 6.8 µm, mean 26.9 × 4.4 µm, had 0 to 6 pseudosepta, with 0 to 3 pseudosepta observed in 88% of conidia. Conidia of e4 measured 20.3 to 103.4 µm × 2.0 to 7.9 µm, mean 41.9 × 4.8 µm, had 0 to 6 pseudosepta, with 2 to 5 pseudosepta observed in 90% of conidia. These isolates were identified as Corynespora cassiicola based on morphology (Ellis 1971). DNA of the two isolates (D1 and e4) was extracted by the cetyltrimethylammonium bromide (CTAB) method, and internal transcribed spacer (ITS) region of rDNA, translation elongation factor 1 alpha (TEF1-α), and beta-tubulin (TUB2) gene were amplified, using the primers ITS1/ITS4 (Bandi et al. 2022), EF1-728F/EF-986R (Wang et al. 2021), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences of ITS OQ991339 (524 bp) and OR044050 (533 bp) shared 99.8% identity to C. cassiicola, with a 99% coverage to MT228951 (536 bp) and OQ991340 (546 bp) in GenBank. Sequences of TEF1-α OR047441 (304 bp) and OR047443 (306 bp) shared 99.3% identity to C. cassiicola, with a 98% and 99% coverage to ON381927 (300 bp) and ON381933 (301 bp) in GenBank, respectively. Sequences of TUB2 OR047449 (427 bp) and OR047451 (427 bp) shared 99.53% identity to C. cassiicola, with a 99% and 98% coverage to MN604075 (442 bp) in GenBank, respectively. Phylogenetic trees were computed with ITS, TEF1-α, and TUB2 sequences in MEGA 11 using the Neighbor-Joining (NJ) method (Fig. 2). The results showed that the two isolates were C. cassiicola with more than 90% bootstrap support (1000 replicates). Nine 2-year-old seedlings of S. baicalensis were used for the pathogenicity assay. Three leaves from each plant were punctured with flame-sterilized needles, and inoculated with mycelial plugs (5 mm in diameter) of D1 and e4. Plants inoculated with sterile PDA plugs were used as control. All the inoculated seedlings were incubated at 25 oC and 90% relative humidity. About 3 to 4 days after inoculation, similar symptoms to those observed in the field were present on leaves inoculated with D1 and e4, while no symptoms were observed in the uninoculated control seedlings (Supplementary Fig. 1). Isolates with vigorous, downy, and white to grayish-green aerial mycelia were reisolated from the diseased leaves inoculated with D1 and e4 and identified as C. cassiicola by DNA sequencing, fulfilling Koch's postulates. Based on morphological and multilocus phylogenetic results, these isolates were identified as C. cassiicola, a pathogen that threatens several important crops (Dixon et al. 2009; Zhang et al. 2018; Xie et al. 2021). To our knowledge, this is the first report of C. cassiicola as the causal pathogen of septoria leaf spot on S. baicalensis in China, which poses a potential threat to the production of S. baicalensis.

ZIC2 induces pro-tumor macrophage polarization in nasopharyngeal carcinoma by activating the JUNB/MCSF axis.

Nasopharyngeal carcinoma (NPC) is a common malignant epithelial tumor of the head and neck that often exhibits local recurrence and distant metastasis. The molecular mechanisms are understudied, and effective therapeutic targets are still lacking. In our study, we found that the transcription factor ZIC2 was highly expressed in NPC. Although ZIC family members play important roles in neural development and carcinogenesis, the specific mechanism and clinical significance of ZIC2 in the tumorigenesis and immune regulation of NPC remain elusive. Here, we first reported that high expression of ZIC2 triggered the secretion of MCSF in NPC cells, induced M2 polarization of tumor-associated macrophages (TAMs), and affected the secretion of TAM-related cytokines. Mechanistically, ChIP-seq and RNA-seq analyses identified JUNB as a downstream target of ZIC2. Furthermore, ZIC2 was significantly enriched in the promoter site of JUNB and activated JUNB promoter activity, as shown by ChIP-qPCR and luciferase assays. In addition, JUNB and MCSF participated in ZIC2-induced M2 TAMs polarization. Thus, blocking JUNB and MCSF could reverse ZIC2-mediated M2 TAMs polarization. Moreover, Kaplan-Meier survival analyses indicated that high expression of ZIC2, JUNB, and CD163 was positively associated with a poor prognosis in NPC. Overexpression of ZIC2 induced tumor growth in vivo, with the increase of JUNB, MCSF secretion, and CD163. In summary, our study implies that ZIC2 induces M2 TAM polarization, at least in part through regulation of JUNB/MCSF and that ZIC2, JUNB, and CD163 can be utilized as prognostic markers for NPC and as therapeutic targets for cancer immunotherapy.

Study of 43 SLE patients with autoimmune liver cirrhosis: emphasis on clinical features and differences from lupus without cirrhosis.

OBJECTIVE: To investigate the clinical characteristics of systemic lupus erythematosus accompanied by autoimmune liver cirrhosis (SLE-ALC) patients and differences from the non-cirrhosis group. METHODS: Forty-three patients with SLE-ALC were enrolled in this study from 2653 patients with SLE in Peking University People's Hospital. A descriptive case-control study was performed between SLE-ALC patients and the entry time-matched non-cirrhosis group. RESULTS: Among the 43 SLE-ALC patients, 41 (95.3%) were female. Eight patients (18.6%) were first found to have cirrhosis and then diagnosed with SLE. Eighteen patients (41.9%) had jaundice and 27 (62.8%) had esophageal and gastric varices. The age of SLE-ALC patients was 51.1 ± 17.2 years, which was significantly older than the non-cirrhosis group (P < 0.001). Lung involvement was more common as initial manifestations in SLE-ALC patients during the SLE course (P=0.027). Compared with the non-cirrhosis group, SLE-ALC patients had worse liver function. A significantly higher rate of hematological system involvement (anemia, leucopenia, and thrombocytopenia) and a higher level of immunoglobulins were observed in SLE-ALC patients (P<0.05). Moreover, SLE-ALC patients displayed a lower positive rate of anti-double-stranded DNA and anti-ribosomal P protein (P<0.05). The most common radiologic manifestations are ascitic fluid (72.1%) and splenomegaly (71.4%) in SLE-ALC patients. Six SLE-ALC patients underwent liver biopsy, and interface hepatitis was present in all patients. CONCLUSIONS: Cirrhosis is rare in SLE patients but is manifested as a unique pattern of clinical features characterized by late-onset age, lung involvement, high immunoglobulins, and impaired liver function.

[Sensitivity baseline establishment and resistance risk assessment of Botrytis cinerea from Panax ginseng to prochloraz].

This study aimed to establish the baseline sensitivity of Botrytis cinerea from Panax ginseng to prochloraz, and ensure the fitness of prochloraz-resistant mutants and the cross-resistance of B. cinerea to prochloraz and commonly used fungicides for the prevention and control of gray mold including boscalid, pyraclostrobin, iprodione, and pyrimethanil. The sensitivity of B. cinerea from P. ginseng to fungicides was determined by the mycelial growth rate method. The prochloraz-resistant mutants were screened out through fungicide domestication and ultraviolet(UV) induction. The fitness of resistant mutants was determined through the stability of subculture, mycelial growth rate, and pathogenicity test. The cross-resistance between prochloraz and the four fungicides was determined by Person correlation analysis. The results showed that all B. cinerea strains tested were sensitive to prochloraz, and the EC_(50) value ranged from 0.004 8 to 0.062 9 µg·mL~(-1), with an average of 0.022 µg·mL~(-1). The sensitivity frequency distribution diagram showed that 89 B. cinerea strains were located within the main peak with a continuous single peak curve, and the average EC_(50) value of 0.018 µg·mL~(-1) was taken as the baseline sensitivity of B. cinerea to prochloraz. The fungicide domestication and UV induction obtained 6 resistant mutants, among which 2 strains were unstable and the other 2 strains showed decreased resistance after multiple generations of culture. Furthermore, the mycelial growth rate and spore yield of all resistant mutants were lower than those of their parents, and the pathogenicity of most mutants was lower than that of their parents. In addition, prochloraz had no obvious cross-resistance with boscalid, pyraclostrobin, iprodione, and pyrimethanil. In conclusion, prochloraz has great potential for controlling gray mold in P. ginseng, and the resistance risk of B. cinerea to prochloraz is low.

Efficiency of percutaneous transhepatic cholangioscopy in the treatment of biliary complications after liver transplantation.

BACKGROUND: Percutaneous transhepatic cholangioscopy (PTCS) has provided an alternative therapeutic option for handling refractory biliary complications in liver transplanted recipients. This study aimed to evaluate short-term PTCS efficiency in the management of biliary complications following liver transplantation. METHODS: Clinical data of 25 patients who received therapeutic PTCS due to biliary complications after liver transplantation were retrospectively analyzed. RESULTS: Therapeutic PTCS was successfully performed in 25 patients. Biliary complications were anastomotic strictures in seven cases, intrahepatic cholangiolithiasis in four cases, extra-and intrahepatic cholangiolithiasis in three cases, choledocholithiasis complicated with anastomotic strictures in four cases, intrahepatic cholangiolithiasis complicated with non-anastomotic strictures in one case, intrahepatic cholangiolithiasis complicated with anastomotic strictures in five cases, intrahepatic cholangiolithiasis complicated with anastomotic strictures and ischemic cholangitis in one case. The median time between liver transplantation and first PTCS was 24 months, and median times of PTCS was 2.6. Clinical manifestations were significantly improved in most patients after PTCS, and biliary complications were successfully managed through PTCS in 15 cases, which were partially effective in eight cases and ineffective in two cases. PTCS was more effective in tackling anastomotic strictures and cholangiolithiasis. CONCLUSION: PTCS was an effective therapeutic modality for treating refractory biliary complications following liver transplantation.

Disease predisposition of human leukocyte antigen class II genes influences the gut microbiota composition in patients with primary biliary cholangitis.

Background: The human leukocyte antigen (HLA) susceptibility gene is the main genetic risk factor for primary biliary cholangitis (PBC). The prognosis of patients with PBC is linked to gut microbiota dysbiosis. However, whether the HLA alleles are associated with the gut microbiota distribution and disease severity remains unknown. Methods: A cohort of 964 Chinese patients with PBC was enrolled at Beijing YouAn Hospital, Beijing, China. High-resolution genotyping of the HLA class I and class II loci from 151 of these patients was performed using sequence-based PCR. Stool samples were collected from 43 of the 151 fully HLA-typed patients to analyze their microbiota compositions via 16S RNA gene sequencing. Results: Of the 964 patients, the male:female ratio was 114:850, and 342 of these patients (35.5%) had already developed liver cirrhosis (LC) before enrollment. Patients with PBC showed a significantly higher frequency of HLA DRB1*08:03 than did the controls (21.2% vs. 9.0%, P=0.0001). HLA-DRB1*03:01, DRB1*07:01, DRB1*14:05, and DRB1*14:54 frequencies were also increased but did not reach significance after Bonferroni's correction. Conversely, the DQB1*03:01 frequency was significantly lower in patients with PBC than in the controls (24.5% vs. 39.2%, P=0.0010). The patients' gut microbiota were analyzed from four perspectives. The microbial community abundances were significantly lower in FHRAC-positive patients (patients with a combination of five HLA DRB1 high-risk alleles) than in FHRAC-negative patients (P<0.05). Of the top 10 microbial genera, Lachnospiraceae_incertae_sedis was higher in the FHRAC-positive patients than in the FHRAC-negative patients (P<0.05). linear discriminant analysis (LDA) effect-size (LEfSe) analysis showed different microbes at different levels in the FHRAC-negative patients but not in the FHRAC-positive patients. DQB1*03:01-positive patients contained mostly Lactobacillaceae at the family level. A comparison of the FHRAC-positive patients with and without liver cirrhosis showed that the abundances of Veillonella were significantly higher in patients with cirrhosis and FHRAC than in those without cirrhosis and are FHRAC-negative. Conclusion: The HLA class II genes may influence the gut microbiota compositions in patients with PBC. Differential gut microbiota were expressed at different taxonomic levels. Some bacterial abundances may be increased in FHRAC-positive patients with PBC and cirrhosis.

Tenofovir Alafenamide for Pregnant Chinese Women With Active Chronic Hepatitis B: A Multicenter Prospective Study.

BACKGROUND & AIMS: Data on long-term tenofovir alafenamide (TAF) therapy for pregnant women with active chronic hepatitis B (CHB) (immune clearance and reactivation phases, currently and previously diagnosed) and their infants are lacking. METHODS: Pregnant women with active CHB treated with TAF and tenofovir disoproxil fumarate (TDF) were enrolled in this multicenter prospective study, and infants received immunoprophylaxis. The primary outcomes were rates of adverse (safety) events in pregnant women and defects in infants and fetuses. The secondary outcomes were virologic responses in pregnant women, infants' safety, hepatitis B surface antigen (HBsAg) status, and growth conditions. RESULTS: One hundred three and 104 pregnant women were enrolled and 102 and 104 infants were born in the TAF and TDF groups, respectively. In the TAF group, the mean age, gestational age, alanine aminotransferase level, and viral loads at treatment initiation were 29.3 years, 1.3 weeks, 122.2 U/L, and 5.1 log10 IU/mL, respectively. TAF was well-tolerated, and the most common adverse event was nausea (29.1%) during a mean of 2 years of treatment. Notably, 1 (1.0%) TAF-treated pregnant woman underwent induced abortion due to noncausal fetal cleft lip and palate. No infants in either group had birth defects. In the TAF group, the hepatitis B e antigen seroconversion rate was 20.7% at postpartum month 6, infants had normal growth parameters, and no infants were positive for HBsAg at 7 months. The TDF group had comparable safety and effectiveness profiles. CONCLUSIONS: TAF administered throughout or beginning in early pregnancy is generally safe and effective for pregnant women with active CHB and their infants.

Using two-step cluster analysis to classify inpatients with primary biliary cholangitis based on autoantibodies: A real-world retrospective study of 537 patients in China.

Background: A variety of autoantibodies have been detected in primary biliary cholangitis (PBC), while the presence of autoantibody clusters and their clinical significance have not been fully understood. We aimed at defining autoantibody clusters and to better understand the clinical features and prognosis of PBC patients based on autoantibody clusters under real-world conditions. Methods: We retrospectively analyzed 788 inpatients with PBC evaluated between October 2008 and July 2019, and included 537 patients. Nineteen autoantibodies which were measured routinely were investigated for cluster analysis. Two-step clustering, Kaplan-Meier survival, and Cox regression analyses were used. Results: Five clusters were defined. A cluster of antinuclear antibodies (ANA) and anti-gp210 positive patients were identified with a high rate of cirrhosis at baseline and low survival rate; a cluster of ANA, anti-centromere antibodies (ACA) and/or anti-CENP-B female dominant patients with older disease onset, low level of platelet count at baseline, high rate of hepatic decompensation, and low survival rate was also characterized; and another cluster of anti-mitochondrial antibodies (AMA) and/or AMA-M2, anti-Ro52 and a high rate of anti-gp210 positive patients were identified with a high proportion of male patients and low survival rate. A subgroup of patients with anti-SSA and/or anti-SSB coexists with SjS was also identified; patients with only AMA and/or AMA-M2-positive with a benign clinical outcome and relatively high complication of non-alcoholic fatty liver disease (NAFLD) were also identified. Only anti-gp210 was considered as a significant predictor for poor outcomes especially in patients with cirrhosis. Conclusion: Clustering methods allow the identification of distinct autoantibody profiles of PBC that form clinical subsets and can be useful for personalized approaches to diagnosis, clinical management, and the prediction of clinical outcomes. Anti-gp210 was the strongest predictive factor for poor outcomes especially in PBC patients with cirrhosis under real-world conditions.

Clinical characteristics and HLA genotypes in Chinese patients with anti-SLA/LP-positive autoimmune hepatitis.

BACKGROUND: Anti-soluble liver antigen/liver pancreas (anti-SLA/LP) is a highly specific serological marker for the diagnosis of autoimmune hepatitis (AIH). The aim of the present study was to define the clinical characteristics and human leucocyte antigen (HLA) genotypes of Chinese patients with anti-SLA/LP positive AIH. METHODS: Ninety-one AIH patients who were anti-SLA/LP positive were enrolled in this case control study. Clinical information was obtained through reviewing patients' clinical notes. High-resolution genotyping of HLA-A, B, C, DRB1, and DQB1 alleles was performed by sequence-based typing polymerase chain reaction on 62 of the 91 patients. Data from 500 healthy patients were used as baseline controls. RESULTS: Anti-SLA/LP-positive AIH patients were characterized as follows: adults (age 20-80 years), female (88%), and frequent anti-nuclear antibody positivity (91%). Genetically, compared with the controls, HLA-B*35:01 and C*08:01 were significantly more frequent in patients. The frequencies of HLA-B*08:01, B*40:02, DRB1*04:01, DRB1*04:05, DRB1*14:01, and DRB1*16:02 increased, and the frequency in DRB1*15:01 decreased in patients, but did not reach significance after Bonferroni's correction. Patients with other autoimmune diseases had a higher DRB1*04:05 and DQB1*04:01 allele carrier frequency than those without. DRB1*04:05 and DQB1*04:01 alleles were found at increased frequency in patients with decompensated liver disease than those with compensated liver disease. CONCLUSIONS: Chinese anti-SLA/LP-positive AIH patients have some distinct clinical characteristics than other populations reported in the literature. The presence of certain specific HLA alleles could potentially increase the risk of developing anti-SLA/LP-positive AIH or other autoimmune disease and decompensated liver disease in the Chinese population.

Tenofovir Alafenamide to Prevent Perinatal Hepatitis B Transmission: A Multicenter, Prospective, Observational Study.

BACKGROUND: Few safety and effectiveness results have been published regarding the administration of tenofovir alafenamide fumarate (TAF) during pregnancy for the prevention of mother-to-child transmission (MTCT) of hepatitis B virus (HBV). METHODS: In this multicenter prospective observational study, pregnant women with HBV DNA levels higher than 200 000 IU/mL who received TAF or tenofovir disoproxil fumarate (TDF) from gestational weeks 24-35 to delivery were 1:1 enrolled and followed until postpartum month 6. Infants received immunoprophylaxis. The primary endpoint was the safety of mothers and infants. The secondary endpoint was the hepatitis B surface antigen (HBsAg)-positive rate at 7 months for infants. RESULTS: In total, 116 and 116 mothers were enrolled, and 117 and 116 infants were born, in the TAF and TDF groups, respectively. TAF was well tolerated during a mean treatment duration of 11.0 weeks. The most common maternal adverse event was nausea (19.0%). One (0.9%), 3 (2.6%), and 9 (7.8%) mothers had abnormal alanine aminotransferase levels at delivery and at postpartum months 3 and 6, respectively. The TDF group had safety profiles that were comparable to those of the TAF group. No infants had birth defects in either group. The infants' physical and neurological development at birth and at 7 months in the TAF group were comparable with those in the TDF group. The HBsAg positive rate was 0% at 7 months in all 233 infants. CONCLUSIONS: Antiviral prophylaxis with TAF was determined to be generally safe for both mothers and infants and reduced the MTCT rate to 0%.

Single-cell transcriptomic analysis defines the interplay between tumor cells, viral infection, and the microenvironment in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy with a complex tumor ecosystem. How the interplay between tumor cells, EBV, and the microenvironment contributes to NPC progression and immune evasion remains unclear. Here we performed single-cell RNA sequencing on ~104,000 cells from 19 EBV+ NPCs and 7 nonmalignant nasopharyngeal biopsies, simultaneously profiling the transcriptomes of malignant cells, EBV, stromal and immune cells. Overall, we identified global upregulation of interferon responses in the multicellular ecosystem of NPC. Notably, an epithelial-immune dual feature of malignant cells was discovered and associated with poor prognosis. Functional experiments revealed that tumor cells with this dual feature exhibited a higher capacity for tumorigenesis. Further characterization of the cellular components of the tumor microenvironment (TME) and their interactions with tumor cells revealed that the dual feature of tumor cells was positively correlated with the expression of co-inhibitory receptors on CD8+ tumor-infiltrating T cells. In addition, tumor cells with the dual feature were found to repress IFN-γ production by T cells, demonstrating their capacity for immune suppression. Our results provide new insights into the multicellular ecosystem of NPC and offer important clinical implications.

Short-term Peginterferon-Induced High Functional Cure Rate in Inactive Chronic Hepatitis B Virus Carriers With Low Surface Antigen Levels.

BACKGROUND: None of the current guidelines recommend antiviral therapy for inactive hepatitis B virus (HBV) carriers (IHCs). METHODS: In this real-world, multicenter, nonrandomized study, 32 participants meeting the inclusion criteria were enrolled 1:1 for treatment with peginterferon α-2b or monitoring without treatment based on participant preference. The expected treatment duration was 48 weeks. The primary end point was hepatitis B surface antigen (HBsAg) loss. The HBV vaccine could be injected after HBsAg loss. RESULTS: All patients had HBsAg levels of <20 IU/mL. The mean baseline HBsAg levels were 6.6 IU/mL and 5.8 IU/mL in the treated and untreated groups, respectively. Fifteen (93.8%) participants achieved HBsAg loss, 5 obtained HBsAg seroconversion after undergoing a mean of 19.7 weeks of therapy in the treated group, and no one in the follow-up group achieved HBsAg loss during a mean follow-up time of 12.6 months (P < .0001). Generally, the therapy was well tolerated. Nine of 11 individuals who exhibited HBsAg loss benefited from receiving the HBV vaccine. CONCLUSIONS: This study provides justification for further studies of short-course peginterferon α-2b for the functional cure of IHCs with low HBsAg levels. Additionally, HBV vaccine injection is beneficial after interferon-induced HBsAg loss.

ZIC2 is downregulated and represses tumor growth via the regulation of STAT3 in breast cancer.

Although early detection and systemic therapies have improved the diagnosis and clinical cure rate of breast cancer, breast cancer remains the most frequently occurring malignant cancer in women due to a lack of sufficiently effective treatments. Thus, to develop potential targeted therapies and thus benefit more patients, it is helpful to understand how cancer cells work. ZIC family members have been shown to play important roles in neural development and carcinogenesis. In our study, we found that ZIC2 is downregulated in breast cancer tissues at both the mRNA and protein levels. Low expression of ZIC2 was correlated with poor outcome in breast cancer patients and serves as an independent prognostic marker. Furthermore, overexpression of ZIC2 repressed, whereas knockdown of ZIC2 promoted, cell proliferation and colony formation ability in vitro and tumor growth in vivo. Using ChIP-seq and RNA-seq analysis, we screened and identified STAT3 as a potential target for ZIC2. ZIC2 bound to the STAT3 promoter and repressed the promoter activities of STAT3. ZIC2 knockdown induced the expression of STAT3, increasing the level of phosphorylated STAT3. These results suggest that ZIC2 regulates the transcription of STAT3 by directly binding to the STAT3 promoter. Additionally, interfering STAT3 with siRNAs or inhibitors abrogated the oncogenic effects induced by decreased ZIC2. Taken together, our results indicate that ZIC2 serves as a useful prognostic marker in breast cancer and acts as a tumor suppressor by regulating STAT3, implying that STAT3 inhibitors might provide an alternative treatment option for breast cancer patients with ZIC2 downregulation.

Fluctuations of antimitochondrial antibodies and anti-gp210 antibody in a patient with primary biliary cholangitis and Sjögren syndrome with subsequent autoimmune hemolytic anemia: A case report.

RATIONALE: Primary biliary cholangitis (PBC) is a rare autoimmune cholestatic liver disease. It is often associated with extrahepatic autoimmune disorders. However, the concurrence of PBC and Sjögren syndrome (SS) with the subsequent onset of autoimmune hemolytic anemia (AIHA) is extremely rare. PATIENT CONCERNS: This study investigated a 60-year-old woman admitted to our hospital with complaints of xerostomia for 5 years, pruritus for 3 years, and abnormal liver function for 3 months. DIAGNOSES: The patient was suffering from typical clinical PBC and SS, and developed decompensated liver cirrhosis after 32 months of ursodeoxycholic acid (UDCA) therapy. In May 2018, she was readmitted to the hospital with a high fever of 39 °C, coughing, and sever fatigue without remission after 3 days of cephalosporin antibiotic therapy. During the clinical course of PBC, her antimitochondrial antibodies (AMA) titers fluctuated from 1:1000 to negative and then to weakly positive, determined by indirect immunofluorescence (IIF), immunoblotting, and enzyme-linked immunosorbent assay (ELISA) based on recombinant mitochondrial antigens; furthermore, her titers of anti-gp210, an antinuclear antibody (ANA), increased sharply. Laboratory tests and imaging were performed to diagnose PBC and SS in September 2015. However, she was subsequently diagnosed with AIHA after 32 months of UDCA therapy based on the identification of pancytopenia, increased reticulocyte (RET) count, and a positive result from the direct Coombs test. INTERVENTIONS: UDCA, hepatic protectant, albumin infusion, chest drainage, rational antibiotic use, diuretics, and methylprednisolone were used to treat the patient. OUTCOMES: Liver cirrhosis was complicated by the development of AIHA, which became severe at 42 months of follow-up. LESSONS: This is the first case report showing a patient with comorbid PBC and SS, as well as the sequential development of AIHA with decreased AMA and increased anti-gp210 titers; this may have been due to immunodeficiency. These findings stress the importance of the serological screening of ANA profile, as well as repeated measurement of ANA and AMA to track PBC progression and prognosis.

Occult HBV infection in patients with autoimmune hepatitis: A virological and clinical study.

BACKGROUND/PURPOSE: Occult HBV infection (OBI) could have serious clinical consequences in patients receiving immunosuppressive therapy. We aimed to investigate the prevalence of OBI in Chinese patients with autoimmune hepatitis (AIH) and to analyze its clinical and virological features. METHODS: 103 AIH cases were enrolled. Hepatitis B virus (HBV) serological markers were screened by chemiluminescence. HBV-DNA were detected by nest-PCR and real-time PCR. HBV genotyping and mutation analysis were performed by Sanger sequencing. RESULTS: Twenty-four out of 103 (23.30%) AIH patients had OBI as evidenced by positive HBV-DNA and negative hepatitis B surface antigen (HBsAg). HBV genotype C is the predominant genotype (57.89%), which had more amino acid (AA) substitutions in S region than that of B-genotype group (P = 0.001). The distribution of AA substitution in the 'α' determinant region between genotype C and B were significantly different (P = 0.042). In addition to those already reported OBI-associated AA substitutions (e.g., sG145R and sV184A), some new OBI-associated AA substitutions (e.g., sV106A, sC137* and sL176P) were found in AIH patients in our study. Three out of 24 (12.50%) OBI patients were diagnosed as decompensated cirrhosis, one patient with S deletion mutation and two patients with HBV extensive AA substitutions. CONCLUSIONS: There was a higher proportion of AIH patients with OBI than the general population in China, which can be either seropositive or seronegative-OBI in AIH patients is associated with some specific AA substitutions. The presence of deletion mutations and the extent of AA substitutions in the HBV S region may have predictive clinical implications.

Xijiao Dihuang Decoction () and Rehmannia glutinosa Libosch. protect mice against lipopolysaccharide and tumor necrosis factor alpha-induced acute liver failure.

OBJECTIVE: To investigate the hepatoprotective effect of Xijiao Dihuang Decoction (, XJDHD) on lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNF-α)-induced acute liver failure (ALF) as well as the underlying mechanism of action, and to clarify the key herbs and components of XJDHD. METHODS: LPS/D-galactosamine (D-GalN) or TNF-α/D-GalN were intraperitoneally injected into C57BL/6J mice to induce ALF. Simultaneously, XJDHD or its individual herbs and components were orally administered. Survival rates, transaminase levels in serum, and hepatic histology were examined to evaluate the effects of XJDHD. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and real-time polymerase chain reaction were additionally performed to expound the mechanism underlying the anti-apoptotic activity of XJDHD. RESULTS: Oral administration of XJDHD protected mice from lethal liver failure induced by LPS and TNF-α, with notable amelioration of liver injury in histology and a significant decrease in transaminase levels in serum. XJDHD significantly inhibited apoptosis of hepatocytes and enhanced expression of the antiapoptosis genes, c-Flip, Iap1, Gadd45b and A20. In addition, Rehmannia glutinosa Libosch. was identified as the key herb of XJDHD and galactose as the effective component of Rehmannia glutinosa Libosch. that protects against ALF. CONCLUSIONS: XJDHD inhibits TNF-α-induced apoptosis of hepatocytes by promoting the expression of nuclear factor κ B-regulated anti-apoptotic genes. Rehmannia glutinosa Libosch. is the effective herb of XJDHD and galactose is an active component in this protection.

Protective Effect of Ganshuang Granules () on Liver Cirrhosis by Suppressing Regulatory T Cells in Mouse Model.

OBJECTIVE: To investigate the potential antifibrotic mechanisms of Chinese medicine Ganshuang Granules (, GSG) and to provide clinical therapeutic evidence of its effects. METHODS: A cirrhotic mouse model was established by intraperitoneally injecting a mixture of CCl4 (40%) and oil (60%) at 0.2 mL per 100 g of body weight twice a week for 12 weeks. After 12-week modeling, GSG was intragastric administrated to the mice for 2 weeks, and the mice were divided into low-, medium- and high-dose groups at doses of 1, 2 and 4 g/(kg·day), respectively. Liver morphology changes were observed using Masson's trichrome staining and B-ultrasound. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA) in serum were detected using an automatic biochemistry analyzer. The expressions of desmin, smooth muscle actin (SMA) and Foxp3 in liver were detected by immunoflfluorescence. The regulatory T cell (Treg) frequency was determined through flflow cytometry analysis. Collagen-I, SMA, IL-6, tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and transforming growth factor ß1 (TGF-ß1) expression levels were measured using quantitative polymerase chain reaction (qPCR). RESULTS: Masson's staining result showed fewer pseudolobule structures and fibrous connective tissue in the GSG-treatment groups than in the spontaneous recovery group. Ultrasonography showed that GSG treatment reduced the number of punctate hyperechoic lesions in mice cirrhotic livers. The serum ALT, AST, HA levels were significantly ameliorated by GSG treatment (ALT: F=8.104, P=0.000; AST: F=7.078, P=0.002; and HA: F=7.621, P=0.001). The expression levels of collagen-I and SMA in the cirrhotic livers were also attenuated by GSG treatment (collagen-I: F=3.938, P=0.011; SMA: F=4.115, P=0.009). Tregs, which were elevated in the fibrotic livers, were suppressed by GSG treatment (F=8.268, P=0.001). The expressions of IL-6, TNF-α and IL-1ß increased, and TGF-ß levels decreased in the cirrhotic livers after GSG treatment (IL-6: F=5.457, P=0.004; TNF-α: F=6.023, P=0.002; IL-1ß: F=6.658, P=0.001; and TGF-ß1: F=11.239, P=0.000). CONCLUSIONS: GSG promoted the resolution/regression of cirrhosis and restored liver functions in part by suppressing Treg cell differentiation, which may be mediated by hepatic stellate cells.