Purpose: Carbapenem-resistant Enterobacterales (CRE) infection is an urgent threat to human health. This study aimed to develop and validate a novel multiplex real-time PCR (multi-qPCR) assay for the detection of the blaKPC, blaNDM, blaIMP, blaOXA-48-like, and blaVIM genes in CRE isolates and clinical samples, as well as to compare it with three phenotypic methods. Methods: The reliability and limit of detection (LOD) of the multi-qPCR assay were evaluated. PCR and DNA sequencing were used as the reference methods to identify carbapenemase genes in CRE isolates and clinical samples. The accuracy of the multi-qPCR assay, modified carbapenem inactivation and EDTA-modified carbapenem inactivation method (mCIMandeCIM), carbapenemase inhibitorbased combined disk test (CDT), and colloidal gold-based immunochromatographic test was compared with the reference methods with 182 isolates of CRE. Furthermore, 112 clinical samples were collected to validate the efficacy of this multi-qPCR assay. Results: The standard deviations (CVs) of intra-assay and inter-assay of the multi-qPCR assay were <= 0.53% and <= 2.04% for detecting the five major carbapenemase genes, respectively; while the LOD ranged from 2x102 copies/mL to 8x102 copies/mL. PCR and DNA sequencing confirmed 168 out of 182 CRE isolates producing carbapenemase(s): KPC (n = 93), NDM (n = 46), IMP (n = 8), OXA-48-like (n = 14), VIM (n = 1), KPC&NDM (n = 5), and KPC&NDM&IMP (n = 1). The accuracy of mCIMandeCIM, CDT, Colloidal Gold, and the multi-qPCR assay was 96.2%, 89.6%, 100%, and 100% respectively for detecting carbapenemase(s) producers. Moreover, the sensitivity and specificity of the multi-qPCR assay were all 100% for the detection of each carbapenemase gene in clinical samples, compared with PCR and sequencing. Conclusion: For clinical isolate detection, the multi-qPCR assay is comparable to Colloidal Gold, and superior to mCIMandeCIM and CDT; while for clinical samples detection, it also shows excellent performance. Therefore, the multi-qPCR assay has great potential for clinical diagnosis.

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Purpose: Yiqi Liangxue Jiedu prescription (YLJP), a Chinese medicine that is commonly used to prevent liver cancer and is authorized by a national patent (patent No. ZL202110889980.5) has a therapeutic effect on precancerous lesions; however, the underlying mechanism remains unclear. This study is aimed at determining the clinical therapeutic efficacy of YLJP in patients with precancerous liver lesions and to explore and validate its possible effector mechanism. Patients and Methods: The 1-year incidence of hepatocellular carcinoma (HCC) was retrospectively analyzed in 241 patients with cirrhosis complicated by abnormal alpha-fetoprotein precancer. Network pharmacological analysis, molecular docking, and molecular dynamics simulation were used to explore the key targets and compounds of YLJP in treating HCC. Immunohistochemical methods were used to detect the expression of key proteins in tumor and cirrhotic tissues. Finally, the mechanism underlying the effects of YLJP was verified in rats with precancerous lesions. Results: The 1-year incidence of HCC was lower in the YLJP group than in the Western medicine group. The Wnt pathway protein, CTNNB1, is a key target of YLJP in preventing and treating HCC, and the canonical Wnt pathway is the key signaling pathway and is overexpressed in human liver tumors. In vivo experiments showed that YLJP significantly inhibited the canonical Wnt pathway and reduced the abnormal differentiation of hepatic oval cells. The binding of CTNNB1 to oleanolic acid, stigmasterol, and beta-sitosterol was found to be stable, indicating the action of these compounds in treating HCC. Conclusion: YLJP reduces the 1-year incidence of HCC, with its mechanism likely due to oleanolic acid, beta-sitosterol, and stigmasterol inhibition of the CTNNB1 activation of the (3-catenin protein, which in turn regulates the Wnt signaling pathway and prevents the abnormal differentiation of hepatic oval cells into cancer cells, thus delaying the occurrence and progression of the disease.

Few studies focused on human papillomavirus (HPV) in male patients. This study aimed to explore the detection rate and genotyping of HPV among male patients in Beijing to provide a reference for formulating prevention strategies for HPV infection. The cross-sectional study was conducted in Beijing Chaoyang Hospital from November 2015 to March 2023. It covered male patients from the urology and dermatology departments. Fifteen high-risk HPV genotypes were detected by the multiplex real-time polymerase chain reaction method. The overall detection rate of HPV was 25.19% (1288/5114, 95% confidence interval [CI] 24.00%-26.38%), of which the single infection rate was 16.99% (869/5114, 95% CI 15.97%-18.05%) and the co-infection rate was 8.19% (419/5114, 95% CI 7.46%-8.98%). The detection rate of HPV was 40.77% (521/1278), 35.58% (58/163), 32.69% (101/309), 31.91% (60/188), 12.63% (299/2367), and 32.35% (131/405) among male patients with balanitis, warts, rash, urethritis, prostatitis, and other urinary inflammation, respectively (P < 0.001). The top five HPV genotypes were HPV-52, HPV-58, HPV-16, HPV-51, and HPV-66. After the first positive HPV test, the proportion of male patients who turned negative was 22.47% within 3 months, 26.40% within 3-6 months, 24.72% within 6-12 months, 17.98% within 12-24 months, and 8.43% more than 24 months. The detection rate of HPV was high among male patients from the urology and dermatology departments in Beijing, which should be considered to develop HPV vaccines with better prevention effects.

Background: Intrarenal arteriolar disease is a major risk factor for poor prognosis in immunoglobulin A nephropathy (IgAN). The morphologic factor sonic hedgehog (SHH) plays an important role in a variety of vascular diseases, so it may be directly or indirectly involved in the process of renal arteriolar disease. The purpose of this study was to investigate the correlation between serum SHH levels and renal arteriole disease in patients with IgAN. Methods: Subjects with primary IgAN diagnosed by renal biopsy performed between October 2018 and August 2019 at the First Medical Center of the Chinese PLA General Hospital were recruited. Blood specimens were collected from the patients within 1 week before renal biopsy after they signed an informed consent form, and healthy controls were recruited for blood specimen collection during the same period. The concentration of serum SHH was measured by enzyme-linked immunosorbent assay in this population. Results: Serum SHH levels were significantly lower in the IgAN group than in the control group. 41 of the 94 subjects diagnosed with IgAN had severe renal arteriolosclerosis and, compared to their less severely affected counterparts, were older, more hypertensive, and characterized by lower levels of SHH, higher levels of tubular atrophy/interstitial fibrosis and a higher Lee's classification. Serum SHH concentration was found to be an independent predictor of severe intrarenal arteriolosclerosis in IgAN subjects after correction using multivariate analysis. Conclusion: In this study, serum SHH levels were found to be significantly lower in patients with IgAN than in healthy subjects. Serum SHH may serve as a noninvasive biomarker of intrarenal arteriolosclerosis in patients with IgAN.

Background and Objectives: The relationship between circulating retinol-binding protein 4 (RBP4) levels and hepatitis C virus (HCV) infection remains unclear. This study aims to systematically assess RBP4 expression in patients with HCV and its correlation with disease severity. Materials and Methods: We searched the Embase, PubMed, and Cochrane databases for relevant studies up to 1 January 2024. This study was registered on PROSPERO (CRD42023489051). Results: Our analysis included eight studies with 2612 participants (1152 controls and 1282 patients with HCV). Overall, RBP4 levels did not significantly differ between patients with HCV and controls (SMD: −0.36; 95% CI: −0.94, 0.23; p = 0.23). However, in a subgroup of Asian subjects, patients with HCV showed significantly lower RBP4 levels (SMD: −0.40; 95% CI: −0.49, −0.31; p = 0.10). Additionally, a negative correlation between RBP4 levels and disease severity was observed across all studied populations. Conclusions: RBP4 levels may vary due to HCV genotype, ethnicity, and environmental factors. In the context of HCV infection, RBP4 levels appear to reflect the severity of disease progression. Our findings indicate that RBP4 could serve as a biomarker for HCV disease progression. Further research is needed to elucidate the complex mechanisms of RBP4 in HCV infection.

The most prevalent primary glomerulonephritis and leading cause of end-stage renal disease worldwide is IgA nephropathy (IgAN). More and more studies are describing urinary microRNA (miRNA) as a non-invasive marker for a variety of renal diseases. We screened candidate miRNAs based on data from three published IgAN urinary sediment miRNAs chips. In separate confirmation and validation cohorts, we included 174 IgAN patients, 100 patients with other nephropathies as disease controls (DC), and 97 normal controls (NC) for quantitative real-time PCR. A total of three candidate miRNAs, miR-16-5p, Let-7g-5p, miR-15a-5p were obtained. In both the confirmation and validation cohorts, these miRNAs levels were considerably higher in the IgAN than in NC, with miR-16-5p significantly higher than in DC. The area under the ROC curve for urinary miR-16-5p levels was 0.73. Correlation analysis suggested that miR-16-5p was positively correlated with endocapillary hypercellularity (r = 0.164 p = 0.031). When miR-16-5p was combined with eGFR, proteinuria and C4, the AUC value for predicting endocapillary hypercellularity was 0.726. By following the renal function of patients with IgAN, the levels of miR-16-5p were noticeably higher in the IgAN progressors than in the non- progressors (p = 0.036). Urinary sediment miR-16-5p can be used as noninvasive biomarkers for the assessment of endocapillary hypercellularity and diagnosis of IgA nephropathy. Furthermore, urinary miR-16-5p may be predictors of renal progression.

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.

Objectives: The purpose of this study was to investigate the clinicopathological characteristics of primary central nervous system lymphoma (PCNSL). Methods: We collected 41 PCNSL formalin-fixed, paraffin-embedded (FFPE) samples from human immunodefi-ciency virus (HIV)-positive patients and performed HE (haematoxylin-eosin) staining, immunohistochemistry (IHC) staining, in situ hybridization, fluorescence in situ hybridization (FISH). Real-time quantitative polymerase chain reaction (RT-qPCR) was performed in 9 cases of FFPE samples. Meanwhile, we analysed the clinical pathological significance of the results. Results: Seven patients had diffuse large B-cell lymphoma (DLBCL) with germinal centre B-cell (GCB)-like DLBCL, 32 had activated B-cell (ABC)-like DLBCL, and 2 had Burkitt lymphoma (BL). GCB-like DLBCL patients were older at onset (P = 0.040).A lower CD4+ T-cell count and a decrease in cerebrospinal fluid (CSF) glucose content were more frequent in ABC-like DLBCL (P = 0.012, P = 0.006). Overexpression of P53 was more in ABC-like DLBCL (P = 0.041). 73.2 % cases were Epstein-Barr encoding region (EBER) positive, which was more likely in ABC-like DLBCL patients (P = 0.037). EBV DNA were detected in 5/7 EBER-negative DLBCL cases and none (0/2) of the BL cases. All the cases were negative for HHV8 staining. None of the 7 Double expressor lymphoma (DEL) cases had BCL2, BCL6, or c-MYC genetic rearrangements. Conclusions: HIV-related PCNSL showed unique clinical pathological significance. None of EBV detected in HIV -related BL and without HHV8 infectious are new sights in our single-center study of Chinese HIV-related PCNSL patients.

Antibody inhibitors that block PD-1/PD-L1 interaction have been approved for oncological clinics, yielding impressive treatment effects. Small molecules inhibiting PD-1 signalling are at various stages of development, given that small molecular drugs are expected to outperform protein drugs in several ways. Currently, a significant portion of these small molecular inhibitors achieve this purpose by binding to a limited region of the PD-L1 protein, thereby limiting the choice of chemical structures. Alternative strategies for developing small-molecular PD-1 inhibitors are urgently needed to broaden the choice of chemical structures. Here, we report that 6-mercaptopurine (6-MP) inhibits PD-1 signalling, activates T cell function in vitro and in vivo and shrinks tumours by activating cytotoxic T cells. Mechanistically, 6-MP potently inhibited PD-1 signalling by blocking the recruitment of SHP2 by PD-1. Considering that 6-MP is a chemotherapeutic agent already approved by the FDA for childhood leukaemia, our work revealed a novel anti-tumour mechanism for this drug and suggests that 6-MP warrants further clinical evaluation for other tumour types.

Current knowledge regarding the long-term humoral response of people infected with human immunodeficiency virus to the third dose of inactivated coronavirus disease (COVID-19) vaccine is incomplete. As a result, concerns remain about the safety and efficacy of the vaccination. To improve our understanding of the safety and immunogenicity of the COVID-19 inactivated vaccine booster in people living with HIV (PLWH), a prospective study was conducted on participants who had not yet received a third dose of the COVID-19 inactivated vaccine, had no history of SARS-CoV-2 infection, and had received a second dose of the vaccine more than six months prior. The primary safety outcomes included the incidence of adverse reactions, changes in CD4+ T-cell count, viral load, blood routine examination, liver and kidney function examination, blood sugar, and blood lipid examination. The pseudovirus-neutralizing antibody responses to the D614G variant, Delta variant, and Omicron variants BA.5 and BF.7 were evaluated before vaccination, 14 days, 28 days, 3 months, and 6 months after vaccination to evaluate the immune response of PLWH to the injection of inactivated vaccine booster and the safety of the vaccine. In conclusion, COVID-19 vaccine booster shots were effective in PLWH, resulting in an increase in the number of CD4+ T-cells, neutralizing antibodies that lasted up to six months, and higher levels of neutralizing antibodies lasting approximately 3 months. However, the vaccine protection against the two variants of BA.5 and BF.7 was significantly lower than that of D614G and Delta.

Purpose Vaccination reduces the incidence of severe COVID-19 and death and effectively limits viral spread. Concerns have been raised about COVID-19 vaccine responses in the large population of HIV-infected patients. This study aims to explore the safety and immunogenicity of the inactivated COVID-19 vaccine in people living with HIV (PLWH). Patients and Methods All participants in this study already had their second dose of an inactivated COVID-19 vaccine at least 14 days earlier, without a history of SARS-CoV-2 infection. The primary safety outcomes were the incidence of adverse reactions and changes in CD4+ T-cell counts. SARS-CoV-2 IgG and neutralizing antibody responses to the D614G variant and delta variant were measured for immune response assessment. Results Forty-seven HIV-infected patients and 18 healthy donors (HDs) were enrolled in this study. Adverse reactions were mild or self-limiting and were reported in 19.1% of HIV-infected patients. Most PLWH developed antibody responses against the inactivated COVID-19 vaccine. The longitudinal analysis of antibody responses in PLWH (median interval between detection and complete vaccination, 59 days) showed that antibodies were maintained for at least three months, though their titers were reduced. However, the antibody-positive rates in PLWH were significantly lower than those in HDs. Additionally, compared to HDs (Geomean titers (GMT) of 165 for D614G and GMT of 72 for delta), the neutralizing antibody titers against the two variants in PLWH (GMT of 43 for D614G and GMT 13 for delta) were decreased significantly (p = 0.018 and p < 0.001, respectively). HIV-infected patients with CD4+T-cell counts ≤350 cells/μL appeared to exhibit a poor antibody response to inactivated vaccination. Conclusion Inactivated COVID-19 vaccines appear to be efficacious in PLWH. However, antibody responses in HIV-infected patients are inferior to those in healthy individuals, especially PLWH with lower CD4+T-cell counts.

Background: Dual anti-retroviral therapy is the main proven valuable intervention type for treating naïve HIV/AIDS. Currently, no high-quality evidence is available regarding the best dual schemes.Objectives: The aim of this study is to evaluate the effectiveness and safety of PIs/r-based dual therapy in treatment-naïve HIV/AIDS patients by using network meta-analysis.Methods: Randomized controlled trials of PIs/r-based dual therapy in treatment-naïve HIV/AIDS were searched based on Embase, PubMed and Cochrane library database from January 2006 to June 2021. Taking viral suppression rate, CD4+T cell count changes from baseline as the primary indicator and adverse events rate as secondary indicator, the network meta-analysis was performed on Review Manager and STATA software. Heterogeneity was assessed by the Q statistic and I2. We registered our protocol in Prospero with ID CRD42021275466.Results: Among 15 randomized controlled trials (3,497 patients and 7 PIs/r-based dual therapy) were reviewed in this study. According to the forest map, DRV/r + INSTIs was more effective compared to triple therapy (TT) in viral suppression [OR 0.82, 95% CI (0.61–1.11)], in CD4+T cell count changes from baseline [MD 1.9, 95% CI (0.7, 3.1), I2 86%], in adverse events [OR 0.98, 95% CI (0.68–1.39)]. Furthermore, SUCRA ranking analysis indicated that DRV/r + INSTIs was superior to TT in viral suppression (DRV/r + INSTIs 75.5% > TT 41.2%) and in immune construction (DRV/r + INSTIs 67% > TT 42%). In addition, DRV/r + INSTIs was similar to TT in adverse events (DRV/r + INSTIs 54.9% ≈ TT 54.7%).Conclusion: DRV/r + INSTIs was obviously superior to TT in viral suppression and immune reconstruction, and was not higher than TT in adverse events.Systematic Review Registration:https://www.crd.york.ac.uk/prospero/, identifier CRD42021275466

The prognosis of hepatocellular carcinoma (HCC) is extremely poor, of which hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) accounts for the majority in China. Immune checkpoint inhibitors have become an effective immunotherapy method for the treatment of HCC, but they are mainly used for T cells. NK cells play a vital role as the first line of defense against tumors. Therefore, we explored the characteristic expression pattern of immune checkpoints on NK cells of HBV-HCC patients. We analyzed the correlation between the co-expression of TIGIT and TIM-3 and the clinical progress of patients with HBV-HCC. The co-expression of TIGIT and TIM-3 on NK cells is elevated in patients with HBV-HCC. TIGIT+TIM-3+NK cells showed exhausted phenotypic characteristics and displayed dysfunction manifested as weakened killing function, reduced cytokine production, and proliferation function. TIGIT+TIM-3+NK cells participate in NK cells function exhaustion and are closely related to the disease progression of patients with HBV-HCC, suggesting a new target for future immunotherapy.

Background Hydroa Vacciniforme-like Lymphoproliferative Disorder (HV-LPD) is the name given to a group of Epstein-Barr virus (EBV)-associated diseases. It resembles hydroa vacciniforme (HV), the rarest form of photosensitivity, and is a T-cell disorder associated with an Epstein-Barr virus infection. The majority of diagnosed cases occur in East Asia and South America. It is rare in the United States and Europe. Multiple studies have revealed the clinical manifestation of an enlarged liver, but no gold standard such as pathology has yet supported this as a clinical sign of HV-LPD. Case presentation Here, we report a case of a 34-year-old Asian female with definite liver invasion. The patient had complained of a recurring facial rash for many years . The patient was admitted to the hospital because of an enlarged liver. After hospitalization, she was given an EB virus nucleic acid test. The EB virus nucleic acid test was positive, and pathological examination suggested that HV-LPD had invaded the skin, bone marrow, and liver. After being given antiviral treatment, the patient’s symptoms were mitigated. Conclusions Our case confirms the liver damage was caused by HV-LPD and the effectiveness of antiviral treatment.

Abstract PurposeHepatocellular carcinoma (HCC) patients with alpha-fetoprotein (AFP)-negative (<8.78 ng/mL) have special clinicopathologic characteristics and prognosis. The aim of this study was to apply a new method to establish and validate a new model for predicting the prognosis of HCC patients with AFP-negative.Materials and MethodsA total of 410 AFP-negative patients with clinical diagnosed with HCC as a primary cohort; 148 AFP-negative HCC patients as an independent validation cohort. In primary cohort, independent factors for overall survival (OS) by LASSO Cox regression were all contained into the nomogram1; by univariate and multivariate Cox hazard analysis were all contained into the nomogram2. Nomograms performance and discriminative power were assessed with concordance index (C-index) values, area under curve (AUC), Calibration curve and decision curve analyses (DCA). The results were validated in the validation cohort.ResultsThe C-index of nomogram1was 0.708 (95%CI: 0.673-0.743), which was superior to nomogram2 (0.706) and traditional modes (0.606-0.629). The AUC of nomogram1 was 0.736 (95%CI: 0.690-0.778). In the validation cohort, the nomogram1 still gave good discrimination (C-index: 0.752, 95%CI: 0.691-0.813; AUC: 0.784, 95%CI: 0.709-0.847) and good calibration. The calibration curve for probability of OS showed good homogeneity between prediction by nomogram1 and actual observation. DCA demonstrated that nomogram1 was clinically useful. Moreover, patients were divided into three distinct risk groups for OS by the nomogram1: low-risk group, middle-risk group and high-risk group, respectively.ConclusionsNovel nomogram based on LASSO Cox regression presents more accurate and useful prognostic prediction for patients with AFP-negative HCC. This model could help AFP-negative HCC facilitate a personalized prognostic evaluation.

Rationale: Effective targeting therapies are limited in Hepatocellular carcinoma (HCC) clinic. Characterization of tumor suppressor genes (TSGs) and elucidation their signaling cascades could shed light on new strategies for developing targeting therapies for HCC. Methods: We checked genome-wide DNA copy number variation (CNV) of HCC samples, focusing on deleted genes for TSG candidates. Clinical data, in vitro and in vivo data were collected to validate the tumor suppressor functions. Results: Focal deletion of GATA4 gene locus was the most prominent feature across all liver cancer samples. Ectopic expression of GATA4 resulted in senescence of HCC cell lines. Mechanistically, GATA4 exerted tumor suppressive role by orchestrating the assembly of a tumor suppressor enhancing module: GATA4 directly bound and potently inhibited the mRNA transcription activity of β-catenin; meanwhile, β-catenin was recruited by GATA4 to promoter regions and facilitated transcription of GATA4 target genes, which were TSGs per se. Expression of GATA4 was effective to shrink GATA4-deficient HCC tumors in vivo. We also showed that β-catenin inhibitor was capable of shrinking GATA4-deficient tumors. Conclusions: Our study unveiled a previously unnoticed tumor suppressor enhancing module assembled by ectopically expressed GATA4 in HCC cells and denoted a therapeutic opportunity for GATA4 deficient HCC patients. Our study also presented an interesting case that an oncogenic transcription factor conditionally functioned as a tumor suppressor when recruited by a TSG transcription factor.

Hepatocellular carcinoma (HCC) is one of the most common malignant cancers with a high recurrence rate. An effective therapeutic strategy for HCC is, therefore, in urgent demand. This work aims to investigate the in vivo anti-HCC effect of microwave ablation (MWA) followed by tumor lysate activated dendritic cell (DC) vaccine. C57BL/J mice were rechallenged with Hepa1-6 tumor cells with different treatments (single MWA, single DC injection and MWA combined with DC injection). Then tumor growth was monitored and compared. The populations of Treg and Th17 cells were detected by flow cytometry and immunohistochemistry before and after different treatments. The migration of DCs was monitored using optimal imaging. A general volume decrease in the rechallenged tumors was observed both in the MWA and MWA + DC groups. The volume of the rechallenged tumors in the MWA + DC group showed a more significant downward trend compared with that in the MWA group. The migration of DCs from the injected site to the inguinal lymph node region was detected 24 h after injection by optimal imaging. A significant decrease of Treg and Th17 cells in the peripheral blood and lymph nodes was observed in the mice of the MWA + DC group. In summary, MWA combined with DC vaccination showed an effective synergistic therapy for HCC, with few tumor recurrences.

Nonalcoholic steatohepatitis (NASH) has the potential to progress to fibrosis, cirrhosis, and hepatocellular carcinoma. Upregulation of sonic hedgehog (Shh) has been documented in development of NASH through sustained cell stress. At the same time, transforming growth factor-β1 (TGF-β1), which is a central element in fibrogenic reactions in various diseases and sites, has been reported to be associated with hepatic inflammation and fibrotic reaction. To explore crosstalk between Shh and TGF-β1 in the development and progression of NASH, we investigated the expression of both these proteins in 135 human specimens of NASH, 35 fatty liver specimens, 35 specimens of alcoholic steatohepatitis with immunohistochemistry. Shh protein was expressed in the cytoplasm of ballooned hepatocytes with an ubiquitin-like pattern. In addition, a few scattered apoptotic hepatocytes in the inflammatory foci showed homogeneous cytoplasmic Shh expression. TGF-β1 protein was observed mainly in the activated hepatic stellate cells (HSCs) which were located in the inflammatory foci surrounding ballooned hepatocytes. Moreover, the mRNA levels of both Shh and TGF-β1 in the liver biopsy specimens from NASH patients was significantly increased compared with those in fatty liver patients. Statistically, there was a significant association of the expressions of Shh and TGF-β1 proteins in NASH (r=0.6, P<0.05). In addition, increased expression of Shh protein significantly parallels the severity of hepatocellular ballooning, lobular, and portal inflammatory responses and progression of fibrosis in NASH patients. Moreover, we found that much HSCs transformed into myofibroblast-like phenotype and migrated downward to HepG2 hepatocellular carcinoma cells with overexpression of Shh by transwell assay. We also observed overexpression of proteins of Shh and TGF-β1 in cultured activated HSCs with confocal microscopy. These findings strongly suggest there is interplay between Shh and TGF-β1 in hepatic inflammatory reactions. Shh secreted through damaged hepatocytes may result in activation of TGF-β1 and subsequent transformation of HSCs, which together modulate the progression of human NASH.

To the Editor: Hereditary hemochromatosis (HH) is a late-onset autonomic disease characterized by enhanced intestinal absorption of iron and iron overload, which may lead to liver cirrhosis, cardiomyopathy, diabetes, arthritis, and skin pigmentation. In 1996, Feder et al[1] cloned the gene responsible for HH (HFE) and reported that 85% of HH patients were homozygous for the C282Y mutation in the HFE gene. This was subsequently termed type 1 HH. Other inherited forms of HH exist that are non HFE-related, an example of which is juvenile hemochromatosis (JH); two genotypes that induce JH have been reported thus far. One JH genotype involves the hemojuvelin gene (HJV), which was cloned from a Greek family with JH by Papanikolaou et al[2] and is known as type 2A HH. The other involves the human antimicrobial peptide (HAMP) gene, which encodes hepcidin and is termed type 2B HH. JH is a rare autosomal recessive disorder that typically occurs in the first to third decades of life. Its symptoms are more acute and severe than those of classic hemochromatosis. The primary goal of therapy is iron depletion to normalize the body's iron stores and to prevent or decrease organ dysfunction. A 31-year-old man who presented with polyuria, arterial hypertension, and transaminasemia for 8 years, and increased skin pigmentation for 2 years, was admitted. He was initially diagnosed with liver dysfunction and diabetes at age 23. Since then, he was treated with insulin and liver protection therapy, but the effects were not good. When he was admitted to our hospital, he complained of progressive sexual impotence. Laboratory test results revealed remarkable hyperglycemia with glycosuria (13.2 mmol/L) and biochemical liver damage: glutamyl transpeptadase (GGT) 140 U/L, aspartate transaminase (AST) 113 U/L, and alkaline phosphatase (ALP) 162 U/L. His serum ferritin concentration was 8729.2 ng/mL, with a transferrin saturation of 112.4%. To determine the cause of the liver dysfunction, a liver biopsy was performed, which showed ballooning degeneration of hepatocytes. Portal lymphocytic infiltrates were present, with variable periportal inflammatory activity (interface hepatitis). Bridging necrosis was observed [Figure 1A]. Furthermore, fibrous septa and regenerative nodule formation were observed by reticulin stain [Figure 1B]. The Ishak scores for inflammation and fibrosis were 13 and 5, respectively. Iron staining (Perls’ Prussian blue stain) showed diffuse extensive iron deposition in all hepatocytes, Kupffer cells, macrophages, biliary epithelial cells, and vascular endothelial cells [Figure 1C]. Iron was distributed according to a decreasing gradient from periportal to centrolobular areas.Figure 1: Pathological features of biopsy of the liver of the patient. (A) Bridging necrosis was present (H&E, original magnification ×100). (B) Fibrous septa and regenerative nodule formation (Reticulin staining, original magnification ×40). (C) Diffuse extensive iron deposition in all hepatocytes, Kupffer cells, macrophages, biliary epithelium and vascular endothelial cells (Perls’ Prussian blue staining, original magnification ×200).We detected mutations in the HFE, HAMP, TFR2, SLC40A1, and HJV genes in the patient and in his parents, but only two mutations in HJV were identified. The patient was heterozygous for two mutations in HJV: a premature termination mutation (962G>A and 963C>A; C321X) and a mutation in the signal peptide (18G>C; Q6H). His father had the same mutations in the HJV gene, while his mother had one heterozygous mutation in HJV (9G>C; E3D). Due to the slightly lower hemoglobin level (121 g/L), treatment with iron chelators was initiated. Intravenous deferoxamine (2.5 g/d–3.5 g/d) biweekly was administered for approximately 3 years until the serum ferritin decreased to a normal level. At the same time, he was treated with insulin and liver protection therapy. His serum ferritin apparently decreased to a normal level and his AST nearly decreased to a normal level. The skin hyperpigmentation completely disappeared after a 4-month treatment with deferoxamine. The elevated glucose and loss of libido appeared to be unaffected. Liver involvement is a constant feature of HH, but the presence of cirrhosis at initial presentation is less common in JH and is only about 27%.[3] Hepatocellular carcinoma has not been reported in JH, which is primarily because untreated individuals with JH die prematurely as a result of cardiac complications. It has been reported that, when patients present with JH, the incidences of hypogonadism and cardiomyopathy are 94.6% and 43.2%, respectively, and that these are the earliest and most frequent manifestations of JH.[3] Different clinical manifestations could reflect the occurrence of the disease in a different environment or genetic background. Laboratory data for JH is limited because documented cases of JH are rare. However, tests for serum ferritin (SF) concentration and transferrin-iron saturation (TS) should be performed. Several studies have demonstrated that the SF concentration provides a valuable correlation with the degree of iron stores in the body, and thus SF concentration has added value as a predictor of advanced fibrosis and cirrhosis in confirmed HH. Liver biopsy plays an important role in the accurate assessment of iron deposition in JH patients. Furthermore, liver biopsy is currently the gold standard for the diagnosis of fibrosis and cirrhosis. Patients with elevated serum iron levels should be considered for liver biopsy if they have elevated liver enzymes or other clinical evidence of liver disease. Iron deposition in the liver may affect parenchymal cells and mesenchymal cells. In HFE hemochromatosis, iron remains located at the biliary pole of hepatocytes. It is distributed according to a decreasing gradient from periportal to centrolobular areas, which is a typical pattern indicative of parenchymal iron overload. The histologic presentation of iron overload in JH is different and is responsible for marked iron overload of a mixed pattern with parenchymal predominance. The iron deposition in the patient with JH in our study was also of a mixed pattern, which showed diffuse extensive iron deposition in all hepatocytes, Kupffer cells, macrophages, biliary epithelial cells, and vascular endothelial cells. The Deugnier scores of parenchymal and mesenchymal iron deposition were 21 and 17, respectively. Due to the fast progression of iron deposition in JH patients, hepatocellular iron load may increase quickly and finally leading to the redistribution of iron toward mesenchymal cells. Iron accumulates and triggers chronic liver damage that ends in hepatic fibrosis and cirrhosis. The Ishak scores for inflammation and fibrosis in our case were 13 and 5, respectively. Mutations in the HJV gene are the most frequent cause of JH and may be found in either a homozygous or a compound heterozygous state. Various mutations in the HJV gene have been reported. The G320V amino acid change appears to be common in patients originating in Europe.[2] However, the mutation found in our patient was different, as he was heterozygous for 2 mutations: a premature termination mutation (962G>A and 963C>A; C321X) and a mutation in the signal peptide (18G>C; Q6H). We searched two papers on only Chinese JH, and all of those patients had the same two mutations. Therefore, we speculated that these mutations may be hotspots in Chinese patients with JH and that these mutations could cause loss of function of hemojuvelin and impaired iron metabolism.[4,5] A study showed that deferoxamine treatment was as effective as normal phlebotomy treatment with weekly 500-mL blood removals. Due to the slightly lower hemoglobin level in our study, treatment with intravenous deferoxamine was also effective. Thus, deferoxamine treatment is an effective second-line treatment in those patients for whom regular phlebotomy cannot be performed. In conclusion, liver biopsy has an important role in JH patients so that the degree of iron deposition and fibrosis can be accurately assessed. C321X and Q6H mutations may be hotspots in Chinese patients with JH. Iron parameters, cardiac function, sexual function, and liver function are more easily normalized by administration of iron chelators in combination with phlebotomy than phlebotomy alone. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understands that his names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Funding This study was supported by a grant from the Beijing Hospitals Authority Youth Programme (No. QML20181805). Conflicts of interest None.