Blockade of the checkpoint receptor TIGIT prevents NK cell exhaustion and elicits potent anti-tumor immunity.

Checkpoint blockade enhances effector T cell function and has elicited long-term remission in a subset of patients with a broad spectrum of cancers. TIGIT is a checkpoint receptor thought to be involved in mediating T cell exhaustion in tumors; however, the relevance of TIGIT to the dysfunction of natural killer (NK) cells remains poorly understood. Here we found that TIGIT, but not the other checkpoint molecules CTLA-4 and PD-1, was associated with NK cell exhaustion in tumor-bearing mice and patients with colon cancer. Blockade of TIGIT prevented NK cell exhaustion and promoted NK cell-dependent tumor immunity in several tumor-bearing mouse models. Furthermore, blockade of TIGIT resulted in potent tumor-specific T cell immunity in an NK cell-dependent manner, enhanced therapy with antibody to the PD-1 ligand PD-L1 and sustained memory immunity in tumor re-challenge models. This work demonstrates that TIGIT constitutes a previously unappreciated checkpoint in NK cells and that targeting TIGIT alone or in combination with other checkpoint receptors is a promising anti-cancer therapeutic strategy.

Spatial transcriptomics reveals a low extent of transcriptionally active hepatitis B virus integration in patients with HBsAg loss.

OBJECTIVE: Hepatitis B virus (HBV) can integrate into the chromosomes of infected hepatocytes, contributing to the production of hepatitis B surface antigen (HBsAg) and to hepatocarcinogenesis. In this study, we aimed to explore whether transcriptionally active HBV integration events spread throughout the liver tissue in different phases of chronic HBV infection, especially in patients with HBsAg loss. DESIGN: We constructed high-resolution spatial transcriptomes of liver biopsies containing 13 059 tissue spots from 18 patients with chronic HBV infection to analyse the occurrence and relative distribution of transcriptionally active viral integration events. Immunohistochemistry was performed to evaluate the expression of HBsAg and HBV core antigen. Intrahepatic covalently closed circular DNA (cccDNA) levels were quantified by real-time qPCR. RESULTS: Spatial transcriptome sequencing identified the presence of 13 154 virus-host chimeric reads in 7.86% (1026 of 13 059) of liver tissue spots in all patients, including three patients with HBsAg loss. These HBV integration sites were randomly distributed on chromosomes and can localise in host genes involved in hepatocarcinogenesis, such as ALB, CLU and APOB. Patients who were receiving or had received antiviral treatment had a significantly lower percentage of viral integration-containing spots and significantly fewer chimeric reads than treatment-naïve patients. Intrahepatic cccDNA levels correlated well with viral integration events. CONCLUSION: Transcriptionally active HBV integration occurred in chronically HBV-infected patients at different phases, including in patients with HBsAg loss. Antiviral treatment was associated with a decreased number and extent of transcriptionally active viral integrations, implying that early treatment intervention may further reduce the number of viral integration events.

Blockade of T-cell receptor with Ig and ITIM domains elicits potent antitumor immunity in naturally occurring HBV-related HCC in mice.

BACKGROUND AND AIMS: Chronic HBV infection is the leading cause of HCC and a serious health problem in China, East Asia, and North African countries. Effective treatment of HBV-related HCC is currently unavailable. This study evaluated the therapeutic potential of T-cell immunoreceptor with Ig and ITIM domains (TIGIT) blockade in HBV-related HCC. APPROACH AND RESULTS: A mouse model of spontaneous HBV-related HCC was generated by replacing wild-type hepatocytes with HBsAg + hepatocytes (namely HBs-HepR mice). The tumors in HBs-HepR mice were inflammation-associated HCC, similar to HBV-related HCC in patients, which was distinguished from other HCC mouse models, such as diethylnitrosamine-induced HCC, TGF-ß-activated kinase 1 knockout-induced HCC, HCC in a stelic animal model, or NASH-induced HCC. HCC in HBs-HepR mice was characterized by an increased number of CD8 + T cells, whereas the production of IL-2, TNF-α, and interferon-gamma (IFN-γ) by intrahepatic CD8 + T cells was decreased. Increased expression of TIGIT on CD8 + T cells was responsible for functional exhaustion. The therapeutic effect of TIGIT blockade was investigated at the early and middle stages of HCC progression in HBs-HepR mice. TIGIT blockade reinvigorated intrahepatic CD8 + T cells with increased TNF-α and IFN-γ production and an increased number of CD8 + T cells in tumors, thereby slowing the development of HCC in HBs-HepR mice. Blocking PD-L1 did not show direct therapeutic effects or synergize with TIGIT blockade. CONCLUSIONS: Blockade of TIGIT alone enhanced the antitumor activity of CD8 + T cells during the progression of HBV-related HCC in a spontaneous HCC mouse model.

CD11b + CD43 hi Ly6C lo splenocyte-derived macrophages exacerbate liver fibrosis via spleen-liver axis.

BACKGROUND AND AIMS: Monocyte-derived macrophages (MoMFs), a dominant population of hepatic macrophages under inflammation, play a crucial role in liver fibrosis progression. The spleen serves as an extra monocyte reservoir in inflammatory conditions; however, the precise mechanisms of involvement of the spleen in the pathogenesis of liver fibrosis remain unclear. APPROACH AND RESULTS: By splenectomy and splenocyte transfusion, it was observed that splenic CD11b + cells accumulated intrahepatically as Ly6C lo MoMFs to exacerbate CCl 4 -induced liver fibrosis. The splenocyte migration into the fibrotic liver was further directly visualized by spleen-specific photoconversion with KikGR mice and confirmed by CD45.1 + /CD45.2 + spleen transplantation. Spleen-derived CD11b + cells purified from fibrotic livers were then annotated by single-cell RNA sequencing, and a subtype of CD11b + CD43 hi Ly6C lo splenic monocytes (sM-1s) was identified, which was markedly expanded in both spleens and livers of mice with liver fibrosis. sM-1s exhibited mature feature with high expressions of F4/80, produced much ROS, and manifested preferential migration into livers. Once recruited, sM-1s underwent sequential transformation to sM-2s (highly expressed Mif , Msr1 , Clec4d , and Cstb ) and then to spleen-derived macrophages (sMφs) with macrophage features of higher expressions of CX 3 CR1, F4/80, MHC class II, and CD64 in the fibrotic hepatic milieu. Furthermore, sM-2s and sMφs were demonstrated capable of activating hepatic stellate cells and thus exacerbating liver fibrosis. CONCLUSIONS: CD11b + CD43 hi Ly6C lo splenic monocytes migrate into the liver and shift to macrophages, which account for the exacerbation of liver fibrosis. These findings reveal precise mechanisms of spleen-liver axis in hepatic pathogenesis and shed light on the potential of sM-1 as candidate target for controlling liver diseases.

Inadvertent antibiotic exposure during pregnancy may increase the risk for neural tube defects in offspring.

BACKGROUND: As emerging environmental contaminants, antibiotics pose potential threats to human health, in particular to pregnant women and infants. However, the potential harm of inadvertent antibiotic exposure (IAE) is often disregarded in light of the focus on intentional antibiotic use during pregnancy. Currently, little is known about the effects of IAE during pregnancy on fetal neural tube development. METHODS: In this case-control study, we used questionnaire data from 855 subjects to investigate the effects of intentional antibiotic use in early pregnancy on neural tube defects (NTDs). Then we tested for placental antibiotics in mothers who had not intentionally used antibiotics, and the compounds were detected in 379 subjects; these were considered IAE cases. We assessed the association between IAE during pregnancy and fetal NTDs using both multivariable logistic and multi-pollutant exposure models. We also analyzed the correlation between maternal dietary habits and placental antibiotics to explore possible sources of IAE. RESULTS: Only 50 of 855 participants (5.8%) intentionally used antibiotics and such use showed no significant association with NTD risk (odds ratio [OR] = 1.92, confidence interval [95%CI] = [0.66, 5.59]). However, 14 of 15 placental antibiotics were detected in 378 of 379 subjects (99.7%) and multivariable logistic analysis indicated that high levels of placental macrolides were significantly associated with increased NTD risk (4.42 [2.01-10.45]). Multi-pollutant exposure analysis suggested an increase in NTD risk with an increase in exposure to a mixture of placental antibiotics, among which macrolides were the most important contributor. In addition, the level of placental macrolides was positively correlated with the intake frequency of milk. Finally, mothers who drank river, well, or pond water had higher levels of placental macrolides than those who drank only tap water. CONCLUSIONS: Intentional antibiotic use during early pregnancy may not be associated with NTDs, while IAE during pregnancy is associated with higher NTD risk in offspring. Macrolides are crucial risk factors. Milk, and river, well, or pond water may be important sources of IAE.

Dihydrotrichodimerol Purified from the Marine Fungus Acremonium citrinum Prevents NAFLD by Targeting PPARα.

The pathogenesis of nonalcoholic fatty liver disease (NAFLD) is closely linked to the imbalance of lipid and glucose metabolism, in which peroxisome proliferator-activated receptors (PPARs) play essential roles. The clinical trials have shown the beneficial effects of the PPARs' ligands on NAFLD. In this study, we screen the extracts from the marine fungus Acremonium citrinum and identify the natural compounds dihydrotrichodimerol (L1A) and trichodimerol (L1B) as the ligands of PPARs, of which L1A is a dual PPARα/γ agonist, whereas L1B is a selective PPARγ agonist. L1A but not L1B significantly prevents hepatic lipid accumulation in an oleic acid-induced NAFLD cell model as well as in a high-fat-diet-induced NAFLD mouse model. Moreover, L1A potently inhibits hepatic steatosis in a PPARα-dependent manner in another NAFLD mouse model constructed by using a choline-deficient and amino acid-defined diet. Mechanistically, L1A transcriptionally up-regulates the expression of SIRT1 in a PPARα-dependent manner, followed by the activation of AMPK and inactivation of ACC, resulting in the inhibition of lipid anabolism and the increase of lipid catabolism. Taken together, our study reveals a dual ligand of PPARα/γ with a distinct structure and therapeutic effect on NAFLD, providing a potential drug candidate bridging the currently urgent need for the management of NAFLD.

Fentanyl Promoted the Growth of Placenta Trophoblast Cells through Regulating the METTL14 Mediated CCL5 Levels.

Gestational diabetes mellitus (GDM) is an important cause of the increase in incidence rate and mortality of pregnant women and perinatal infants. This study aimed to analyze the role of fentanyl, a µ-opioid agonist, in the GDM progression. The high glucose (HG) treatment HTR8/SVneo cells was used as a GDM model in vitro. The cell viability was assessed with cell counting kit-8 assay. The apoptosis rate was analyzed with flow cytometry and the transwell assay was conducted to test the cell migration and invasion. RT-quantitative PCR (qPCR) assay was performed to determine the relative expressions of related genes. The N6-Methyladenosine (m6A) levels were analyzed with MeRIP analysis. The tumor necrosis factor-α (TNF-α), interleukin 1ß (IL-1ß), and IL-10 levels of the cells were analyzed with commercial kits. The results showed that fentanyl increased the cell viability, migration and invasion, and IL-10 levels, and declined the apoptosis rate, TNF-α and IL-1ß levels of the HG stimulated HTR8/SVneo cells. The chemokine ligand 5 (CCL5) was over expressed in GDM tissues and HG stimulated HTR8/SVneo cells, which was depleted after fentanyl treatment. Over expressed CCL5 neutralized the fentanyl roles in the HG stimulated HTR8/SVneo cells. The methyltransferase-like protein 14 (METTL14) levels was decreased in HG stimulated HTR8/SVneo cells, which was up-regulated after fentanyl treatment. Additionally, METTL14 silenced prominently decreased the m6A and mRNA levels, along with the mRNA stability of CCL5. In conclusion, fentanyl promoted the growth and inhibited the apoptosis of the HG stimulated HTR8/SVneo cells through regulating the METTL14 mediated CCL5 levels.

UGRP1-modulated MARCO+ alveolar macrophages contribute to age-related lung fibrosis.

The aging lungs are vulnerable to chronic pulmonary diseases; however, the underlying mechanisms are not well understood. In this study, we compared the aging lungs of 20-24-month-old mice with the young of 10-16-week-old mice, and found that aging airway epithelial cells significantly upregulated the expression of uteroglobin-related protein 1 (UGRP1), which was responsible for the higher levels of CCL6 in the aging lungs. Alveolar macrophages (AMs) changed intrinsically with aging, exhibiting a decrease in cell number and altered gene expression. Using terminal differentiation trajectories, a population of MARCO+ AMs with the ability to produce CCL6 was identified in the aging lungs. Upregulated UGRP1was demonstrated to modulate CCL6 production of AMs in the UGRP1-MARCO pair in vivo and in vitro. Furthermore, MARCO+ AMs aggravated bleomycin-induced pulmonary fibrosis in a CCL6-dependent manner in the aged mice, and blocking MARCO or neutralizing CCL6 significantly inhibited pulmonary fibrosis, similar to the depletion of AMs. The age-related upregulation of UGRP1 and MARCO+ AMs, involved in the progression of lung fibrosis, was also observed in human lung tissues. Thus, UGRP1 modulated MARCO+ AMs regarding the age-related lung fibrosis in a CCL6-dependent manner, which is key to establishing optimal targeting for the aging population.

Trigeminal Nerve Isolation Application in Microvascular Decompression for Treating Trigeminal Neuralgia: A Retrospective Study.

This study aimed to compare the outcomes of trigeminal nerve isolation (TNI) with conventional microvascular decompression (CMVD) in cases of trigeminal neuralgia (TN). We retrospectively reviewed 143 TN cases who underwent microvascular decompression from January 2017 to January 2020. The surgical management of TNI or CMVD in all patients was randomized. The cases were divided into two groups, one group underwent a TNI and the other one received CMVD. The general data, postoperative outcomes, and complications were reviewed retrospectively. Cases with a narrow cistern of cerebellopontine, short trigeminal nerve root, and arachnoid adhesion were defined as difficult cases. All of the cases were followed up for at least 1 year. Surgical outcomes were assessed and compared between the two groups. In results, we found no significant differences in the general data, duration of hospitalization and blood loss between the two procedures. However, of the 143 cases, 12 cases (17.1%) recurred after surgery in the CMVD group, and four cases (5.5%) recurred after TNI operation. The rates of pain relief were 69 (94.5%) in the CMVD group, and 58 (82.9%) for TNI ( P =0.027). In the TNI group, there was only one difficult case among four no pain-relief cases, while in the CMVD group, 10 difficult cases were found among the 12 no pain-relief cases ( P =0.008). In conclusion, the TNI technique is more effective than the CMVD procedure and could also be performed on patients with classical TN. Future double-blind and randomized controlled trials are necessary to confirm this result.

Identification and validation of quantitative trait loci for the functional stay green trait in common wheat (Triticum aestivum L.) via high-density SNP-based genotyping.

KEY MESSAGE: This study identified QTLs associated with the functional stay green trait by a high-density genetic map. Two large effect QTLs, QSg.sau-2B.1 and QSg.sau-6A.2, were identified in multiple years and one of them was successfully validated. The functional stay green phenotype enables wheat to acclimate to stressful environments and prolongs the effectiveness of photosynthesis during the end-of-crop season. Despite the fact that stay green mutants in wheat have been reported, our knowledge of loci for the functional stay green trait remains limited. In this study, an RIL population containing 371 lines genotyped using the Wheat55K SNP array was used to map QTLs controlling the functional stay green trait in multiple years. In total, 21 and 19 QTLs were mapped using the BIP or MET modules of the ICIM method, respectively. Among them, two QTLs, QSg.sau-2B.1 and QSg.sau-6A.2, were considered large effect QTLs for the stay green trait and explained 11.43% and 15.27% of phenotypic variation on average, respectively. Two KASP markers were developed and tightly linked to QSg.sau-2B.1 and QSg.sau-6A.2, respectively, and the genetic effects of different genotypes in the RIL population were successfully confirmed. QSg.sau-2B.1 was also validated by linked KASP marker in different genetic backgrounds. QSg.sau-2B.1 and QSg.sau-6A.2 may influence heredity of the stay green trait and also exhibited a positive effect on the grain filling content. In the interval where QSg.sau-2B.1 and QSg.sau-6A.2 were located on the Chinese Spring and T. turgidum ssp. dicoccoides reference genomes, several genes associated with the leaf senescence process were identified. Altogether, our results identified two QTLs associated with the functional stay green trait and will be useful for the fine mapping and cloning of genes for stay green in the future.

Identification of a stable major-effect quantitative trait locus for pre-harvest sprouting in common wheat (Triticum aestivum L.) via high-density SNP-based genotyping.

KEY MESSAGE: A major and stable QTL cQSGR.sau.3D, which can explain 33.25% of the phenotypic variation in SGR, was mapped and validated, and cQSGR.sau.3D was found to be independent of GI. In this study, a recombinant inbred line (RIL) population containing 304 lines derived from the cross of Chuan-nong17 (CN17) and Chuan-nong11 (CN11) was genotyped using the Wheat55K single-nucleotide polymorphism array. A high-density genetic map consisting of 8329 markers spanning 4131.54 cM and distributed across 21 wheat chromosomes was constructed. QTLs for whole spike germination rate (SGR) were identified in multiple years. Six and fourteen QTLs were identified using the Inclusive Composite Interval Mapping-Biparental Populations and Multi-Environment Trial methods, respectively. A total of 106 digenic epistatic QTLs were also detected in this study. One of the additive QTLs, cQSGR.sau.3D, which was mapped in the region from 3.5 to 4.5 cM from linkage group 3D-2 on chromosome 3D, can explain 33.25% of the phenotypic variation in SGR and be considered a major and stable QTL for SGR. This QTL was independent of the seeds' germination traits, such as germination index. One Kompetitive Allele-Specific PCR (KASP) marker, KASP-AX-110772653, which is tightly linked to cQSGR.sau.3D, was developed. The genetic effect of cQSGR.sau.3D on SGR in the RIL and natural populations was successfully confirmed. Furthermore, within the interval in which cQSGR.sau.3D is located in Chinese Spring reference genomes, thirty-seven genes were found. cQSGR.sau.3D may provide new resources for pre-harvest sprouting resistance breeding of wheat in the future.

Phloretin ameliorates diabetes-induced endothelial injury through AMPK-dependent anti-EndMT pathway.

Diabetic cardiovascular complications contribute more than half of diabetes mortality. Endothelial damage and subsequent pathological changes play a key role in this process. Phloretin, a plant-derived dihydrochalcone compound, was reported to have the activities in regulating metabolism homeostasis and anti-inflammation. However, its effects and the mechanism on early stage endothelial injury caused by diabetes are not clear yet. In our present study, human umbilical vein endothelial cells (HUVECs) were stimulated by high glucose or advanced glycation end products (AGEs) to induce endothelial damage, and streptozotocin (STZ) -induced diabetes mouse model was used for in vivo study. Our results showed that phloretin effectively reduced endothelial damage marker monocyte chemotactic protein-1 (MCP1) as well as pro-calcification factors bone morphogenetic protein-2 (BMP2) and receptor activator of NF-κB ligand (RANKL) expression, reversed the increased vimentin and decreased CD31 dose-dependently in vitro and in vivo. Phloretin had no effect on blood glucose level. However, it ameliorated endothelial injury and vascular fibrosis in diabetic mice. Further experiments revealed that phloretin could enhance AMP activated protein kinase (AMPK) activation and upregulate peroxidase proliferator activated receptor-gamma coactivator-lα (PGC1α) level, and inhibit the activation of TGFß-Smad2-Snail signalling pathway which was abrogated by AMPK inhibitor, providing a rational mechanism that AMPK activation was required for the effects of phloretin on endothelial injury and endothelial-mesenchymal transformation (EndMT). Our data reveal a new role of phloretin in protection of diabetic endothelial damage via AMPK-dependent anti-EndMT activation, and also provide a potential therapeutic way for diabetic endothelial damage and its subsequent cardiovascular complications.

Elevated concentrations of chromium in maternal serum, umbilical cord serum, and cord tissue are associated with an increased risk for orofacial clefts.

Chromium (Cr) exposure during gestation causes malformations in animal experiments. In this multicenter case-control study, we initially involved 130 orofacial clefts (OFCs) and 260 controls to assess the association between Cr concentration and risk for OFCs. Then, umbilical cord serum (49 vs. 119) and cord tissue (84 vs. 142) were used to validate the association between Cr and OFCs. We found that maternal serum Cr concentrations in OFC cases were significantly higher than those in controls. Compared with the lowest tertile of maternal serum Cr concentration, the highest tertile of Cr increased the risk for OFCs [OR = 2.14 (1.14-4.05)]. In the validation cohort of umbilical cord serum and tissue, higher concentrations of Cr were associated with increased risks for OFCs in a dose-dependent manner (all Ps for trends <0.05). Cr concentrations in maternal serum and cord serum showed a positive correlation. The Cr concentration in cord serum was inversely correlated with egg and milk consumption frequencies, and the Cr concentration in cord tissue was positively associated with indoor coal burning. In conclusion, prenatal Cr exposure is a risk factor for OFCs, and indoor coal burning during pregnancy may be one of the sources of Cr exposure.

Utilization of a Wheat55K SNP array-derived high-density genetic map for high-resolution mapping of quantitative trait loci for important kernel-related traits in common wheat.

KEY MESSAGE: This study mapped QTLs associated with kernel-related traits by high-density genetic map. Five new major and stable QTLs for KL, KDR, SN, and KWPS were mapped in multiple environments. In the present study, a recombinant inbred line population including 371 lines derived from the cross of Chuannong18 and T1208 was genotyped using the Wheat55K single nucleotide polymorphism array. A novel high-density genetic map consisting of 11,583 markers spanning 4192.62 cM and distributed across 21 wheat chromosomes was constructed. QTLs for important kernel-related traits were mapped in multiple environments. A total of 96 and 151 QTLs were mapped by using the ICIM method and the MET method, respectively. And a total of 114 digenic epistatic QTLs were also detected across 21 chromosomes, and the epistatic effects of each trait were analyzed. BLAST analysis showed that 23 QTLs for different kernel-related traits were first time mapped and five of them were major and stable QTLs for kernel diameter ratio (121.34-126.83 cM on 4BS), spike number per square meter (71.32-73.84 cM on 2DS), kernel weight per spike (71.32-75.26 cM on 2DS), and kernel length (16.78-31.64 cM on 6A and 51.63-58.40 cM on 3D), respectively. Fifteen QTL clusters that contained 58 QTLs were also detected, and all most stable QTLs were contained in these QTL clusters. Significant correlations between different traits were detected and discussed. These results lay the foundation for fine mapping and cloning of the gene(s) underlying the stable QTLs detected in this study.

Rare earth elements in umbilical cord and risk for orofacial clefts.

Questions remain about the effects of rare earth elements (REEs) on reproductive health, and no study has explored in utero exposure to REEs and risk of orofacial clefts (OFCs). We recruited subjects from a case-control study conducted in Shanxi Province, China. Concentrations of fifteen REEs were quantified in umbilical cord samples by means of Inductively Coupled Plasma Mass Spectrometry measurements. We employed logistic regression and weighted quantile sum (WQS) regression models to estimate the association between REEs exposures and OFCs. Of 226 subjects included in our study, 34 were cleft lip only, 44 were cleft lip with cleft palate and 6 were cleft palate only. In the logistic regression model, concentrations above the median of all subjects were associated with an increased OFCs risk of 2.35-fold (95% CI: 1.22, 4.53) for Lanthanum and 2.12-fold for Neodymium (95% CI: 1.10, 4.10) adjusting for maternal age, BMI, gestational weeks, sex of infants and passive smoking. In WQS model, a quartile increase in the index resulting in an increase of 3.10 (95% CI: 1.38, 6.96) in the odds of OFC. Lanthanum and Neodymium were suggested to be important factors. The results were largely consistent for OFC subtypes. In conclusion, in utero exposure to mixtures of REEs increased the risk of OFCs. Lanthanum and Neodymium were likely to be important factors in the development of OFCs.

Rare copy number variations of planar cell polarity genes are associated with human neural tube defects.

Select single-nucleotide variants in planar cell polarity (PCP) genes are associated with increased risk for neural tube defects (NTDs). However, whether copy number variants (CNVs) in PCP genes contribute to NTDs is unknown. Considering that CNVs are implicated in several human developmental disorders, we hypothesized that CNVs in PCP genes may be causative factors to human NTDs. DNA from umbilical cord tissues of NTD-affected fetuses and parental venous blood samples were collected. We performed a quantitative analysis of copy numbers of all exon regions in the VANGL1, VANGL2, CELSR1, SCRIB, DVL2, DVL3, and PTK7 genes using a CNVplex assay. Quantitative real-time PCR (qPCR) was carried out to confirm the results of CNV analysis. As a result, 16 CNVs were identified among the NTDs. Of these CNVs, 5 loci were identified in 11 NTD probands with CNVs involving DVL2 (exons 1-15), VANGL1 (exons 1-7, exon 8), and VANGL2 (exons 5-8, exons 7 and 8). One CNV (DVL2 exons 1-15) was a duplication and the remaining 15 CNVs were deletions. Eleven CNVs were confirmed by qPCR. One de novo CNV in VANGL1 and one DVL2 were detected from two cases. Compared with unaffected control populations in 1000 Genome, ExAC, MARRVEL, DGV, and dbVar databases, the frequencies of de novo deletion in VANGL1 (1.14%) and de novo duplication in DVL2 (0.57%) were significantly higher in our NTD subjects (p < 0.05). This study demonstrates that de novo CNVs in PCP genes, notably deletions in VANGL1 and gains in DVL2, could contribute to the risk of NTDs.

Somatic mutations in planar cell polarity genes in neural tissue from human fetuses with neural tube defects.

Extensive studies that have sought causative mutation(s) for neural tube defects (NTDs) have yielded limited positive findings to date. One possible reason for this is that many studies have been confined to analyses of germline mutations and so may have missed other, non-germline mutations in NTD cases. We hypothesize that somatic mutations of planar polarity pathway (PCP) genes may play a role in the development of NTDs. Torrent™ Personal Genome Machine™ (PGM) sequencing was designed for selected PCP genes in paired DNA samples extracted from the tissues of lesion sites and umbilical cord from 48 cases. Sanger sequencing was used to validate the detected mutations. The source and distribution of the validated mutations in tissues from different germ layers were investigated. Subcellular location, western blotting, and luciferase assays were performed to better understand the effects of the mutations on protein localization, protein level, and pathway signaling. ix somatic mutations were identified and validated, which showed diverse distributions in different tissues. Three somatic mutations were novel/rare: CELSR1 p.Gln2125His, FZD6 p.Gln88Glu, and VANGL1 p.Arg374His. FZD6 p.Gln88Glu caused mislocalization of its protein from the cytoplasm to the nucleus, and disrupted the colocalization of CELSR1 and FZD6. This mutation affected non-canonical WNT signaling in luciferase assays. VANGL1 p.Arg374His impaired the co-localization of CELSR1 and VANGL1, increased the protein levels of VANGL1, and influenced cell migration. In all, 7/48 (14.5%) of the studied NTD cases contained somatic PCP mutations. Somatic mutations in PCP genes (e.g., FZD6 and VANGL1) are associated with human NTDs, and they may occur in different stages and regions during embryonic development, resulting in a varied distribution in fetal tissues/organs.

Natural Killer Cell-Derived Interferon-Gamma Promotes Hepatocellular Carcinoma Through the Epithelial Cell Adhesion Molecule-Epithelial-to-Mesenchymal Transition Axis in Hepatitis B Virus Transgenic Mice.

Hepatitis B virus (HBV) is a major risk factor for development of hepatocellular carcinoma (HCC), at least partially due to dysfunctional anti-HBV adaptive immunity; however, the role of innate immune response to HBV in this process is not well understood. In this study, low-dose polyinosinic:polycytidylic acid (poly [I:C]), a natural killer (NK) cell activator (3 µg/g body weight, twice/week for 8 weeks), induced HCC in HBV transgenic (HBs-Tg) mice, with an incidence of 100% after 6 months, while HBs-Tg mice without treatment only had HCC with an incidence of 16.7%. In HBs-Tg mice, poly (I:C) induced liver inflammation with markedly increased infiltrating lymphocytes, along with the concurrently increased apoptosis and proliferation of hepatocytes, leading to the accelerated epithelial-to-mesenchymal transition (EMT) of hepatocytes shown by increased expression of the typical transcriptional factors (Slug, Twist, and mothers against decapentaplegic-interacting protein 1) and phenotypic proteins (vimentin and chemokine [C-X-C motif] receptor 4). The EMT and tumorigenesis in this model depended on the presence of NK cells because depletion of these cells significantly reduced the HCC rate to 28.6%. Further, intrahepatic NK cells highly expressed interferon-gamma (IFN-γ), anti-IFN-γ neutralizing monoclonal antibody might obviously alleviate the hepatitis, and hepatocyte-specific IFN-γ overexpression promoted HCC. Moreover, IFN-γ deficiency in HBs-Tg mice prevented HCC occurring, though hepatic NK cells existed and could be activated, suggesting the critical role of IFN-γ in NK cell-mediated tumorigenesis. In an in vitro experiment, IFN-γ up-regulated epithelial cell adhesion molecule (EpCAM) expression through phosphorylated signal transducer and activator of transcription (p-STAT1) pathway, which was followed by EMT, and p-STAT1 inhibitor might absolutely abolish the expression of EpCAM and EMT in HBV surface antigen-positive hepatocytes. Conclusion: This work demonstrates that NK cell-derived IFN-γ promotes HCC through the EpCAM-EMT axis in HBs-Tg mice, revealing the importance of innate immunity in pathogenesis of HBV-associated HCC.

Hepatic NK cells attenuate fibrosis progression of non-alcoholic steatohepatitis in dependent of CXCL10-mediated recruitment.

BACKGROUND & AIMS: Non-alcoholic steatohepatitis (NASH) is a major cause of chronic liver disease. The precise role of NK cells in the progression of NASH has yet to be elucidated. METHODS: Using methionine- and choline-deficient diets (MCD)-induced NASH model, the role of NK cells was identified in WT mice compared with conventional NK cell-deficient Nfil3-/- mice. RESULTS: After 8 weeks of MCD treatment, NASH was induced as shown by the significant macrovesicular steatosis, necro-inflammation and fibrosis in the liver of WT B6 mice. In MCD-treated WT B6 mice, the number of NK cells was markedly increased in the liver, but decreased in the spleen. Intrahepatic NK cells exhibited high levels of activation, as evidenced by the expression of CD107a and cytokine production of IFN-γ, TGF-ß and IL-10. Lower expression levels of Ki67 indicated a reduction in the proliferation of intrahepatic NK cells after MCD treatment. Increased expression of CXCL10 in the liver early after MCD treatment led to the increased recruitment of CXCR3+ NK cells into the liver. The MCD-treated Nfil3-/- mice showed similar levels of TG and macrovesicular steatosis, thus more inflammatory infiltration and increased collagen deposition in the liver. Furthermore, the depletion of NK cells during MCD-induced NASH caused a significant increase in the infiltration of monocyte-derived macrophages (MoMFs) particularly Ly6Clo subsets towards M2. CONCLUSIONS: Intrahepatic NK cells, recruited through CXCL10-CXCR3 interaction, play a protective role against the fibrosis progression in NASH, which provide us with a better understanding of the immunopathogenesis of NASH.

Effects of an exopolysaccharide from Lactococcus lactis Z-2 on innate immune response, antioxidant activity, and disease resistance against Aeromonas hydrophila in Cyprinus carpio L.

Microbial exopolysaccharides (EPS) from Lactococcus have been found to have an important role in the probiotic activity of this bacterium; however, the immunomodulatory and antioxidant activities have not been fully explored in aquaculture. In the present study, we investigated EPS-2 from Lactococcus lactis Z-2, isolated from healthy common carp, for its immunomodulatory and antioxidant effects and disease resistance against Aeromonas hydrophila in Cyprinus carpio L. We found that the molecular weight of EPS-2 was 18.65 KDa. The monosaccharide composition of this polymer was rhamnose, xylose, mannose, glucose, and galactose at a molar percentage of 13.3%, 14.1%, 18.5%, 27.4%, and 26.7%, respectively. EPS-2 treatment could modulate the immune responses in vitro and in vivo. In vitro tests showed that EPS-2 could significantly enhance the proliferation and phagocytosis activities (P < 0.05) as well as induce the production of nitic oxide (NO), pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), and anti-inflammatory cytokines (IL-10, TGF-ß) (P < 0.05) in head kidney cells. When the fish were gavaged with three different concentrations of EPS-2 (250, 500, 1000 µg/mL) for 7 days and infected with A. hydrophila, different expression patterns of the NO, cytokines, lysozyme (LZM), and alkaline phosphatase (AKP) in the serum and of antioxidants (T-AOC, SOD, CAT, GSH, GSH-Px and MDA) in hepatopancreas were observed. Before infection with A. hydrophila, EPS-2 supplementation significantly up-regulated the NO production, protein levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6), LZM and AKP activities, and levels of antioxidant molecules compared to those in the negative (G1) group (P < 0.05), whereas levels of NO and pro-inflammatory cytokines and LZM and AKP activities were significantly lower than those in the positive (G2) group after infection (P < 0.05). However, whether infected or not, the expression levels of anti-inflammatory cytokines (IL-10, TGF-ß) were significantly increased in the EPS-2 treatment groups (P < 0.05). These results indicate that EPS-2 has immunomodulatory and antioxidant effects on common carp, both in vitro and/or in vivo, and can be applied as a common carp feed supplement to enhance fish immunity and disease resistance against A. hydrophila.