AI-based digital pathology provides newer insights into lifestyle intervention-induced fibrosis regression in MASLD: An exploratory study.

BACKGROUND AND AIMS: Lifestyle intervention is the mainstay of therapy for metabolic dysfunction-associated steatohepatitis (MASH), and liver fibrosis is a key consequence of MASH that predicts adverse clinical outcomes. The placebo response plays a pivotal role in the outcome of MASH clinical trials. Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) microscopy with artificial intelligence analyses can provide an automated quantitative assessment of fibrosis features on a continuous scale called qFibrosis. In this exploratory study, we used this approach to gain insight into the effect of lifestyle intervention-induced fibrosis changes in MASH. METHODS: We examined unstained sections from paired liver biopsies (baseline and end-of-intervention) from MASH individuals who had received either routine lifestyle intervention (RLI) (n = 35) or strengthened lifestyle intervention (SLI) (n = 17). We quantified liver fibrosis with qFibrosis in the portal tract, periportal, transitional, pericentral, and central vein regions. RESULTS: About 20% (7/35) and 65% (11/17) of patients had fibrosis regression in the RLI and SLI groups, respectively. Liver fibrosis tended towards no change or regression after each lifestyle intervention, and this phenomenon was more prominent in the SLI group. SLI-induced liver fibrosis regression was concentrated in the periportal region. CONCLUSION: Using digital pathology, we could detect a more pronounced fibrosis regression with SLI, mainly in the periportal region. With changes in fibrosis area in the periportal region, we could differentiate RLI and SLI patients in the placebo group in the MASH clinical trial. Digital pathology provides new insight into lifestyle-induced fibrosis regression and placebo responses, which is not captured by conventional histological staging.

Serum PRO-C3 is useful for risk prediction and fibrosis assessment in MAFLD with chronic kidney disease in an Asian cohort.

BACKGROUND: Metabolic dysfunction-associated fatty liver disease (MAFLD) is an emerging risk factor for chronic kidney disease (CKD). N-terminal propeptide of collagen type 3 (PRO-C3) is a biomarker of advanced fibrosis in MAFLD and PRO-C3 may be involved in renal fibrosis. We aimed to use PRO-C3 measurements to generate a new algorithmic score to test the prediction of MAFLD with chronic kidney disease (MAFLD-CKD). METHODS: A derivation and independent validation cohort of 750 and 129 Asian patients with biopsy-confirmed MAFLD were included. Serum PRO-C3 concentration was measured and regression analyses were performed to examine associations with MAFLD-CKD. A derivative algorithm for MAFLD-CKD risk prediction was evaluated with receiver operator characteristic (ROC) curve analysis. RESULTS: The study included two Asian cohorts (n = 180 with MAFLD-CKD; mean-eGFR: 94.93 mL/min/1.73 m2; median-urinary albumin-to-creatinine ratio: 6.58 mg/mmol). PRO-C3 was associated with the severity of MAFLD-CKD and independently associated with MAFLD-CKD (adjusted odds ratio = 1.16, 95% confidence interval [CI]: 1.08-1.23, p < .001). A new non-invasive score (termed PERIOD) including PRO-C3 efficiently predicted MAFLD-CKD (AUROC = .842, 95% CI: .805-.875). Accuracy, specificity and negative predictive values were 80.2%, 85.1% and 88.4%, respectively. In the validation cohort, the PERIOD score had good diagnostic performance (AUROC = .807, 95% CI: .691-.893) with similar results in all patient subgroups. In the MAFLD-CKD subgroup, the accuracy for identifying advanced fibrosis was further improved by combining the PRO-C3-based ADAPT with the Agile 3+ scores (AUROC = .90, 95% CI: .836-.964). CONCLUSIONS: The PERIOD score is helpful for accurately predicting the risk of MAFLD-CKD. PRO-C3 can also be used to assess liver fibrosis in people with MAFLD-CKD.

Effect of chemotherapy alone or combined with immunotherapy for locally advanced or metastatic genitourinary small cell carcinoma: a real-world retrospective study.

BACKGROUND: Genitourinary small cell carcinoma is rare, and has a poor prognosis. However, effective treatment options for this disease are limited. We present a study to assess the efficacy of chemotherapy alone or combined with immunotherapy for locally advanced or metastatic genitourinary small cell carcinoma (GSCC). METHODS: We performed a retrospective analysis of patients with locally advanced or metastatic GSCC from Jan 2013 to September 2022 at Sun Yat-sen University Cancer Center. The survival and safety profiles were analyzed. RESULTS: Forty-two GSCC patients were enrolled, which included 20 with chemotherapy plus immunotherapy and 22 with chemotherapy alone. The median follow-up time was 15.13 months (95% CI, 8.84-21.42). The addition of immunotherapy to chemotherapy demonstrated no significant difference in median progression-free survival (p = 0.37). However, the median overall survival (OS) was 22.97 and 14.03 months with immunotherapy plus chemotherapy and chemotherapy alone, respectively (HR = 0.69, 95%CI 0.08-0.55, p = 0.017). Two patients with immunotherapy plus chemotherapy achieved clinical complete remission. The overall response rate for patients receiving chemotherapy combined with immunotherapy was 65%, which was higher in comparison to those treated with chemotherapy alone (50%). Univariate and multivariate analyses demonstrated that chemotherapy combined with immunotherapy independently achieved favorable OS. Four patients experienced immunotherapy-related adverse events, with one developing grade 3 hypothyroidism. CONCLUSIONS: Among patients with locally advanced or metastatic GSCC, immunotherapy combined with chemotherapy might be thought of as a potentially effective treatment option for patients with GSCC.

Hepatocytic ballooning in non-alcoholic steatohepatitis: Dilemmas and future directions.

Hepatocytic ballooning is a key histological feature in the diagnosis of non-alcoholic steatohepatitis (NASH) and is an essential component of the two most widely used histological scoring systems for diagnosing and staging non-alcoholic fatty liver disease (NAFLD) [namely, the NAFLD activity score (NAS), and the steatosis, activity and fibrosis (SAF) scoring system]. As a result of the increasing incidence of NASH globally, the diagnostic challenges of hepatocytic ballooning are unprecedented. Despite the clear pathological concept of hepatocytic ballooning, there are still challenges in assessing hepatocytic ballooning in 'real life' situations. Hepatocytic ballooning can be confused with cellular oedema and microvesicular steatosis. Significant inter-observer variability does exist in assessing the presence and severity of hepatocytic ballooning. In this review article, we describe the underlying mechanisms associated with hepatocytic ballooning. Specifically, we discuss the increased endoplasmic reticulum stress and the unfolded protein response, as well as the rearrangement of the intermediate filament cytoskeleton, the appearance of Mallory-Denk bodies and activation of the sonic Hedgehog pathway. We also discuss the use of artificial intelligence in the detection and interpretation of hepatocytic ballooning, which may provide new possibilities for future diagnosis and treatment.

Synthesis and biological activity assay of novel camptothecin-peptidic conjugates based on PEPT1.

Camptothecin (CPT) and its derivatives are potent candidates for cancer treatment. However, the clinical applications are largely restricted by non-selectivity and severe toxicities. The peptide transporter 1 (PEPT1), which is highly expressed in human intestines, has been found to be overexpressed in several cancer cells. This discovery suggests that PEPT1 has the potential to serve as a therapeutic target for both improving bioavailability and cancer-targeting treatment. Therefore, a prodrug approach for CPT targeting at PEPT1 highly expressed cancer cells was adopted in the present study. Eighteen CPT prodrugs, its peptidic conjugates, were synthesized and the structures were confirmed by NMR and HRMS. The protein expression profiles of PEPT1 in different cell lines were performed using immunofluorescence assay and western blotting analysis. The cytotoxicity of CPT prodrugs and their uptake via competition with Gly-Sar, a typical substrate of PEPT1, were evaluated in both PEPT1-overexpressed and under expressed cells. The results demonstrated that most CPT prodrugs significantly impaired Gly-Sar uptake, suggesting a higher affinity of CPT-peptidic conjugates for PEPT1 and PEPT1 overexpression cells. In addition, these prodrugs demonstrated a higher capability for inhibiting cell growth in PEPT1 highly-expressed cancer cells compared to PEPT1 under expressed cells. These results indicated that this peptidic prodrug strategy might offer great potential for improved tumor selectivity and chemotherapeutic efficacy of CPT.

Efficient production of a cyclic dipeptide (cyclo-TA) using heterologous expression system of filamentous fungus Aspergillus oryzae.

BACKGROUND: Cyclic dipeptides are an important class of natural products owing to their structural diversity and biological activities. In fungi, the cyclo-ring system is formed through the condensation of two α-amino acids via non-ribosomal peptide synthetase (NRPS). However, there are few investigations on the functional identification of this enzyme. Additionally, information on how to increase the production of cyclic dipeptide molecules is relatively scarce. RESULTS: We isolated the Eurotium cristatum NWAFU-1 fungus from Jing-Wei Fu brick tea, whose fermentation metabolites contain echinulin-related cyclic dipeptide molecules. We cloned the cirC gene, encoding an NRPS, from E. Cristatum NWAFU-1 and transferred it into the heterologous host Aspergillus oryzae. This transformant produced a novel metabolite possessing an L-tryptophan-L-alanine cyclic dipeptide backbone (Cyclo-TA). Based on the results of heterologous expression and microsomal catalysis, CriC is the first NRPS characterized in fungi that catalyzes the formation of a cyclic dipeptide from L-tryptophan and L-alanine. After substrate feeding, the final yield reached 34 mg/L. In this study, we have characterized a novel NRPS and developed a new method for cyclic dipeptide production. CONCLUSIONS: In this study we successfully expressed the E. Cristatum NWAFU-1 criC gene in A. oryzae to efficiently produce cyclic dipeptide compounds. Our findings indicate that the A. oryzae heterologous expression system constitutes an efficient method for the biosynthesis of fungal Cyclic dipeptides.

Bioinformatic Prediction of Depression-Related Signaling Pathways Regulated by miR-146a in Peripheral Blood of Patients with Post-Stroke Depression.

It was aimed to explore the differential expression of miR-146a-5p in peripheral blood of patients with post-stroke depression (PSD), and to analyze its mechanism using bioinformatics. Stroke patients were selected as the research objects, and were divided into PSD ones and non-post-stroke depression (N-PSD) ones with the National Institutes of Health stroke scale (NHISS) and Hamilton Depression Scale-17 terms (HAMD-17) scores. Peripheral blood of patients was collected for serum miR-146a-5p detection. Targetscan7.1, miRDB, DIANA TOOLS, and more databases were used to predict the target genes of miR-146a-5p. String11.0 was applied to construct a protein interaction network, and GO and KEGG pathway enrichment analysis of target genes was performed. Compared with that of N-PSD patients, serum miR-146a-5p levels in PSD patients were significantly increased (P<0.05). The receiver operator characteristic (ROC) curve suggested that the sensitivity and specificity of miR-146a-5p in predicting PSD were 0.703 and 0.811, respectively. The human miR-146a-5p sequence was highly conserved, with a total of 43 target genes. It involved analysis of activity, signaling pathways, and transcriptional regulation, as well as related signaling pathways such as Toll-like receptors (TLR), neurotrophic factors, and nuclear factor kappa-B (NF-κB). In conclusion, the expression level of miR-146a-5p was abnormally increased in PSD patients, and it could be taken as a candidate marker for the diagnosis of PSD. miR-146a-5p could affect PSD through signaling pathways of TLRs, neurotrophic factors, and NF-κB.

J-shaped relationship between serum zinc levels and the severity of hepatic necro-inflammation in patients with MAFLD.

BACKGROUND AND AIMS: Zinc is an essential trace element that plays an important role in maintaining health, and affecting gene expression, signal transduction and regulation of apoptosis. It is uncertain whether serum zinc levels are altered in patients with metabolic dysfunction-associated fatty liver disease (MAFLD). We aimed to investigate the association between serum zinc levels and the severity of hepatic necro-inflammation (HN) in patients with MAFLD. METHODS AND RESULTS: Liver disease severity was graded histologically using the NAFLD activity score. HN was defined as the sum of ballooning and lobular inflammation. We used a smooth function regression model to analyze the relationship between serum zinc levels and HN. A total of 561 (76.5% men) patients with biopsy-confirmed MAFLD were enrolled. They had a mean age of 41.3 years, and a mean serum zinc level of 17.0 ± 4.1 µmol/L. Compared to those with mild hepatic necro-inflammation (MHN, grades 0-2; n = 286), patients with severe hepatic necro-inflammation (SHN, grades 3-5; n = 275) had lower serum zinc concentrations (16.3 ± 4.2 vs. 17.6 ± 4.0 µmol/L; p < 0.001). However, a threshold saturation effect analysis showed that there was an inflection in serum zinc levels at 24 µmol/L. After adjustment for potential confounders, serum zinc levels <24 µmol/L were inversely associated with SHN (adjusted-odds ratio 0.88, 95%CI 0.83-0.93; p < 0.001), whereas serum zinc levels >24 µmol/L were positively associated with SHN (adjusted-odds ratio 1.42, 95%CI: 1.03-1.97; p = 0.035). CONCLUSIONS: There is a J-shaped relationship between serum zinc levels and the severity of hepatic necro-inflammation in patients with biopsy-proven MAFLD.

Lipidomic identification of urinary extracellular vesicles for non-alcoholic steatohepatitis diagnosis.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is a usual chronic liver disease and lacks non-invasive biomarkers for the clinical diagnosis and prognosis. Extracellular vesicles (EVs), a group of heterogeneous small membrane-bound vesicles, carry proteins and nucleic acids as promising biomarkers for clinical applications, but it has not been well explored on their lipid compositions related to NAFLD studies. Here, we investigate the lipid molecular function of urinary EVs and their potential as biomarkers for non-alcoholic steatohepatitis (NASH) detection. METHODS: This work includes 43 patients with non-alcoholic fatty liver (NAFL) and 40 patients with NASH. The EVs of urine were isolated and purified using the EXODUS method. The EV lipidomics was performed by LC-MS/MS. We then systematically compare the EV lipidomic profiles of NAFL and NASH patients and reveal the lipid signatures of NASH with the assistance of machine learning. RESULTS: By lipidomic profiling of urinary EVs, we identify 422 lipids mainly including sterol lipids, fatty acyl lipids, glycerides, glycerophospholipids, and sphingolipids. Via the machine learning and random forest modeling, we obtain a biomarker panel composed of 4 lipid molecules including FFA (18:0), LPC (22:6/0:0), FFA (18:1), and PI (16:0/18:1), that can distinguish NASH with an AUC of 92.3%. These lipid molecules are closely associated with the occurrence and development of NASH. CONCLUSION: The lack of non-invasive means for diagnosing NASH causes increasing morbidity. We investigate the NAFLD biomarkers from the insights of urinary EVs, and systematically compare the EV lipidomic profiles of NAFL and NASH, which holds the promise to expand the current knowledge of disease pathogenesis and evaluate their role as non-invasive biomarkers for NASH diagnosis and progression.

Bufalin inhibits human diffuse large B-cell lymphoma tumorigenesis by inducing cell death through the Ca2+/NFATC1/cMYC pathway.

The 5-year survival rate of diffuse large B-cell lymphoma (DLBCL) can reach 60%. However, nearly half of patients undergo relapse/refractory issues with a survival period of less than 2 years. New therapeutic approaches are therefore needed to improve chemotherapy efficacy and patient survival. Bufalin (BF), isolated from the traditional Chinese medicine Chansu, has been reported to play an anticancer role in multiple cancer cell types. However, there are few reports of the effects of BF on the growth of DLBCL. In the present study, we demonstrated that BF exerts antitumor activity in DLBCL cells, both in vitro and in vivo. Treatment of DLBCL cells with BF resulted in increased proliferation and apoptosis in a dose- and time-dependent manner. Daily intraperitoneal injection of 1.5 mg/kg BF significantly delayed DLBCL xenograft growth in NOD/SCID mice without affecting body weight. Bioinformatics analysis showed that BF may regulate NFATC1 protein and affect expression of its downstream gene, cMYC. Our results suggest that BF can attenuate NFATC1 translocation by reducing the intracellular calcium concentration; BF may also have a low synergistic effect with cyclosporin A. In conclusion, we demonstrated that BF exerts antitumor activity that is mediated at least in part by the Ca2+/NFATC1/cMYC pathway. Our findings suggest that BF can be effectively applied as a novel potential therapeutic agent for DLBCL.

TA allele of rs2070673 in the CYP2E1 gene is associated with lobular inflammation and nonalcoholic steatohepatitis in patients with biopsy-proven nonalcoholic fatty liver disease.

BACKGROUND AND AIM: Cytochrome P450 2E1 (CYP2E1) plays a role in lipid metabolism, and by increasing hepatic oxidative stress and inflammation, the upregulation of CYP2E1 is involved in development of nonalcoholic steatohepatitis (NASH). We aimed to explore the relationship between CYP2E1-333A>T (rs2070673) and the histological severity of nonalcoholic fatty liver disease (NAFLD). METHODS: We studied 438 patients with biopsy-proven NAFLD. NASH was defined as NAFLD Activity Score ≥ 5 with existence of steatosis, ballooning, and lobular inflammation. CYP2E1-333A>T (rs2070673) was genotyped by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Serum cytokines related to inflammation were measured by the Bio-plex 200 system to investigate possible mediating factors involved in the process. RESULTS: The TA genotype of rs2070673 had a higher prevalence of moderate/severe lobular inflammation (27.6% vs 20.3% vs 13.3%, P < 0.01) and NASH (55.7% vs 42.4% vs 40.5%, P < 0.01) compared with the AA and TT genotypes, respectively. In multivariable regression modeling, the heterozygote state TA was associated with moderate/severe lobular inflammation (adjusted odds ratio: 2.31, 95% confidence interval 1.41-3.78, P < 0.01) or NASH (adjusted odds ratio: 1.82, 95% confidence interval 1.22-2.69, P < 0.01), independently of age, sex, common metabolic risk factors, and presence of liver fibrosis. Compared with no-NASH, NASH patients had significantly higher levels of serum interleukin-1 receptor antagonist, interleukin-18, and interferon-inducible protein-10 (IP-10), whereas only IP-10 was increased with the rs2070673 TA variant (P = 0.01). Mediation analysis showed that IP-10 was responsible for ~60% of the association between the rs2070672 and NASH. CONCLUSIONS: The TA allele of rs2070673 is strongly associated with lobular inflammation and NASH, and this effect appears to be largely mediated by serum IP-10 levels.

[Protective effects of five different types of Dendrobium on CCl4-induced liver injury in mice].

This study aims to investigate the protective effect of Dendrobium huoshanense, D.officinale(Huoshan), D.officinale(Yunnan), D.moniliforme and D. henanense on CCl4-induced hepatic damage in mice. C57BL/6 mice were randomly divided into control group, model group, high-dose(7.5 g•kg⁻¹) and low-dose (1.25 g•kg⁻¹) groups of the five Dendrobium. Each group was intragastrically administered with drugs for 2 weeks. The control group was intraperitoneally injected with Olive oil solution, while the other groups were intraperitoneally given 0.5%CCl4combined with Olive oil solution 2 h later after the last administration. Subsequently, ALT and AST activities in serum, SOD activities and MDA contents in liver tissues were determined in all groups 16 h later after administration. The liver index was calculated, and hepatic histopathological examination was performed. The mRNA expressions of IL-1ß, IL-6 and TNF-α were analyzed by Real-time PCR. Compared with the CCl4 model group, the activities of ALT and AST in serum decreased significantly in the five different Dendrobium groups. Meanwhile, in liver tissues, the levels of MDA reduced obviously, while the SOD activities markedly increased. Furthermore, liver tissue damage induced by CCl4 was ameliorated according to the histopathological examination. IL-1ß, IL-6 and TNF-α mRNA expressions in D.huoshanense-treated liver tissues were significantly decreased. In conclusion, the five different Dendrobium groups showed hepatoprotective effects on CCl4-induced acute liver injury in mice. However, there were differences among Dendrobium of different types and origins. The protect effect of D.huoshanense is the most obvious, and the order of the protective effect of the other Dendrobium from high to low is D.officinale(Yunnan), D. officinale(Huoshan), D.henanense and D.moniliforme. The differences between the different types of Dendrobium might be related to their chemical components.

Manganese Dioxide Nanoplatform with a Hollow Rhombic Dodecahedron Morphology for Drug Delivery.

Manganese dioxide (MnO2) is considered as a promising drug carrier material suitable for the tumor microenvironment while lacking conducive structures for drug loading. Herein, we construct a MnO2 nanoplatform with a hollow rhombic dodecahedral morphology for drug delivery. In this work, we obtained zeolitic imidazolate framework nanoparticles (ZIF-90 NPs) via a coordination reaction. Furthermore, the drug-loading nanoparticles (ZIF-90/DOX NPs) were obtained by Schiff's base reaction and then selected as a sacrificial template to obtain the hollow nanoplatforms (ZIF-90@MnO2 NPs). Moreover, the photothermal effect and multiresponsive drug release behaviors were revealed by loading the photothermal agent IR-820 and the anticancer drug doxorubicin hydrochloride (DOX). Our study demonstrates that the ZIF-90@MnO2 NPs loaded with photosensitizers exhibited excellent photothermal conversion performance. Benefiting from the hollow structure and redox activity, remarkable drug loading and release performances of ZIF-90@MnO2 NPs were achieved. It is shown that ZIF-90@MnO2 NPs achieved a satisfactory drug-loading efficiency (up to ca. 69.7%) for DOX. More promisingly, the ZIF-90@MnO2 NPs exhibited significant glutathione (GSH)/pH-responsive drug release and degradation performances. Overall, this work highlights the potential of controlled drug release of nanocarriers and offers unique insights into the design of nanocarriers with hollow structures.

Imaging and Histopathological Features Of Primary Thymic Neuroendocrine Tumor.

OBJECTIVES: To investigate CT, MRI, and PET/CT features with histopathological findings of primary thymic neuroendocrine tumor. MATERIALS AND METHODS: All 9 cases with pathologically proven primary thymic neuroendocrine tumors were reviewed retrospectively. Among them, 7 underwent enhanced CT, 1 with MRI (enhanced) and another with PET/CT scan. Multiple characters were examined, including tumor location, contour, CT attenuation, enhancement pattern, involvement of surrounding structure and lymphadenopathy. RESULTS: Among 9 patients studied, 7 (77%) masses were located in the anterior superior mediastinum, 1 in the anterior superior-middle mediastinum, and 1 in the anterior and middle mediastinum. The maximum diameter (longitudinal) ranged from 4.2 to 23 cm (mean ± standard deviation, 9.5 cm ± 2.8). Four masses had irregular, 3 had lobulated, and 2 had smooth contours, while 8 masses had clear margins and 1 had an ill-defined margin. Six masses showed heterogeneous attenuation with necrotic/cystic component (n=5), calcification (n=2) and hemorrhage(n=1), and 3 showed homogeneous attenuation on the non-enhanced image. After contrast administration, 8 masses showed heterogeneous attenuation, and 1 showed homogeneous attenuation with tumor vessels visible in 4 masses. Among all, 8 masses showed strong enhancement, and 1 showed moderate enhancement in comparison to muscles in the anterior thoracic wall on enhanced images. Involvement of adjacent mediastinal structures was observed in 5 cases. Immunohistochemical analysis showed that the tumor cells were positive for CgA, Syn, CK, CD56 and EMA. CONCLUSION: Primary NETs are large masses located anterior superior mediastinum, irregular in contour, showing heterogeneous attenuation with necrotic/cystic component and strong heterogeneous enhancement with tumor vessels, compressing local mediastinal structures. In addition, immunohistochemical examination is required in such a diagnosis.

A Population-Based and Clinical Cohort Validation of the Novel Consensus Definition of Metabolic Hyperferritinemia.

CONTEXT: There is limited data on the clinical significance of metabolic hyperferritinemia (MHF) based on the most recent consensus. OBJECTIVE: We aimed to validate the clinical outcomes of MHF in the general population and patients with biopsy-proven metabolic dysfunction-associated fatty liver disease (MAFLD). METHODS: The NHANES database and PERSONS cohort were included. MHF was defined as elevated serum ferritin with metabolic dysfunction (MD) and stratified into different grades according to ferritin (grade 1: 200 [females]/300 [males]-550 ng/mL; grade 2: 550-1000 ng/mL; grade 3: >1000 ng/mL). The clinical outcomes, including all-cause death, comorbidities, and liver histology, were compared between non-MHF and MHF in adjusted models. RESULTS: In NHANES, compared with non-MHF with MD, MHF was related to higher risks of advanced fibrosis (P = .036), elevated albumin-creatinine ratio (UACR, P = .001), and sarcopenia (P = .013). Although the association between all grades of MHF and mortality was insignificant (P = .122), grades 2/3 was associated with increased mortality (P = .029). When comparing with non-MHF without MD, the harmful effects of MHF were more significant in mortality (P < .001), elevated UACR (P < .001), cardiovascular disease (P = .028), and sarcopenia (P < .001). In the PERSONS cohort, MHF was associated with more advanced grades of steatosis (P < .001), lobular inflammation (P < .001), advanced fibrosis (P = .017), and more severe hepatocellular iron deposition (P < .001). CONCLUSION: Both in the general population and in at-risk individuals with MAFLD, MHF was related with poorer clinical outcomes.

Higher consumption of animal organ meat is associated with a lower prevalence of nonalcoholic steatohepatitis.

Background: Animal organ meat (offal) is a food with high nutrient density that is popular in different parts of the world, but its relationship with nonalcoholic steatohepatitis (NASH) is unclear. We aimed to examine whether daily animal organ meat consumption is associated with the presence of NASH in individuals with nonalcoholic fatty liver disease (NAFLD). Methods: A total of 136 Chinese adults with biopsy-proven NAFLD were included. Definite NASH was defined as NAFLD activity score ≥4 and at least one point for steatosis, ballooning, and lobular inflammation. Daily animal organ meat consumption was estimated using a self-administered validated food frequency questionnaire. Logistic regression analysis was performed to assess the association between animal organ meat intake and liver disease severity. Results: The 136 participants (80.9% men) of the study had a mean ± standard deviation (SD) age of 39.0±12.5 years and body mass index of 27.4±3.6 kg/m2. Prevalence of definite NASH was 65.4%. Daily median organ meat consumption was 1.30 g/1,000 kcal. Animal organ meat consumption was inversely associated with the presence of NASH even after adjustment of demographics, lifestyle variables, metabolic and dietary factors, as well as liver fibrosis stage; adjusted-odds ratios (95% confidence intervals) for NASH were 0.15 (0.03, 0.69) for the highest tertile and 0.18 (0.05, 0.70) for the medium tertile, compared to the lowest (reference) tertile of animal organ meat intake (P value for trend =0.024). Conclusions: Our results suggest for the first time that higher animal organ meat consumption is associated with a lower prevalence of NASH in Chinese individuals with biopsy-proven NAFLD.

AutoFibroNet: A deep learning and multi-photon microscopy-derived automated network for liver fibrosis quantification in MAFLD.

BACKGROUND: Liver fibrosis is the strongest histological risk factor for liver-related complications and mortality in metabolic dysfunction-associated fatty liver disease (MAFLD). Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) is a powerful tool for label-free two-dimensional and three-dimensional tissue visualisation that shows promise in liver fibrosis assessment. AIM: To investigate combining multi-photon microscopy (MPM) and deep learning techniques to develop and validate a new automated quantitative histological classification tool, named AutoFibroNet (Automated Liver Fibrosis Grading Network), for accurately staging liver fibrosis in MAFLD. METHODS: AutoFibroNet was developed in a training cohort that consisted of 203 Chinese adults with biopsy-confirmed MAFLD. Three deep learning models (VGG16, ResNet34, and MobileNet V3) were used to train pre-processed images and test data sets. Multi-layer perceptrons were used to fuse data (deep learning features, clinical features, and manual features) to build a joint model. This model was then validated in two further independent cohorts. RESULTS: AutoFibroNet showed good discrimination in the training set. For F0, F1, F2 and F3-4 fibrosis stages, the area under the receiver operating characteristic curves (AUROC) of AutoFibroNet were 1.00, 0.99, 0.98 and 0.98. The AUROCs of F0, F1, F2 and F3-4 fibrosis stages for AutoFibroNet in the two validation cohorts were 0.99, 0.83, 0.80 and 0.90 and 1.00, 0.83, 0.80 and 0.94, respectively, showing a good discriminatory ability in different cohorts. CONCLUSION: AutoFibroNet is an automated quantitative tool that accurately identifies histological stages of liver fibrosis in Chinese individuals with MAFLD.

LEARN algorithm: a novel option for predicting non-alcoholic steatohepatitis.

Background: There is an unmet need for accurate non-invasive methods to diagnose non-alcoholic steatohepatitis (NASH). Since impedance-based measurements of body composition are simple, repeatable and have a strong association with non-alcoholic fatty liver disease (NAFLD) severity, we aimed to develop a novel and fully automatic machine learning algorithm, consisting of a deep neural network based on impedance-based measurements of body composition to identify NASH [the bioeLectrical impEdance Analysis foR Nash (LEARN) algorithm]. Methods: A total of 1,259 consecutive subjects with suspected NAFLD were screened from six medical centers across China, of which 766 patients with biopsy-proven NAFLD were included in final analysis. These patients were randomly subdivided into the training and validation groups, in a ratio of 4:1. The LEARN algorithm was developed in the training group to identify NASH, and subsequently, tested in the validation group. Results: The LEARN algorithm utilizing impedance-based measurements of body composition along with age, sex, pre-existing hypertension and diabetes, was able to predict the likelihood of having NASH. This algorithm showed good discriminatory ability for identifying NASH in both the training and validation groups [area under the receiver operating characteristics (AUROC): 0.81, 95% CI: 0.77-0.84 and AUROC: 0.80, 95% CI: 0.73-0.87, respectively]. This algorithm also performed better than serum cytokeratin-18 neoepitope M30 (CK-18 M30) level or other non-invasive NASH scores (including HAIR, ION, NICE) for identifying NASH (P value <0.001). Additionally, the LEARN algorithm performed well in identifying NASH in different patient subgroups, as well as in subjects with partial missing body composition data. Conclusions: The LEARN algorithm, utilizing simple easily obtained measures, provides a fully automated, simple, non-invasive method for identifying NASH.

N-terminal propeptide of type 3 collagen-based sequential algorithm can identify high-risk steatohepatitis and fibrosis in MAFLD.

BACKGROUND AND AIMS: With metabolic dysfunction-associated fatty liver disease (MAFLD) incidence and prevalence sharply increasing globally, there is an urgent need for non-invasive diagnostic tests to accurately screen high-risk MAFLD patients for liver inflammation and fibrosis. We aimed to develop a novel sequential algorithm based on N-terminal propeptide of type 3 collagen (PRO-C3) for disease risk stratification in patients with MAFLD. METHODS: A derivation and independent validation cohort of 327 and 142 patients with biopsy-confirmed MAFLD were studied. We compared the diagnostic performances of various non-invasive scores in different disease states, and a novel sequential algorithm was constructed by combining the best performing non-invasive scores. RESULTS: For patients with high-risk progressive steatohepatitis (i.e., steatohepatitis + NAFLD activity score ≥ 4 + F ≥ 2), the AUROC of FAST score was 0.801 (95% confidence interval (CI): 0.739-0.863), and the negative predictive value (NPV) was 0.951. For advanced fibrosis (≥ F3) and cirrhosis (F4), the AUROCs of ADAPT and Agile 4 were 0.879 (95%CI 0.825-0.933) and 0.943 (95%CI 0.892-0.994), and the NPV were 0.972 and 0.992. Sequential algorithm of ADAPT + Agile 4 combination was better than other combinations for risk stratification of patients with severe fibrosis (AUROC = 0.88), with similar results in the validation cohort. Meanwhile, in all subgroup analyses (stratifying by sex, age, diabetes, NAS, BMI and ALT), ADAPT + Agile 4 had a good diagnostic performance. CONCLUSIONS: The new sequential algorithm reliably identifies liver inflammation and fibrosis in MAFLD, making it easier to exclude low-risk patients and recommending high-risk MAFLD patients for clinical trials and emerging pharmacotherapies.

Secondary bile acids improve risk prediction for non-invasive identification of mild liver fibrosis in nonalcoholic fatty liver disease.

BACKGROUND: Dysregulated bile acid (BA) metabolism has been linked to steatosis, inflammation, and fibrosis in nonalcoholic fatty liver disease (NAFLD). AIM: To determine whether circulating BA levels accurately stage liver fibrosis in NAFLD. METHODS: We recruited 550 Chinese adults with biopsy-proven NAFLD and varying levels of fibrosis. Ultra-performance liquid chromatography coupled with tandem mass spectrometry was performed to quantify 38 serum BAs. RESULTS: Compared to those without fibrosis, patients with mild fibrosis (stage F1) had significantly higher levels of secondary BAs, and increased diastolic blood pressure (DBP), alanine aminotransferase (ALT), body mass index, and waist circumstance (WC). The combination of serum BAs with WC, DBP, ALT, or Homeostatic Model Assessment for Insulin Resistance performed well in identifying mild fibrosis, in men and women, and in those with/without obesity, with AUROCs 0.80, 0.88, 0.75 and 0.78 in the training set (n = 385), and 0.69, 0.80, 0.61 and 0.69 in the testing set (n = 165), respectively. In comparison, the combination of BAs and clinical/biochemical biomarkers performed less well in identifying significant fibrosis (F2-4). In women and in non-obese subjects, AUROCs were 0.75 and 0.71 in the training set, 0.65 and 0.66 in the validation set, respectively. However, these AUROCs were higher than those observed for the fibrosis-4 index, NAFLD fibrosis score, and Hepamet fibrosis score. CONCLUSIONS: Secondary BA levels were significantly increased in NAFLD, especially in those with mild fibrosis. The combination of serum BAs and clinical/biochemical biomarkers for identifying mild fibrosis merits further assessment.