The potential mechanism of treating IC/BPS with hyperbaric oxygen by reducing vascular endothelial growth inhibitor and hypoxia-inducible factor-1α.

Background/aim: To investigate the roles of vascular endothelial growth inhibitor (VEGI) and hypoxia-inducible factor-1α (HIF-1α) in the treatment of refractory interstitial cystitis/bladder pain syndrome (IC/BPS) with hyperbaric oxygen (HBO). Materials and methods: A total of 38 patients were included. They were assessed before and 6 months after HBO treatment. Three-day voiding diaries were recorded, and O'leary-Sant scores, visual analog scale (VAS) scores, quality of life (QoL) scores, pelvic pain, and urgency/frequency (PUF) scores were evaluated. Bladder capacity was assessed by cystoscopy. Bladder mucosa was collected for Western blot, qRT-PCR, and immunofluorescence staining to compare the expression of VEGI and HIF-1α before and after treatment. Results: Compared with before treatment, patients showed significant improvements in 24-h voiding frequency (15.32 ± 5.38 times), nocturia (3.71 ± 1.80 times), O'leary-Sant score (20.45 ± 5.62 points), VAS score (41.76 ± 17.88 points), QoL score (3.03 ± 1.44 points), and PUF score (19.95 ± 6.46 points) after treatment (p < 0.05). There was no significant difference in bladder capacity before and after treatment (p ≥ 0.05). The expression levels of VEGI and HIF-1α protein and mRNA were significantly decreased 6 months after treatment compared with before treatment. Immunofluorescence staining results showed that the double positive expression of VEGI and HIF-1α protein in bladder tissue of IC/BPS patients after HBO treatment quantitatively decreased significantly. Conclusion: This study identified a possible mechanism by which VEGI and HIF-1α expression decreased after HBO treatment due to hypoxia reversal, which improved symptoms in IC/BPS patients.

Clinical and pathological investigation of patients with severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory coronavirus 2 (SARS-CoV-2), has become a pandemic. This study addresses the clinical and immunopathological characteristics of severe COVID-19. METHODS: Sixty-nine patients with COVID-19 were classified into severe and nonsevere groups to analyze their clinical and laboratory characteristics. A panel of blood cytokines was quantified over time. Biopsy specimens from 2 deceased cases were obtained for immunopathological, ultrastructural, and in situ hybridization examinations. RESULTS: Circulating cytokines, including IL-8, IL-6, TNF-α, IP10, MCP1, and RANTES, were significantly elevated in patients with severe COVID-19. Dynamic IL-6 and IL-8 were associated with disease progression. SARS-CoV-2 was demonstrated to infect type II and type I pneumocytes and endothelial cells, leading to severe lung damage through cell pyroptosis and apoptosis. In severe cases, lymphopenia, neutrophilia, depletion of CD4+ and CD8+ T lymphocytes, and massive macrophage and neutrophil infiltrates were observed in both blood and lung tissues. CONCLUSIONS: A panel of circulating cytokines could be used to predict disease deterioration and inform clinical interventions. Severe pulmonary damage was predominantly attributed to both cytopathy caused by SARS-CoV-2 and immunopathologic damage. Strategies that prohibit pulmonary recruitment and overactivation of inflammatory cells by suppressing cytokine storm might improve the outcomes of patients with severe COVID-19.

SARS-CoV-2 infection of the liver directly contributes to hepatic impairment in patients with COVID-19.

Background & Aims: Liver enzyme abnormalities are common in patients with coronavirus disease 2019 (COVID-19). Whether or not severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can lead to liver damage per se remains unknown. Herein, we reported the clinical characteristics and liver pathological manifestations of COVID-19 patients with liver enzyme abnormalities. Methods: We analyzed 156 patients diagnosed with COVID-19 from 2 designated centers in China and compared clinical features between patients with or without elevated aminotransferases. Postmortem liver biopsies were obtained from 2 cases who had elevated aminotransferases. We investigated the patterns of liver impairment by electron microscopy, immunohistochemistry, TUNEL assay and pathological studies. Results: Sixty-four out of 156 (41.0%) patients with COVID-19 had elevated aminotransferases. The median levels of alanine aminotransferase were 50 U/L vs. 19 U/L, respectively, aspartate aminotransferase were 45.5 U/L vs. 24 U/L, respectively in abnormal and normal aminotransferase groups. Liver enzyme abnormalities were associated with disease severity, as well as a series of laboratory tests including higher alveolar-arterial oxygen partial pressure difference, higher gamma-glutamyltransferase, lower albumin, decreased CD4+ T cells and B lymphocytes. Ultrastructural examination identified typical coronavirus particles, characterized by spike structures, in the cytoplasm of hepatocytes in 2 COVID-19 cases. SARS-CoV-2-infected hepatocytes displayed conspicuous mitochondrial swelling, endoplasmic reticulum dilatation and glycogen granule decrease. Histologically, massive hepatic apoptosis and some binuclear hepatocytes were observed. Taken together, both ultrastructural and histological evidence indicated a typical lesion of viral infection. Immunohistochemical results showed scarce CD4+ and CD8+ lymphocytes. No obvious eosinophil infiltration, cholestasis, fibrin deposition, granuloma, massive central necrosis, or interface hepatitis were observed. Conclusions: SARS-CoV-2 infection in the liver directly contributes to hepatic impairment in patients with COVID-19. Hence, a surveillance of viral clearance in liver and long-term outcome of COVID-19 is required. Lay summary: Liver enzyme abnormalities are common in patients with coronavirus disease 2019 (COVID-19). We reported the clinical characteristics and liver pathological manifestations of COVID-19 patients with elevated liver enzymes. Our findings suggested that SARS-CoV-2 infection of the liver is a crucial factor contributing to hepatic impairment in patients with COVID-19.

Polypharmacology of Berberine Based on Multi-Target Binding Motifs.

Background: Polypharmacology is emerging as the next paradigm in drug discovery. However, considerable challenges still exist for polypharmacology modeling. In this study, we developed a rational design to identify highly potential targets (HPTs) for polypharmacological drugs, such as berberine. Methods and Results: All the proven co-crystal structures locate berberine in the active cavities of a redundancy of aromatic, aliphatic, and acidic residues. The side chains from residues provide hydrophobic and electronic interactions to aid in neutralization for the positive charge of berberine. Accordingly, we generated multi-target binding motifs (MBM) for berberine, and established a new mathematical model to identify HPTs based on MBM. Remarkably, the berberine MBM was embodied in 13 HPTs, including beta-secretase 1 (BACE1) and amyloid-ß1-42 (Aß1-42). Further study indicated that berberine acted as a high-affinity BACE1 inhibitor and prevented Aß1-42 aggregation to delay the pathological process of Alzheimer's disease. Conclusion: Here, we proposed a MBM-based drug-target space model to analyze the underlying mechanism of multi-target drugs against polypharmacological profiles, and demonstrated the role of berberine in Alzheimer's disease. This approach can be useful in derivation of rules, which will illuminate our understanding of drug action in diseases.

Expressions of programmed death (PD)-1 and PD-1 ligand (PD-L1) in cervical intraepithelial neoplasia and cervical squamous cell carcinomas are of prognostic value and associated with human papillomavirus status.

AIM: The programmed death 1/programmed death 1 ligand (PD-1/PD-L1) pathway can decrease the immune clearance effects of antigen-presenting cells and T lymphocytes to promote immune evasion of cervical cancer cells. However, the effects of this pathway on cervical intraepithelial neoplasia (CIN) progression and squamous cell carcinoma (SCC) metastasis are not clear. We herein investigated whether human papillomavirus infection could affect PD-1 and PD-L1 expression in CIN, and whether their expression is associated with CIN progression and SCC metastasis. METHODS: We collected paraffin-embedded samples from two cohorts of patients: (i) CIN samples from cohort I (40 women who tested positive or negative for high-risk human papillomavirus [HR-HPV] with grades 0, I, and II-III CIN); and (ii) paired primary and metastatic tumor samples from cohort II (20 SCC patients with or without metastasis). Immunohistochemistry was used to detect expressions of PD-L1 in tumor cells and PD-1 in tumor-associated macrophages and tumor-infiltrating lymphocytes. We also measured P16INK4a expression and interferon-γ levels in the cervical tissues. RESULTS: The most common HPV type seen in both cohorts of patients was HPV16, followed by HPV18. Increase in PD-L1 and PD-1 expression was positively correlated with HPV-positivity, increase in CIN grade, and tumor metastasis. Furthermore, upregulation of the PD-1/PD-L1 pathway was associated with decreased expression of the pro-inflammatory cytokine, interferon-γ and increased expression of P16INK4a . CONCLUSION: Expression of PD-L1 and PD-1 could be used as clinical prognostic biomarkers for evaluating CIN and cervical cancer because of its positive correlation with CIN progression and tumor metastasis.

Effects of cancer-testis antigen, TFDP3, on cell cycle regulation and its mechanism in L-02 and HepG2 cell lines in vitro.

TFDP3, also be known as HCA661, was one of the cancer-testis antigens, which only expressed in human tissues. The recent researches about TFDP3 mostly focused on its ability to control the drug resistance and apoptosis of tumor cells. However, the role of TFDP3 in the progress of the cell cycle is rarely involved. In this study, we examined the expression of TFDP3 in human liver tissues firstly. After that, we detect the expression of TFDP3 at the RNA level and protein level in L-02 cell line and HepG2 cell line, and the location of TFDP3 was defined by immunofluorescence technique. Furthermore, we synchronized the cells to G1 phase, S phase and G2 phase, and arrested cell mitosis. The localization of TFDP3 and co-localization with E2F1 molecules in different phases of hepatocyte lines. Finally, TFDP3 gene knockout was performed on L-02 and HepG2 cell lines, and detected the new cell cycles by flow cytometry. The result showed that the expression of TFDP3 molecule is negative in normal liver tissue, but positive in immortalized human hepatocyte cell line, and the expression level is lower than in hepatocellular carcinoma cell line. The expression level of TFDP3 was in the dynamic change of L-02 and HepG2 cell lines, and was related to the phase transition. TFDP3 can bind to E2F1 molecule to form E2F/TFDP3 complex; and the localizations of TFDP3 and E2F1 molecules and the co-localization were different in different phases of cell cycle in the nucleus and cytoplasm, which indicated that the E2F/TFDP3 complex involved in the process of regulating the cell cycle. By knocking down the TFDP3 expression level in L-02 and HepG2 cell lines, the cell cycle would be arrested in S phase, which confirmed that TFDP3 can be a potential target for tumor therapy.

Efficacy of real-time PCR-based detection of Helicobacter pylori infection and genotypic resistance-guided quadruple therapy as the first-line treatment for functional dyspepsia with Helicobacter pylori infection.

BACKGROUND: The eradication rate of Helicobacter pylori is steadily decreasing because of increasing resistance to clarithromycin. According to the new version of Maastricht IV guidelines, molecular tests can be performed as a substitute for bacterial culture and the standard clarithromycin susceptibility test for the detection of H. pylori and clarithromycin resistance directly on gastric biopsy samples. OBJECTIVE: To evaluate the clinical efficacy of H. pylori detection using a molecular test and treatment outcomes of the clarithromycin-based genotypic resistance test. MATERIALS AND METHODS: A total of 385 patients diagnosed with functional dyspepsia were recruited in this clinical trial. Total DNA was extracted from formalin-fixed paraffin-embedded samples and prepared for a molecular test and H. pylori detection was performed simultaneously by modified Giemsa staining. Genotypically sensitive patients with positive H. pylori were treated by quadruple therapy: bismuth potassium citrate, rabeprazole, amoxicillin, and clarithromycin (BRAC) and genotypically resistant individuals were treated by bismuth potassium citrate, rabeprazole, amoxicillin, and furazolidone (BRAF) twice daily for 7 consecutive days. The eradication rate of H. pylori was assessed using the C-urea breath test at 6 weeks after treatment. RESULTS: The prevalence of H. pylori infection in functional dyspepsia patients was 35.3% (136/385), 29.1% for women (53/182) and 40.9% for men (83/203). The sensitivities of real-time PCR and histological examinations were 95.6% (130/136) and 69.9% (95/136). Forty-one samples were found to be positive by real-time PCR alone and six by histological examination alone, the majority of which (32/41, 5/6) were identified as grade 1 multiplicity of infection. The overall resistance rate to clarithromycin was 37.7% (49/130): 37.3% (19/51) for women and 38.0% for men (30/79). Eighty-nine patients with positive H. pylori detected by both real-time PCR and histological examinations received quadruple therapies. For the intention-to-treat analysis, the eradication rates of BRAC and BRAF were 98% (52/53) and 92% (33/36), or 100% (52/52) and 94% (33/35) for per-protocol analysis. CONCLUSION: Real-time PCR is efficacious for H. pylori detection and genotypic resistance-guided quadruple therapy has a high efficacy in treating functional dyspepsia with H. pylori infection.

RIP1-dependent Bid cleavage mediates TNFα-induced but Caspase-3-independent cell death in L929 fibroblastoma cells.

L929 fibroblastoma cells (L929-A) and L929 fibrosarcoma cells (L929-N) are different cell lines that are commonly used to study the cytotoxicity of tumor necrosis factor alpha (TNFα). TNFα has been reported to induce necrosis in both of these cell lines. However, comparing the TNFα-induced cell death in these two cell lines, we found that, unlike the L929-N cells that show typical RIP3-dependent necrosis, TNFα-induced cell death in L929-A cells is pan-caspase inhibitor Z-VAD-FMK (Z-VAD)-sensitive, which does not depend on RIP3. We also confirmed that the cell death signal in the L929-A cells was initiated through cytosol-preassembled ripoptosome and that the knockdown of either Caspase-8 or RIP1 protein blocked cell death. Compared with the L929-N cells, the L929-A cell line had lower levels of constitutive and inducible TNFα autocrine production, and the pan-caspase inhibitors Z-VAD or Q-VD did not kill the L929-A cells as they affect the L929-N cells. Moreover, the L929-A cells expressed less RIP3 protein than the L929-N cells; therefore, TNFα failed to induce RIP3-dependent necroptosis. In addition, the ripoptosome-mediated cell death signal was transduced to the mitochondria through Caspase-8-mediated and RIP1 kinase activity-dependent Bid cleavage. The RIP1 kinase inhibitor Necrostatin-1 (Nec-1) or Caspase-8 knockdown completely blocked Bid cleavage, and the knockdown of Bid or Bax/Bak prevented TNFα-induced cell death in the L929-A cells. Although the activation of Bax/Bak decreased the mitochondrial membrane potential, the levels of mitochondrial intermembrane space proteins, including cytochrome-c (cyt-C) and Smac, declined, and western blotting and immunofluorescence staining analysis did not determine whether these proteins were redistributed to the cytosol. In addition, the mitochondrial outer membrane protein Tom20 was also reduced, indicating that the reduced mitochondria proteins may be induced by the reduced mitochondria numbers. No efficient cyt-C release was observed; therefore, the limited activation and cleavage of downstream caspases, including Caspase-9, Caspase-7, Caspase-6 and Caspase-3, was insufficient to kill the cells. The Caspase-9, Caspase-6 and Caspase-3/7 inhibitors or Caspase-9 and -3 knockdown also failed to block cell death, and the overexpression of Bcl-2 also did not abrogate cell death. Moreover, the dead cells showed necrotic-like but not apoptotic characteristics under transmission electronmicroscopy, and these features were significantly different from mitochondrial apoptosis, indicating that the effector caspases were not the executioners of cell death. These new discoveries show that TNFα-induced cell death in L929-A cells is different than typical RIP3-dependent necrosis and Caspase-8/Caspase-3-mediated apoptosis. These results highlight that caution is necessary when using different L929 cells as a model to investigate TNFα-induced cell death.

Adoptive transfer of bone marrow-derived dendritic cells decreases inhibitory and regulatory T-cell differentiation and improves survival in murine polymicrobial sepsis.

A decrease in the number of dendritic cells (DCs) is a major cause of post-sepsis immunosuppression and opportunistic infection and is closely associated with poor prognosis. Increasing the number of DCs to replenish their numbers post sepsis can improve the condition. This therapeutic approach could improve recovery after sepsis. Eighty C57BL/6 mice were subjected to sham or caecal ligation and puncture (CLP) surgery. Mice were divided into four groups: (i) Sham + vehicle, (ii) Sham + DC, (iii) CLP + vehicle, and (iv) CLP + DC. Bone-marrow-derived DCs (BMDCs) were administered at 6, 12 and 24 hr after surgery. After 3 days, we assessed serum indices of organ function (alanine aminotransferase, aspartate aminotransferase, creatinine, amylase and lipase), organ tissue histopathology (haematoxylin and eosin staining), cytokine [interferon-γ (IFN-γ), tumour necrosis factor-α, interleukin-12p70 (IL-12p70), IL-6 and IL-10] levels in the serum, programmed death-1 (PD-1) expression on T cells, regulatory T-cell differentiation in the spleen, and the survival rate (monitored for 7 days). BMDC transfer resulted in the following changes: a significant reduction in damage to the liver, kidney and pancreas in the CLP-septic mice as well as in the pathological changes seen in the liver, lung, small intestine and pancreas; significantly elevated levels of the T helper type 1 (Th1) cytokines IFN-γ and IL-12p70 in the serum; decreased levels of the Th2 cytokines IL-6 and IL-10 in the serum; reduced expression of PD-1 molecules on CD4(+) T cells; reduced the proliferation and differentiation of splenic suppressor T cells and CD4(+)  CD25(+)  Foxp3(+) regulatory T cells, and a significant increase in the survival rate of the septic animals. These results show that administration of BMDCs may have modulated the differentiation and immune function of T cells and contributed to alleviate immunosuppression, hence reducing organ damage and mortality post sepsis. Hence, the immunoregulatory effect of BMDC treatment has potential for the treatment of sepsis.

N-acetylcysteine administration is associated with reduced activation of NF-kB and preserves lung dendritic cells function in a zymosan-induced generalized inflammation model.

PURPOSE: In severe sepsis, functional impairment and decreased numbers of dendritic cells (DCs) are essential reasons for immune function paralysis, secondary organ infection, and organ failure. We investigated the effects of N-acetylcysteine (NAC) administration on protecting lung DCs function in a zymosan-induced generalized inflammation (ZIGI) model. METHODS: ZIGI was initiated in 80 Balb/c mice by intraperitoneal injection of zymosan (ZYM; 900 mg/kg). Mice were divided into 4 groups: (1) SHAM+Vehicle; (2) SHAM+NAC; (3) ZYM+Vehicle; and (4) ZYM+NAC. NAC (100 mg/kg) was administered at different time after ZYM injection. After 48 h, we assessed: lung tissue pathological changes; arterial blood gas values; purified lung DCs surface expressions of MHC-II/I-A(d) and co-stimulatory molecules CD80, CD83, and CD86; lung DCs mRNA levels of chemokine receptors CCR5 and CCR7; lung DCs apoptosis; lung DCs ultrastructure by transmission electron microscopy; lung DCs NF-kB transcription factor activity; and LPS-stimulated lung DCs in vitro production of IL-12 and IL-10 were examined. RESULTS: NAC treatment resulted in: significant improvements in ZYM-induced lung tissue damage and impaired lung function; inhibited lung DCs ZYM-induced increased expression of MHC-II/I-A(d), CD83, and CD86, but not CD80; reduced lung DCs ZYM-induced CCR5 and CCR7 mRNA levels; suppressed ZYM-induced lung DCs apoptosis; ameliorated ZYM-induced lung DCs ultrastructural abnormalities; inhibited ZYM-induced lung DCs NF-κB activity; and enhanced lung DCs production of IL-12 and inhibited their production of IL-10. CONCLUSIONS: Repeated injections of NAC during the early stage of severe sepsis effectively inhibited lung DCs activation and their apoptosis, which could preserve DCs function.