Multi-objective Bayesian optimization with enhanced features for adaptively improved glioblastoma partitioning and survival prediction.

Glioblastoma, an aggressive brain tumor prevalent in adults, exhibits heterogeneity in its microstructures and vascular patterns. The delineation of its subregions could facilitate the development of region-targeted therapies. However, current unsupervised learning techniques for this task face challenges in reliability due to fluctuations of clustering algorithms, particularly when processing data from diverse patient cohorts. Furthermore, stable clustering results do not guarantee clinical meaningfulness. To establish the clinical relevance of these subregions, we will perform survival predictions using radiomic features extracted from them. Following this, achieving a balance between outcome stability and clinical relevance presents a significant challenge, further exacerbated by the extensive time required for hyper-parameter tuning. In this study, we introduce a multi-objective Bayesian optimization (MOBO) framework, which leverages a Feature-enhanced Auto-Encoder (FAE) and customized losses to assess both the reproducibility of clustering algorithms and the clinical relevance of their outcomes. Specifically, we embed the entirety of these processes within the MOBO framework, modeling both using distinct Gaussian Processes (GPs). The proposed MOBO framework can automatically balance the trade-off between the two criteria by employing bespoke stability and clinical significance losses. Our approach efficiently optimizes all hyper-parameters, including the FAE architecture and clustering parameters, within a few steps. This not only accelerates the process but also consistently yields robust MRI subregion delineations and provides survival predictions with strong statistical validation.

EEF1B2 regulates bone marrow-derived mesenchymal stem cells bone-fat balance via Wnt/ß-catenin signaling.

The pathological advancement of osteoporosis is caused by the uneven development of bone marrow-derived mesenchymal stem cells (BMSCs) in terms of osteogenesis and adipogenesis. While the role of EEF1B2 in intellectual disability and tumorigenesis is well established, its function in the bone-fat switch of BMSCs is still largely unexplored. During the process of osteogenic differentiation, we observed an increase in the expression of EEF1B2, while a decrease in its expression was noted during adipogenesis. Suppression of EEF1B2 hindered the process of osteogenic differentiation and mineralization while promoting adipogenic differentiation. On the contrary, overexpression of EEF1B2 enhanced osteogenesis and strongly inhibited adipogenesis. Furthermore, the excessive expression of EEF1B2 in the tibias has the potential to mitigate bone loss and decrease marrow adiposity in mice with osteoporosis. In terms of mechanism, the suppression of ß-catenin activity occurred when EEF1B2 function was suppressed during osteogenesis. Our collective findings indicate that EEF1B2 functions as a regulator, influencing the differentiation of BMSCs and maintaining a balance between bone and fat. Our finding highlights its potential as a therapeutic target for diseases related to bone metabolism.

CRIP1 regulates osteogenic differentiation of bone marrow stromal cells and pre-osteoblasts via the Wnt signaling pathway.

With the aging of the global demographic, the prevention and treatment of osteoporosis are becoming crucial issues. The gradual loss of self-renewal and osteogenic differentiation capabilities in bone marrow stromal cells (BMSCs) is one of the key factors contributing to osteoporosis. To explore the regulatory mechanisms of BMSCs differentiation, we collected bone marrow cells of femoral heads from patients undergoing total hip arthroplasty for single-cell RNA sequencing analysis. Single-cell RNA sequencing revealed significantly reduced CRIP1 (Cysteine-Rich Intestinal Protein 1) expression and osteogenic capacity in the BMSCs of osteoporosis patients compared to non-osteoporosis group. CRIP1 is a gene that encodes a member of the LIM/double zinc finger protein family, which is involved in the regulation of various cellular processes including cell growth, development, and differentiation. CRIP1 knockdown resulted in decreased alkaline phosphatase activity, mineralization and expression of osteogenic markers, indicating impaired osteogenic differentiation. Conversely, CRIP1 overexpression, both in vitro and in vivo, enhanced osteogenic differentiation and rescued bone mass reduction in ovariectomy-induced osteoporosis mice model. The study further established CRIP1's modulation of osteogenesis through the Wnt signaling pathway, suggesting that targeting CRIP1 could offer a novel approach for osteoporosis treatment by promoting bone formation and preventing bone loss.

Decreased expression of GEM in osteoarthritis cartilage regulates chondrogenic differentiation via Wnt/ß-catenin signaling.

BACKGROUND: GEM (GTP-binding protein overexpressed in skeletal muscle) is one of the atypical small GTPase subfamily members recently identified as a regulator of cell differentiation. Abnormal chondrogenesis coupled with an imbalance in the turnover of cartilaginous matrix formation is highly relevant to the onset and progression of osteoarthritis (OA). However, how GEM regulates chondrogenic differentiation remains unexplored. METHODS: Cartilage tissues were obtained from OA patients and graded according to the ORASI and ICRS grading systems. The expression alteration of GEM was detected in the Grade 4 cartilage compared to Grade 0 and verified in OA mimic culture systems. Next, to investigate the specific function of GEM during these processes, we generated a Gem knockdown (Gem-Kd) system by transfecting siRNA targeting Gem into ATDC5 cells. Acan, Col2a1, Sox9, and Wnt target genes of Gem-Kd ATDC5 cells were detected during induction. The transcriptomic sequencing analysis was performed to investigate the mechanism of GEM regulation. Wnt signaling pathways were verified by real-time PCR and immunoblot analysis. Finally, a rescue model generated by treating Gem-KD ATDC5 cells with a Wnt signaling agonist was established to validate the mechanism identified by RNA sequencing analysis. RESULTS: A decreased expression of GEM in OA patients' cartilage tissues and OA mimic chondrocytes was observed. While during chondrogenesis differentiation and cartilage matrix formation, the expression of GEM was increased. Gem silencing suppressed chondrogenic differentiation and the expressions of Acan, Col2a1, and Sox9. RNA sequencing analysis revealed that Wnt signaling was downregulated in Gem-Kd cells. Decreased expression of Wnt signaling associated genes and the total ß-CATENIN in the nucleus and cytoplasm were observed. The exogenous Wnt activation exhibited reversed effect on Gem loss-of-function cells. CONCLUSION: These findings collectively validated that GEM functions as a novel regulator mediating chondrogenic differentiation and cartilage matrix formation through Wnt/ß-catenin signaling.

A+T rich interaction domain protein 3a (Arid3a) impairs Mertk-mediated efferocytosis in cholestasis.

BACKGROUND & AIMS: Macrophages are key elements in the pathogenesis of cholestatic liver diseases. Arid3a plays a prominent role in the biologic properties of hematopoietic stem cells, B lymphocytes and tumor cells, but its ability to modulate macrophage function during cholestasis remains unknown. METHODS: Gene and protein expression and cellular localization were assessed by q-PCR, immunohistochemistry, immunofluorescence staining and flow cytometry. We generated myeloid-specific Arid3a knockout mice and established three cholestatic murine models. The transcriptome was analyzed by RNA-seq. A specific inhibitor of the Mertk receptor was used in vitro and in vivo. Promoter activity was determined by chromatin immunoprecipitation-seq against Arid3a and a luciferase reporter assay. RESULTS: In cholestatic murine models, myeloid-specific deletion of Arid3a alleviated cholestatic liver injury (accompanied by decreased accumulation of macrophages). Arid3a-deficient macrophages manifested a more reparative phenotype, which was eliminated by in vitro treatment with UNC2025, a specific inhibitor of the efferocytosis receptor Mertk. Efferocytosis of apoptotic cholangiocytes was enhanced in Arid3a-deficient macrophages via upregulation of Mertk. Arid3a negatively regulated Mertk transcription by directly binding to its promoter. Targeting Mertk in vivo effectively reversed the protective phenotype of Arid3a deficiency in macrophages. Arid3a was upregulated in hepatic macrophages and circulating monocytes in primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC). Mertk was correspondingly upregulated and negatively correlated with Arid3a expression in PBC and PSC. Mertk+ cells were located in close proximity to cholangiocytes, while Arid3a+ cells were scattered among immune cells with greater spatial distances to hyperplastic cholangiocytes in PBC and PSC. CONCLUSIONS: Arid3a promotes cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes by macrophages during cholestasis. The Arid3a-Mertk axis is a promising novel therapeutic target for cholestatic liver diseases. IMPACT AND IMPLICATIONS: Macrophages play an important role in the pathogenesis of cholestatic liver diseases. This study reveals that macrophages with Arid3a upregulation manifest a pro-inflammatory phenotype and promote cholestatic liver injury by impairing Mertk-mediated efferocytosis of apoptotic cholangiocytes during cholestasis. Although we now offer a new paradigm to explain how efferocytosis is regulated in a myeloid cell autonomous manner, the regulatory effects of Arid3a on chronic liver diseases remain to be further elucidated.

Multi-Modal Learning for Predicting the Genotype of Glioma.

The isocitrate dehydrogenase (IDH) gene mutation is an essential biomarker for the diagnosis and prognosis of glioma. It is promising to better predict glioma genotype by integrating focal tumor image and geometric features with brain network features derived from MRI. Convolutional neural networks show reasonable performance in predicting IDH mutation, which, however, cannot learn from non-Euclidean data, e.g., geometric and network data. In this study, we propose a multi-modal learning framework using three separate encoders to extract features of focal tumor image, tumor geometrics and global brain networks. To mitigate the limited availability of diffusion MRI, we develop a self-supervised approach to generate brain networks from anatomical multi-sequence MRI. Moreover, to extract tumor-related features from the brain network, we design a hierarchical attention module for the brain network encoder. Further, we design a bi-level multi-modal contrastive loss to align the multi-modal features and tackle the domain gap at the focal tumor and global brain. Finally, we propose a weighted population graph to integrate the multi-modal features for genotype prediction. Experimental results on the testing set show that the proposed model outperforms the baseline deep learning models. The ablation experiments validate the performance of different components of the framework. The visualized interpretation corresponds to clinical knowledge with further validation. In conclusion, the proposed learning framework provides a novel approach for predicting the genotype of glioma.

Nonalcoholic steatohepatitis critically rewires the ischemia/reperfusion-induced dysregulation of cardiolipins and sphingolipids in mice.

Background: Lipid dysregulation plays a fundamental role in nonalcoholic steatohepatitis (NASH), which is an emerging critical risk factor that aggravates hepatic ischemia/reperfusion (I/R) injury. However, the specific lipids that mediate the aggressive I/R injury in NASH livers have not yet been identified. Methods: The mouse model of hepatic I/R injury on NASH was established on C56B/6J mice by first feeding the mice with a Western-style diet to induce NASH, then the NASH mice were subjected to surgical procedures to induce hepatic I/R injury. Untargeted lipidomics were performed to determine hepatic lipids in NASH livers with I/R injury through ultra-high performance liquid chromatography coupled with mass spectrometry. The pathology associated with the dysregulated lipids was examined. Results: Lipidomics analyses identified cardiolipins (CL) and sphingolipids (SL), including ceramides (CER), glycosphingolipids, sphingosines, and sphingomyelins, as the most relevant lipid classes that characterized the lipid dysregulation in NASH livers with I/R injury. CER were increased in normal livers with I/R injury, and the I/R-induced increase of CER was further augmented in NASH livers. Metabolic pathway analysis revealed that the enzymes involved in the synthesis and degradation of CER were highly upregulated in NASH livers with I/R injury, including serine palmitoyltransferase 3 (Sptlc3), ceramide synthase 2 (Cers2), neutral sphingomyelinase 2 (Smpd3), and glucosylceramidase beta 2 (Gba2) that produced CER, and alkaline ceramidase 2 (Acer2), alkaline ceramidase 3 (Acer3), sphingosine kinase 1 (Sphk1), sphingosine-1-phosphate lyase (Sgpl1), and sphingosine-1-phosphate phosphatase 1 (Sgpp1) that catalyzed the degradation of CER. CL were not affected by I/R challenge in normal livers, but CL was dramatically reduced in NASH livers with I/R injury. Consistently, metabolic pathway analyses revealed that the enzymes catalyzing the generation of CL were downregulated in NASH-I/R injury, including cardiolipin synthase (Crls1) and tafazzin (Taz). Notably, the I/R-induced oxidative stress and cell death were found to be aggravated in NASH livers, which were possibly mediated by the reduction of CL and accumulation of CER. Conclusions: The I/R-induced dysregulation of CL and SL were critically rewired by NASH, which might potentially mediate the aggressive I/R injury in NASH livers.

Structural connectome quantifies tumour invasion and predicts survival in glioblastoma patients.

Glioblastoma is characterized by diffuse infiltration into the surrounding tissue along white matter tracts. Identifying the invisible tumour invasion beyond focal lesion promises more effective treatment, which remains a significant challenge. It is increasingly accepted that glioblastoma could widely affect brain structure and function, and further lead to reorganization of neural connectivity. Quantifying neural connectivity in glioblastoma may provide a valuable tool for identifying tumour invasion. Here we propose an approach to systematically identify tumour invasion by quantifying the structural connectome in glioblastoma patients. We first recruit two independent prospective glioblastoma cohorts: the discovery cohort with 117 patients and validation cohort with 42 patients. Next, we use diffusion MRI of healthy subjects to construct tractography templates indicating white matter connection pathways between brain regions. Next, we construct fractional anisotropy skeletons from diffusion MRI using an improved voxel projection approach based on the tract-based spatial statistics, where the strengths of white matter connection and brain regions are estimated. To quantify the disrupted connectome, we calculate the deviation of the connectome strengths of patients from that of the age-matched healthy controls. We then categorize the disruption into regional disruptions on the basis of the relative location of connectome to focal lesions. We also characterize the topological properties of the patient connectome based on the graph theory. Finally, we investigate the clinical, cognitive and prognostic significance of connectome metrics using Pearson correlation test, mediation test and survival models. Our results show that the connectome disruptions in glioblastoma patients are widespread in the normal-appearing brain beyond focal lesions, associated with lower preoperative performance (P < 0.001), impaired cognitive function (P < 0.001) and worse survival (overall survival: hazard ratio = 1.46, P = 0.049; progression-free survival: hazard ratio = 1.49, P = 0.019). Additionally, these distant disruptions mediate the effect on topological alterations of the connectome (mediation effect: clustering coefficient -0.017, P < 0.001, characteristic path length 0.17, P = 0.008). Further, the preserved connectome in the normal-appearing brain demonstrates evidence of connectivity reorganization, where the increased neural connectivity is associated with better overall survival (log-rank P = 0.005). In conclusion, our connectome approach could reveal and quantify the glioblastoma invasion distant from the focal lesion and invisible on the conventional MRI. The structural disruptions in the normal-appearing brain were associated with the topological alteration of the brain and could indicate treatment target. Our approach promises to aid more accurate patient stratification and more precise treatment planning.

Serum Biomarkers for Autoimmune Hepatitis Type 1: the Case for CD48 and a Review of the Literature.

In autoimmune hepatitis (AIH), the persisting inflammation contributes to fibrosis progression, for which conventional biochemical markers manifest relatively unsatisfactory prediction. Herein, we assessed the value of serum CD48 (sCD48) as an indicator for inflammation and fibrosis in AIH type 1. The levels of sCD48 were detected first in an exploratory cohort using ELISA. In this cohort, compared with healthy controls (4.90 ng/mL, P < 0.0001), primary biliary cholangitis (7.32 ng/mL, P < 0.0001), and non-alcoholic fatty liver disease (7.76 ng/mL, P < 0.0001), sCD48 levels were elevated in AIH (12.81 ng/mL) and correlated with histological inflammation and fibrosis. Further using multivariate logistic regression analysis, sCD48 was identified as an independent predictor for both significant inflammation (G3-4) and advanced fibrosis (S3-4). Two predictive scores, based on sCD48, were constructed for diagnosing significant inflammation and advanced fibrosis (sCD48-AIH-SI and sCD48-AIH-AF, respectively). Using these data as a premise, predictive abilities were subsequently evaluated and verified in a validation cohort. In the exploratory cohort, the area under the receiver operating characteristic curve of sCD48 and sCD48-AIH-SI, for significant inflammation, were 0.748 and 0.813, respectively. Besides, during treatment follow-up, sCD48 levels gradually decreased from immunosuppression initiation to re-evaluation biopsy, in parallel with aspartate transaminase, total sera IgG, and fibrosis-4 score. For AIH patients in a re-evaluation biopsy cohort, sCD48 could predict significant fibrosis (S2-4). Further using immunohistochemistry, hepatic CD48 expression was elevated in AIH patients and decreased after treatment. In conclusion, sCD48 and sCD48-based predictive scores predict histological inflammation and fibrosis in AIH-1. Detecting sCD48 might help in the clinical management of AIH.

Prognostic significance of MRI-based late-course tumor volume in locoregionally advanced nasopharyngeal carcinoma.

BACKGROUND: To validate tumor volume-based imaging markers for predicting local recurrence-free survival (LRFS) in locoregionally advanced nasopharyngeal carcinoma patients, who underwent induction chemotherapy followed by definitive intensity-modulated radiotherapy. METHODS: We enrolled 145 patients with stage III-IVA nasopharyngeal carcinoma in this retrospective study. Pre-treatment tumor volume (Vpre) and late-course volume (LCV) were measured based on the MRIs scanned before treatment and during the first 3 days in the sixth week of radiotherapy, respectively. The volume regression rate (VRR) was calculated according to Vpre and LCV. Receiver operating characteristic (ROC) curves were used to identify the cut-off best separating patient subgroups in assessing the prognostic value of Vpre, LCV and VRR. The Kaplan-Meier method was used for survival analysis. Prognostic analyses were performed using univariate and multivariate COX proportional hazard models. RESULTS: The LCV was 5.3 ± 0.5 (range 0-42.1) cm3; The VRR was 60.4 ± 2.2% (range 2.9-100.0). The median follow-up period was 36 months (range 6-98 months). The cut-off value of LCV determined by the ROC was 6.8 cm3 for LRFS prediction (sensitivity 68.8%; specificity 79.8%). The combination of LCV and VRR for LRFS prediction (AUC = 0.79, P < 0.001, 95% CI 0.67-0.90), LCV (AUC = 0.74, P = 0.002, 95% CI 0.60-0.88) and Vpre (AUC = 0.71, P = 0.007, 95% CI 0.56-0.85) are better than T category (AUC = 0.64, P = 0.062, 95% CI 0.50-0.79) alone. Patients with LCV ≤ 6.8 cm3 had significantly longer LRFS (P < 0.001), disease-free survival (DFS, P < 0.001) and overall survival (OS, P = 0.005) than those with LCV > 6.8 cm3. Multivariate Cox regression showed LCV was the only independent prognostic factor for local control (HR = 7.80, 95% CI 2.69-22.6, P < 0.001). CONCLUSIONS: LCV is a promising prognostic factor for local control and chemoradiosensitivity in patients with locoregionally advanced NPC. The LCV, and the combination of LCV with VRR are more robust predictors for patient survival than T category.

Manifold Roles of Ceramide Metabolism in Non-Alcoholic Fatty Liver Disease and Liver Cancer.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic disorder manifested in hepatic fat accumulation (hepatic steatosis) in the absence of heavy alcohol use. NAFLD consists of four major stages ranging from simple steatosis or non-alcoholic fatty liver (NAFL) to more advanced stages, non-alcoholic steatohepatitis (NASH), fibrosis, and cirrhosis. NFLAD may further advance to hepatocellular carcinoma (HCC). Primary causes of NAFLD are obesity and obesity-associated insulin resistance (IR). As a result of the obesity pandemic, NAFLD has become one of the most common liver disorders worldwide and both the incidence and mortality rate of HCC that develops from NAFLD are increasing steadily. As treatment options are not available for advanced NAFLD, a better understanding of the molecular mechanisms for NAFLD development and progression is urgently needed. Emerging evidence suggests that dysregulation of the metabolism of sphingolipids contributes to development and progression of NAFLD and NAFLD-associated HCC. The present chapter summarizes roles of bioactive sphingolipids, ceramides, sphingosine, and sphingosine-1-phosphate (S1P) and their metabolizing enzymes in NAFLD and HCC.

Clinical, Dermoscopic, Ultrasonographic, and Histopathologic Correlations in Kaposi's Sarcoma Lesions and Their Differential Diagnoses: A Single-Center Prospective Study.

(1) Background: Kaposi's sarcoma (KS) is an angioproliferative neoplasm typically appearing as angiomatous patches, plaques, and/or nodules on the skin. Dermoscopy and ultrasonography have been suggested as an aid in the diagnosis of KS, but there is little evidence in the literature, especially regarding its possible differential diagnoses. Our aim is to describe and compare the clinical, dermoscopic, and ultrasonographic features of KS and KS-like lesions. (2) Methods: we conducted a prospective study on 25 consecutive patients who were first referred to our tertiary care center from January to May 2021 for a possible KS. (3) Results: 41 cutaneous lesions were examined by means of dermoscopy, Doppler ultrasonography, and pathology, 32 of which were KS-related, while the remaining 9 were lesions with clinical resemblance to KS. On dermoscopy, a purplish-red pigmentation, scaly surface, and the collarette sign were the most common features among KS lesions (81.3%, 46.9%, and 28.1%, respectively). On US, all 9 KS plaques and 21 KS nodules presented a hypoechoic image. Dermoscopic and Doppler ultrasonographic findings of KS-like lesions, such as cherry angioma, venous lake, glomus tumor, pyogenic granuloma, and angiosarcoma were also analyzed. (4) Conclusions: dermoscopy and Doppler ultrasonography can be useful to better assess the features of KS lesions and in diagnosing equivocal KS-like lesions.

Radiological tumor classification across imaging modality and histology.

Radiomics refers to the high-throughput extraction of quantitative features from radiological scans and is widely used to search for imaging biomarkers for prediction of clinical outcomes. Current radiomic signatures suffer from limited reproducibility and generalizability, because most features are dependent on imaging modality and tumor histology, making them sensitive to variations in scan protocol. Here, we propose novel radiological features that are specially designed to ensure compatibility across diverse tissues and imaging contrast. These features provide systematic characterization of tumor morphology and spatial heterogeneity. In an international multi-institution study of 1,682 patients, we discover and validate four unifying imaging subtypes across three malignancies and two major imaging modalities. These tumor subtypes demonstrate distinct molecular characteristics and prognoses after conventional therapies. In advanced lung cancer treated with immunotherapy, one subtype is associated with improved survival and increased tumor-infiltrating lymphocytes compared with the others. Deep learning enables automatic tumor segmentation and reproducible subtype identification, which can facilitate practical implementation. The unifying radiological tumor classification may inform prognosis and treatment response for precision medicine.

Bovine Oviduct Epithelial Cell-Derived Culture Media and Exosomes Improve Mitochondrial Health by Restoring Metabolic Flux during Pre-Implantation Development.

Oviduct flushing is enriched by a wide variety of nutrients that guide the 3-4 days journey of pre-implantation embryo through the oviduct as it develops into a competent blastocyst (BL). However, little is known about the specific requirement and role of these nutrients that orchestrate the early stages of embryonic development. In this study, we aimed to characterize the effect of in vitro-derived bovine oviduct epithelial cell (BOECs) secretion that mimics the in vivo oviduct micro-fluid like environment, which allows successful embryonic development. In this study, the addition of an in vitro derived BOECs-condition media (CM) and its isolated exosomes (Exo) significantly enhances the quality and development of BL, while the hatching ability of BLs was found to be high (48.8%) in the BOECs-Exo supplemented group. Surprisingly, BOECs-Exo have a dynamic effect on modulating the embryonic metabolism by restoring the pyruvate flux into TCA-cycle. Our analysis reveals that Exo treatment significantly upregulates the pyruvate dehydrogenase (PDH) and glutamate dehydrogenase (GLUD1) expression, required for metabolic fine-tuning of the TCA-cycle in the developing embryos. Exo treatment increases the influx into TCA-cycle by strongly suppressing the PDH and GLUD1 upstream inhibitors, i.e., PDK4 and SIRT4. Improvement of TCA-cycle function was further accompanied by higher metabolic activity of mitochondria in BOECs-CM and Exo in vitro embryos. Our study uncovered, for the first time, the possible mechanism of BOECs-derived secretion in re-establishing the TCA-cycle flux by the utilization of available nutrients and highlighted the importance of pyruvate in supporting bovine in vitro embryonic development.

Cytotoxic KLRG1 expressing lymphocytes invade portal tracts in primary biliary cholangitis.

Primary biliary cholangitis (PBC) is a chronic autoimmune liver disease with an immunopathogenesis that includes highly differentiated cytotoxic T cell infiltration in portal areas. We have taken advantage of a large and well-defined cohort of patients with PBC, AIH, chronic hepatitis virus, and healthy controls to study for the presence of highly differentiated T cells which express the killer cell lectin-like receptor G1 (KLRG1). Such studies were performed using both liver and peripheral blood mononuclear cells. In particular, gene expression data (GSE79850) from 16 PBC patients stratified according to future risk of liver transplantation were analyzed for markers of highly differentiated cytotoxic T cells. Liver biopsy samples from 44 PBC patients were studied by immunohistochemistry and a separate cohort of PBC blood samples were studied by flow cytometry. Gene expression data demonstrated correlation of increased KLRG1 and cytotoxic lymphocyte molecules, such as granzyme B (GZMB) and perforin (PRF1), to disease severity as measured by future risk of liver transplantation. Immunohistochemistry demonstrated abundant infiltration of KLRG1+ cells into liver portal areas (mean of 45% of infiltrating cells, range 25-75%) positively correlated with hepatic inflammatory (r = 0.47, p = 0.001) and hepatic fibrosis (r = 0.34, p = 0.021) scores. KLRG1+ lymphocyte liver portal area infiltration was positively correlated with serum alkaline phosphatase (r = 0.45, p = 0.005) and GGT (r = 0.40, p = 0.014), and AST (r = 0.35, p = 0.033) levels. Mononuclear blood flow cytometry studies showed KLRG1+ lymphocytes had greater levels of cytotoxic molecules (granzyme B and perforin), inflammatory cytokines (IFN-γ and TNF-α) and inflammatory chemokine receptors (CCR5 and CX3CR1) than KLRG1-counterparts. However, clearly the most significant data was that found in liver with the intense portal infiltrates that are unique to PBC. Conclusion: Highly cytotoxic KLRG1+ lymphocytes have invaded PBC liver portal areas. Liver KLRG1 gene expression and the abundance of KLRG1+ lymphocytes are positively correlated with disease biomarkers used as clinical trial outcome measures (liver transplantation and serum alkaline phosphatase), suggesting the targeting of KLRG1+ lymphocytes as a rational approach for PBC therapeutic drug development.

Histologically proven AMA positive primary biliary cholangitis but normal serum alkaline phosphatase: Is alkaline phosphatase truly a surrogate marker?

BACKGROUND AND AIMS: The most highly directed and specific autoantibody in human immunopathology is the serologic hallmark of primary biliary cholangitis (PBC), antimitochondrial antibodies (AMAs). However the clinical significance of finding a positive AMA, with normal alkaline phosphatase (ALP) remains enigmatic. METHODS: We took advantage of 169 consecutive outpatients who were identified as having a positive AMA, but normal ALP levels between January 2012 and January 2018. A liver biopsy was performed on 67/169 of these AMA positive normal ALP patients. RESULTS: In all 169 patients we reconfirmed the AMA and also performed anti-gp210 and anti-sp100, liver stiffness (LSM) assessed by vibration-controlled transient elastography (VCTE), an abdominal computed tomography (CT) scan, and either a magnetic resonance imaging (MRI) or ultrasound. The liver biopsies were reviewed by two unbiased observers. 87.6% of the 169 patients were females with a mean age of 46; the median AMA titer 1:320; an elevated serum IgM was found in 53.3%. Importantly, in patients with a liver biopsy, 55（82.1%）out of 67 had varying degrees of cholangitis activity, diagnostic of PBC. CONCLUSION: In patients who were AMA-positive but had normal ALP levels, more than 80% were associated with histological classic PBC. These data emphasize the importance of a positive AMA, even with a normal ALP and also question the role of ALP as a sole surrogate marker of cholangitis.

Mucosal-Associated Invariant T Cells Improve Nonalcoholic Fatty Liver Disease Through Regulating Macrophage Polarization.

Mucosal-associated invariant T (MAIT) cells, a novel population of innate-like lymphocytes, have been involved in various inflammatory and autoimmune diseases. However, their role in the development of nonalcoholic fatty liver disease (NAFLD) remains unclear. In this study, we investigated the alterations of phenotype and immunological function of MAIT cells in NAFLD. Analysis of PBMCs in 60 patients with NAFLD and 48 healthy controls (HC) revealed that circulating MAIT cell frequency decreased in NAFLD, especially in the patients with higher serum levels of γ-glutamyl transferase or total triglyceride. Functional alterations of circulating MAIT cells were also detected in NAFLD patients, such as the increased production of IL-4 whereas the decreased production of IFN-γ and TNF-α. Furthermore, elevated expression of CXCR6 was observed in circulating MAIT cells of patients. Meanwhile, we found an increased number of MAIT cells in the livers of NAFLD, and the number was even greater in patients with higher NAFLD activity score. Moreover, activated MAIT cells induced monocytes/macrophages differentiation into M2 phenotype in vitro. Additionally, MAIT cells were enriched and displayed Th2 type cytokines profile in livers of wild type mice fed with methionine and choline deficient diet (MCD). Notably, mice deficient of MAIT cells exhibited more severe hepatic steatosis and inflammation upon MCD, accompanied with more CD11c+ proinflammatory macrophages (M1) and less CD206+ anti-inflammatory macrophages (M2) in livers. Our results indicate that MAIT cells protect against inflammation in NAFLD through producing regulatory cytokines and inducing anti-inflammatory macrophage polarization, which may provide novel therapeutic strategies for NAFLD.

The Immunobiology of Receptor Activator for Nuclear Factor Kappa B Ligand and Myeloid-Derived Suppressor Cell Activation in Immunoglobulin G4-Related Sclerosing Cholangitis.

The primary function of myeloid-derived suppressor cells (MDSCs) is reflected in their immune modulatory role in several immune-mediated diseases. In immunoglobulin G4 (IgG4)-related disease (IgG4-RD), it has been hypothesized that there are selective regulatory defects that lead to a T helper 2 (Th2) bias immune response. Herein we have taken advantage of a large cohort of patients with IgG4-related sclerosing cholangitis (IgG4-SC), the most common extrapancreatic involvement of IgG4-RD, as well as controls consisting of primary sclerosing cholangitis, autoimmune hepatitis, and healthy volunteers, to study MDSCs. We report dramatically increased levels of receptor activator for nuclear factor kappa B ligand (RANKL) expression in serum and liver from patients with IgG4-SC compared to both liver-disease and healthy controls. Moreover, in IgG4-SC liver, RANKL-secreting cells specifically colocalized with cluster of differentiation 38-positive plasma cells and MDSCs, particularly monocytic MDSCs, and express the RANKL receptor in liver. Similarly, the frequency and number of peripheral blood MDSCs were significantly increased. Importantly, serum expression levels of RANKL were inversely correlated with the serum level of gamma-glutamyltransferase but significantly positively correlated with the frequency of MDSCs. Moreover, we confirmed that RANKL induced the expansion and activation of MDSCs through the RANKL/RANK/nuclear factor kappa B signal pathway. Of note, RANKL-treated MDSCs suppressed T-cell proliferation and induced Th2 differentiation. Conclusion: Our data suggest that plasma cell-derived RANKL induces the expansion and activation of MDSCs, which suppress T-cell proliferation and contribute to the Th2-type response characteristic of IgG4-SC.

A Common Variant in CLDN14 is Associated with Primary Biliary Cirrhosis and Bone Mineral Density.

Primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, has been associated with increased incidence of osteoporosis. Intriguingly, two PBC susceptibility loci identified through genome-wide association studies are also involved in bone mineral density (BMD). These observations led us to investigate the genetic variants shared between PBC and BMD. We evaluated 72 genome-wide significant BMD SNPs for association with PBC using two European GWAS data sets (n = 8392), with replication of significant findings in a Chinese cohort (685 cases, 1152 controls). Our analysis identified a novel variant in the intron of the CLDN14 gene (rs170183, Pfdr = 0.015) after multiple testing correction. The three associated variants were followed-up in the Chinese cohort; one SNP rs170183 demonstrated consistent evidence of association in diverse ethnic populations (Pcombined = 2.43 × 10(-5)). Notably, expression quantitative trait loci (eQTL) data revealed that rs170183 was correlated with a decline in CLDN14 expression in both lymphoblastoid cell lines and T cells (Padj = 0.003 and 0.016, respectively). In conclusion, our study identified a novel PBC susceptibility variant that has been shown to be strongly associated with BMD, highlighting the potential of pleiotropy to improve gene discovery.