Development and validation of a novel death risk stratification scale in patients with hemorrhagic fever with renal syndrome: a 14-year ambispective cohort study.

OBJECTIVES: To develop and validate a simple and effective death risk stratification scale for hemorrhagic fever with renal syndrome (HFRS). METHODS: In this ambispective cohort study, we investigated the epidemiological and clinical data of 2245 patients with HFRS (1873 enrolled retrospectively and constituting the training cohort, 372 prospectively recruited as the validation cohort) from September 2008 to December 2021, and identified independent risk factors for 30-day death of HFRS. Using logistic regression analysis, a nomogram prediction model was established and was further simplified into a novel scoring scale. Calibration plot, receiver operating characteristic curve, net reclassification index, integrated discrimination index, and decision curve analysis were used to assess the calibration, discrimination, precision, and clinical utility in both training and validation cohorts. RESULTS: Of 2245 patients with HFRS, 132 (5.9%) died during hospitalization. The nomogram prediction model and scoring scale were developed using six predictors: comorbid hypertension, hypotensive shock, hypoxemia, neutrophils, aspartate aminotransferase, and activated partial thromboplastin time. Both the scale and nomogram were well calibrated (near-diagonal calibration curves) and demonstrated significant predictive values (areas under receiver operating characteristic curves >0.9, sensitivity and specificity >90% in the training cohort and >84% in the validation cohort). The simplified scoring scale demonstrated equivalent discriminative ability to the nomogram, with net reclassification index and integrated discrimination index of 0.022 and 0.007 in the training cohort, 0.126 and 0.022 in the validation cohort. Decision curve analysis graphically represented significant clinical utility and comparable net benefits of the nomogram and scoring scale across a range of threshold probabilities. DISCUSSION: This evidence-based, factor-weighted, accurate score could help clinicians swiftly stratify HFRS mortality risk and facilitate the implementation of patient triage and tiered medical services during epidemic peaks.

Alterations of the gut microbial community structure modulates the Th17 cells response in a rat model of asphyxial cardiac arrest.

Th17 cells triggered inflammation is a critical element in cerebral ischemic injury, and the gut microbiota intricately impacts T lymphocytes. Nevertheless, it remains unclear whether the gut microbiota involves in cardiac arrest/cardiopulmonary resuscitation (CA/CPR) induced-brain injury through Th17 cells. The present study investigated the interaction between gut microbiota and Th17 cells in a rat model. We observed that CA/CPR induced the alterations of the gut microbial community structure, and elevated the level of IL-17 in the serum, and a slight infiltration of Th17 cells into the brain. The Th17 cells were increased significantly in the peripheral blood, 28.33 ± 6.18% of these Th17 cells were derived from the Peyer's patches of small intestine. Furthermore, fecal microbiota transplantation (FMT) from rats with CA/CPR induced Th17 cell response, promoting hippocampal cell apoptosis and declining learning ability and memory in recipient rats. Taken together, CA/CPR-induced alterations of the gut microbial community structure stimulated Th17 cell response which aggravated brain injury.

LPS stimulation during HCV infection induces MMP/TIMP1 imbalance in macrophages.

Introduction. During chronic hepatitis C virus (HCV) infections, HCV antigens establish cross-tolerance of endotoxins, but additional lipopolysaccharide (LPS) stimulation effects in this condition are poorly understood.Aim. This study aims to investigate the effects of the upregulated LPS on MMP and TIMP expression during chronic hepatitis C infection.Methodology. In the present study, we analysed the effect of HCV antigens and LPS stimulation on peripheral blood mononuclear cells (PBMCs) both in vivo and in vitro. Macrophages from HCV patients were isolated and their association with endotoxin tolerance was examined. MMP/TIMP1 expression and the related signalling pathways in macrophages were analysed. The macrophage and Huh7.5 cell co-culture model was used to analyse the effects of the cross-tolerance on collagen I deposition.Results. LPS levels were found to be significantly higher in HCV patients, particularly in those with HCV-induced liver fibrosis. In addition, although LPS serum level was occasionally upregulated in the patients, it did not induce intense immune response in PBMCs due to endotoxin cross-tolerance, and this was measured according to the changes in IL-6 and TNF-α levels. However, TIMP1 expression increased significantly during stimulation, exhibiting a tolerance/resistance phenotype, which was associated with TGF-ß/Erk activation in macrophages. However, MMP levels did not increase due to endotoxin tolerance, which ultimately led to MMP/TIMP imbalance and influenced the deposition of collagen I.Conclusion. Increased LPS stimulation of macrophage during HCV antigen-induced endotoxin cross-tolerance contributes to MMP/TIMP1 imbalance and collagen I deposition.

[Mechanism for the inhibition of proliferation and promotion of apoptosis in Huh7.5 cells by iron overload].

Objective To investigate the effect of iron overload on biological activity and apoptosis in Huh7.5 cells. Methods Huh7.5 cells were cultured in the medium supplemented with 50, 100, 200 µmol/L ferric ammonium citrate (FAC). Fluorescence microscopy was employed to determine cell iron load labeled by Phen Green FL; proliferation activity of Huh7.5 cells was evaluated by MTT assay; protein and mRNA levels of transferrin receptor (TfR1), TfR2, divalent metal transporter 1 (DMT1) and ferroportin 1 (FPN1) in Huh7.5 cells were detected by Western blotting and real-time PCR, respectively; cell reactive oxygen species (ROS) labeled by dichlorofluorescin diacetate (DCFH-DA) and cell apoptosis labeled by annexinV-FITC/PI were analyzed by flow cytometry. Results FAC treatment increased intracellular iron load in a dose-dependent manner. Compared with control group, mRNA and protein expressions of TfR1, TfR2 and DMT1 were down-regulated, while mRNA and protein expression of FPN1 was significantly up-regulated in FAC treated groups. With the increasing dose of FAC, intracellular ROS level increased significantly and cell proliferation activity decreased significantly. The cell apoptosis rate in FAC treated groups were remarkably higher than that in control group, but after antioxidant N-acetylcysteine (NAC) was added, the cell apoptosis in FAC treated group was inhibited obviously. Conclusion Iron overload can inhibit the proliferation and promote the apoptosis of Huh7.5 cells through oxidative stress.

Modulation of Tim-3 Expression by Antigen-Dependent and -Independent Factors on T Cells from Patients with Chronic Hepatitis B Virus Infection.

T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) was up-regulated on viral specific T cells and contributed to T cells exhaustion during chronic hepatitis B virus (HBV) infection. However, modulation of Tim-3 expression was still not fully elucidated. To evaluate the potential viral and inflammatory factors involved in the inductor of Tim-3 expression on T cells, 76 patients with chronic HBV infection (including 40 chronic hepatitis B [CHB] and 36 asymptomatic HBV carriers [AsC]) and 40 of normal controls (NCs) were enrolled in this study. Tim-3 expressions on CD4+ and CD8+ T cells were assessed in response to HBV-encoding antigens, HBV peptide pools, and common γ-chain (γc) cytokines stimulation by flow cytometry. HBV peptides and anti-CD3/CD28 directly induced Tim-3 expression on T cells. γc cytokines also drive Tim-3 up-regulations on both CD4+ and CD8+ T cells in patients with chronic HBV infection. However, γc cytokines did not enhance the Tim-3 inductions by either anti-CD3/CD28 or HBV peptides stimulation. Furthermore, γc cytokines-mediated Tim-3 induction could not be abrogated by γc cytokine receptor-neutralizing antibodies. The current results suggested that elevation of Tim-3 expression on T cells could be regulated by both antigen-dependent and -independent manner in patients with chronic HBV infection. The role of γc cytokines in modulation of inhibitory pathway might be evaluated as immunotherapies in humans.

T-bet-mediated Tim-3 expression dampens monocyte function during chronic hepatitis C virus infection.

Hepatitis C virus (HCV) induces a high rate of chronic infection via dysregulation of host immunity. We have previously shown that T-cell immunoglobulin and mucin domain protein-3 (Tim-3) is up-regulated on monocyte/macrophages (M/Mφ) during chronic HCV infection; little is known, however, about the transcription factor that controls its expression in these cells. In this study, we investigated the role of transcription factor, T-box expressed in T cells (T-bet), in Tim-3 expression in M/Mφ in the setting of HCV infection. We demonstrate that T-bet is constitutively expressed in resting CD14+ M/Mφ in the peripheral blood. M/Mφ from chronically HCV-infected individuals exhibit a significant increase in T-bet expression that positively correlates with an increased level of Tim-3 expression. Up-regulation of T-bet is also observed in CD14+ M/Mφ incubated with HCV+ Huh7.5 cells, as well as in primary M/Mφ or monocytic THP-1 cells exposed to HCV core protein in vitro, which is reversible by blocking HCV core/gC1qR interactions. Moreover, the HCV core-induced up-regulation of T-bet and Tim-3 expression in M/Mφ can be abrogated by incubating the cells with SP600125 - an inhibitor for the c-Jun N-terminal kinase (JNK) signalling pathway. Importantly, silencing T-bet gene expression decreases Tim-3 expression and enhances interleukin-12 secretion as well as signal transducer and activator of transcription 1 phosphorylation. These data suggest that T-bet, induced by the HCV core/gC1qR interaction, enhances Tim-3 expression via the JNK pathway, leading to dampened M/Mφ function during HCV infection. These findings reveal a novel mechanism for Tim-3 regulation via T-bet during HCV infection, providing new targets to combat this global epidemic viral disease.

Further characterization of ocular safety profile of commercially available preserved and preservative-free triamcinolone acetonide.

PURPOSE: To characterize the safety profile of triamcinolone acetonide (TA) for intraocular application. METHODS: In vitro cell viability assay was performed on 2 types of human ocular cells to evaluate the cytotoxicity of the simulated different vitreal concentrations (from a 1:15 dilution as if injected into 1.5 mL of rabbit vitreous to 1:50 dilution as if injected into 5 mL of human vitreous) of preservative and excipient (supernatant) from Kenalog-40. In vivo 35 guinea pigs were used for evaluating either a dose of intravitreal triamcinolone acetonide (Kenalog-40 and Triesence) or the supernatant of Kenalog-40. The animal eyes were monitored by biomicroscopy, ophthalmoscopy, tonometry, electroretinography, and histology. RESULTS: A ≥ 1:15 dilution of triamcinolone acetonide supernatant from Kenalog-40 did not show cytotoxicity on cultured human pigment epithelial (retinal pigment epithelium) cells or Müller cells. In vivo, neither intravitreal 6 µL (0.248 mg, equivalent to 4 mg in 0.1 mL for human eyes) nor 18 µL (0.744 mg, equivalent to 4 mg in 0.1 mL for rabbit eyes and equivalent to 12 mg in 0.1 mL for human eyes) of triamcinolone acetonide suspension showed ocular toxicity. No significant difference was noted between Kenalog-40 and Triesence clinically and histopathologically. CONCLUSION: The equivalent triamcinolone acetonide doses to 0.1 mL (4 or 12 mg) intravitreal injection for human eye were found safe in guinea pig eyes. No significant difference was noted for 0.1 mL intravitreal injection between Kenalog-40 and Triesence.

Epigenetics, microRNAs, and carcinogenesis: functional role of microRNA-137 in uveal melanoma.

PURPOSE: MicroRNAs (miRNAs) can contribute to tumorigenesis by acting as either oncogenes or tumor suppressor genes. The authors' previous studies on miR-34a showed that miRNA can influence the growth of uveal melanoma cells. In this study, they investigated the role of miR-137 in the pathogenesis of uveal melanoma. METHODS: Real-time RT-PCR was used to screen the expression levels of miR-137 in uveal melanocytes and uveal melanoma cell lines. Cell proliferation was examined by MTS assay and cell cycle was analyzed by flow cytometry. The target genes of miR-137 were predicted by bioinformatics and confirmed using a luciferase reporter assay. The expression of MITF, CDK6, and cell cycle regulatory proteins was determined by Western blot analysis. The ability to increase miR-137 expression by epigenetic drugs was tested using real-time RT-PCR. RESULTS: miR-137 expression was lower in uveal melanoma cell lines than in uveal melanocytes. Ectopic transfection of miR-137 into uveal melanoma cells induced G1 cell cycle arrest, leading to a significant decrease in cell growth. Overexpression of miR-137 downregulated MITF, a transcription factor with oncogenic activity. Moreover, the introduction of miR-137 downregulated the oncogenic tyrosine kinase protein receptor c-Met and cell cycle-related proteins, including CDK6. One avenue to increase the expression levels of miR-137 was through treatment with a DNA hypomethylating agent, 5-aza-2'-deoxycytidine, and a histone deacetylase inhibitor, trichostatin A. CONCLUSIONS: The results showed that miR-137 can act as a tumor suppressor in uveal melanoma cell proliferation through downregulation of the targets MITF and CDK6. miR-137 may be epigenetically silenced during uveal melanoma tumorigenesis.