RPL9 acts as an oncogene by shuttling miRNAs through exosomes in human hepatocellular carcinoma cells.

The exosomal pathway is an essential mechanism that regulates the abnormal content of microRNAs (miRNAs) in hepatocellular carcinoma (HCC). The directional transport of miRNAs requires the assistance of RNA­binding proteins (RBPs). The present study found that RBPs participate in the regulation of miRNA content through the exosomal pathway in HCC cells. First, differential protein expression profiles in the serum exosomes of patients with HCC and benign liver disease were detected using mass spectrometry. The results revealed that ribosomal protein L9 (RPL9) was highly expressed in serum exosomes of patients with HCC. In addition, the downregulation of RPL9 markedly suppressed the proliferation, migration and invasion of HCC cells and reduced the biological activity of HCC­derived exosomes. In addition, using miRNA microarrays, the changes in exosomal miRNA profiles in HCC cells caused by RPL9 knockdown were examined. miR­24­3p and miR­185­5p were most differentially expressed, as verified by reverse transcription­quantitative PCR. Additionally, using RNA immunoprecipitation, it was found that RPL9 was directly bound to the two miRNAs and immunofluorescence assays confirmed that RPL9 was able to carry miRNAs into recipient cells via exosomes. Overexpression of miR­24­3p in cells increased the accumulation of miR­24­3p in exosomes and simultaneously upregulated RPL9. Excessive expression of miR­24­3p in exosomes also increased their bioactivity. Exosome­mediated miRNA regulation and transfer require the involvement of RBPs. RPL9 functions as an oncogene, can directly bind to specific miRNAs and can be co­transported to receptor cells through exosomes, thereby exerting its biological functions. These findings provide a novel approach for modulating miRNA profiles in HCC.

Glycerol kinase enzyme is a prognostic predictor in esophageal carcinoma and is associated with immune cell infiltration.

The influence of lipid metabolism on tumorigenesis and progression has garnered significant attention. However, the role of Glycerol Kinase (GK), a key enzyme in glycerol metabolism, in Esophageal Carcinoma (ESCA) remains unclear. To further elucidate the relationship between GK and ESCA, we investigated GK expression levels using database information. Controlled studies employing immunohistochemistry were conducted on clinical ESCA tumor samples and normal specimens, confirming GK's elevated expression in ESCA. Analysis of The Cancer Genome Atlas (TCGA) data via Kaplan-Meier (KM) survival plots revealed that increased GK expression correlates with poorer ESCA patient outcomes, particularly in overall survival (OS) and disease-specific survival (DSS). Multiple regression analysis indicated that elevated GK expression is an independent risk factor affecting ESCA prognosis. Statistical analysis of prognostic data from clinical samples further corroborated this finding. Moreover, there appears to be a significant correlation between GK expression and immune infiltration, specifically involving certain T and B lymphocytes. In conclusion, elevated GK expression in ESCA is strongly linked to poor prognosis and increased immune cell infiltration, highlighting its potential as an independent prognostic biomarker and a viable therapeutic target.

Risk-based lung cancer screening in heavy smokers: a benefit-harm and cost-effectiveness modeling study.

BACKGROUND: Annual screening through low-dose computed tomography (LDCT) is recommended for heavy smokers. However, it is questionable whether all individuals require annual screening given the potential harms of LDCT screening. This study examines the benefit-harm and cost-effectiveness of risk-based screening in heavy smokers and determines the optimal risk threshold for screening and risk-stratified screening intervals. METHODS: We conducted a comparative cost-effectiveness analysis in China, using a cohort-based Markov model which simulated a lung cancer screening cohort of 19,146 heavy smokers aged 50 ~ 74 years old, who had a smoking history of at least 30 pack-years and were either current smokers or had quit for < 15 years. A total of 34 risk-based screening strategies, varying by different risk groups for screening eligibility and screening intervals (1-year, 2-year, 3-year, one-off, non-screening), were evaluated and were compared with annual screening for all heavy smokers (the status quo strategy). The analysis was undertaken from the health service perspective with a 30-year time horizon. The willingness-to-pay (WTP) threshold was adopted as three times the gross domestic product (GDP) of China in 2021 (CNY 242,928) per quality-adjusted life year (QALY) gained. RESULTS: Compared with the status quo strategy, nine risk-based screening strategies were found to be cost-effective, with two of them even resulting in cost-saving. The most cost-effective strategy was the risk-based approach of annual screening for individuals with a 5-year risk threshold of ≥ 1.70%, biennial screening for individuals with a 5-year risk threshold of 1.03 ~ 1.69%, and triennial screening for individuals with a 5-year risk threshold of < 1.03%. This strategy had the highest incremental net monetary benefit (iNMB) of CNY 1032. All risk-based screening strategies were more efficient than the status quo strategy, requiring 129 ~ 656 fewer screenings per lung cancer death avoided, and 0.5 ~ 28 fewer screenings per life-year gained. The cost-effectiveness of risk-based screening was further improved when individual adherence to screening improved and individuals quit smoking after being screened. CONCLUSIONS: Risk-based screening strategies are more efficient in reducing lung cancer deaths and gaining life years compared to the status quo strategy. Risk-stratified screening intervals can potentially balance long-term benefit-harm trade-offs and improve the cost-effectiveness of lung cancer screenings.

Spontaneous expulsion of a duodenal lipoma after endoscopic biopsy: A case report.

BACKGROUND: Gastrointestinal (GI) lipomas are benign submucosal tumors of mature adipocytes that arise mainly in the colon and stomach, sometimes in the ileum and jejunum, and rarely in the duodenum. Patients with symptomatic lipomas require endoscopic or surgical treatment. Spontaneous expulsion of lipomas after biopsy is a rare condition that has limited case reports. CASE SUMMARY: A 56-year-old man presented to our hospital with intermittent postprandial epigastric fullness. Esophagogastroduodenoscopy (EGD) revealed a 10-mm soft yellowish submucosal lesion with the "pillow sign," located in the second portion of duodenum. Endoscopic ultrasonography (EUS) using a 12-MHz catheter probe showed a hyperechoic, homogenous, and round solid lesion (OLYMPUS EUS EU-ME2, UM-DP12-25R, 12-MHz radial miniprobe, Olympus Corporation, Tokyo, Japan). Deep biopsy was performed using the bite-on-bite technique with forceps. Histological examination was compatible with submucosal lipoma. The lesion spontaneously expelled 12 d after the biopsy. Follow-up EUS performed after 2 mo confirmed this condition. CONCLUSION: Deep biopsy could lead to spontaneous GI lipoma expulsion. This might be the first step in lipoma diagnosis and treatment.

Simultaneous determination of vitamin D metabolites 25(OH)D3 and 1α,25(OH)2D3 in human plasma using liquid chromatography tandem mass spectrometry.

Background: Although measurement of 25(OH)D3 is a routine analytical method to determine plasma vitamin D status, 1α,25(OH)2D3 is the biologically active form. Hence, simultaneous measurement of 25(OH)D3 and 1α,25(OH)2D3 could provide better insight into vitamin D status and pharmacokinetics. However, 1α,25(OH)2D3 has a low plasma concentration, making its quantification challenging for most analytical techniques. Here, we demonstrate use of liquid chromatography tandem mass spectrometry (LC-MSMS) for the development of a simple and rapid method for the simultaneous quantification of 25(OH)D3 and 1α,25(OH)2D3. Methods: Samples were purified from 250 µL human plasma. Chromatography was performed on an analytical column, under gradient conditions using a mobile phase consisting of methanol-lithium acetate. The mass detector was operated in positive multiple reaction monitoring mode. The established method was validated according to the guidance issued by ICH and FDA. Furthermore, a clinical study was performed using this method to detect the plasma concentrations of 1α,25(OH)2D3 after oral administration of calcitriol. Results and conclusion: The method was acceptably linear over the concentration ranges of 20-1200 pg/mL for 1α,25(OH)2D3 and 1-60 ng/mL for 25(OH)D3, respectively, with correlation coefficients of r2 > 0.993. Both the inter-assay and intra-assay precision was < 15%, and the analytical recoveries were within 100% ± 10%, with no significant matrix effect or carryover. Thereby, we, provide a facile method for the simultaneous detection of vitamin D metabolites in plasma.

p62 Promotes Malignancy of Hepatocellular Carcinoma by Regulating the Secretion of Exosomes and the Localization of ß-Catenin.

BACKGROUND: p62 is a multi-domain protein and participates in a variety of cellular biological activities. p62 is also related to tumor malignancy. However, the underlying molecular mechanism of p62 regulating the progression of hepatocellular carcinoma (HCC) remains unclear. METHODS: The expression levels of p62 in HCC tissues and adjacent non-tumor tissues were confirmed using the TCGA dataset and immunohistochemistry. Stable p62-overexpressing HepG2 cells and p62-knockdown MHCC97H cells were established with lentiviral vectors. Cell proliferation, migration, and invasion assays were carried out to investigate the role of p62 in HCC cells and HCC-derived exosomes. The relationship between p62 and ß-catenin was investigated by immunofluorescence and co-immunoprecipitation assays. Male nude mice (BALB/c-nu/nu) were used to establish the xenograft tumors. RESULTS: We found that p62 was significantly upregulated in HCC, and a high level of p62 indicated the promotion of malignancy including cell proliferation, migration, and invasion. Exosomes derived from p62-overexpressing HepG2 also demonstrated the ability to promote tumor malignancy. Immunofluorescence and co-immunoprecipitation assays indicated that p62 interacts with ß-catenin and regulates the localization of ß-catenin to affect the intercellular junction. p62 also promotes tumor growth of HCC and down-regulates the expression of ß-catenin in vivo. CONCLUSIONS: The results of this study concluded that p62 promotes the malignancy of HCC by regulating the secretion of exosomes and the localization of ß-catenin. These findings may provide new ideas for the diagnosis and treatment of HCC.

Evaluation on two types of paramyosin vaccines for the control of Haemaphysalis longicornis infestations in rabbits.

BACKGROUND: Haemaphysalis longicornis is an obligate hematophagous ectoparasite that transmits a variety of pathogens causing life-threatening diseases in humans and animals. Paramyosin (Pmy) is not only an invertebrate-specific myofibrillar protein but also an important immunomodulatory protein. Therefore, it is one of the ideal candidate antigens for vaccines. METHODS: We conducted two vaccine trials to evaluate the protective efficacy of Pmy recombinant protein (rPmy) and peptide vaccine (KLH-LEE). Each rabbit was immunized with three doses of rPmy or KLH-LEE adjuvanted with Freund's complete/incomplete at 500 µg/dose at 2-week intervals before challenge with 40 female H. longicornis/rabbit. PBS plus adjuvant, Trx or KLH was used as control group. The antibodies of rabbits were detected by ELISA. Then, female ticks were fed on the rabbits until detachment. RESULTS: ELISA results showed that both vaccines induced rabbits to produce antibodies. Compared with the Trx group, the engorgement weight, oviposition and hatchability of the rPmy group decreased by 8.87%, 26.83% and 38.86%, respectively. On the other hand, engorgement weight, oviposition and hatchability of female ticks in the KLH-LEE group correspondingly resulted in 27.03%, 53.15% and 38.40% reduction compared with that of the KLH group. Considering the cumulative effect of vaccination on the evaluated parameters, results showed 60.37% efficacy of the rPmy vaccine formulation and 70.86% efficacy in the KLH-LEE group. CONCLUSIONS: Pmy and particularly epitope LEE have potential for further development of an effective candidate vaccine to protect the host against tick infection. GRAPHIC ABSTARCT.

Gene cloning, analysis and effect of a new lipocalin homologue from Haemaphysalis longicornis as a protective antigen for an anti-tick vaccine.

Haemaphysalis longicornis is distributed worldwide and transmits a variety of pathogens, causing human and animal disease. Use of chemical acaricides, as a primary tick control method, has several disadvantages, including acaricide resistance, environmental damage and residue accumulation in livestock. Development of a livestock vaccination aimed at a tick protective antigen could be an effective, labor-saving and environmentally-friendly method of reducing tick infestation and transmission of tick-borne pathogens. Lipocalins are low molecular weight proteins that play important roles in blood feeding, immune response and reproduction in ticks. In our study, the open reading frame (ORF) of a lipocalin homologue from H. longicornis (HlLIP) was successfully cloned, which consisted of 387 bp encoding a protein of 128 amino acids. The HlLIP protein sequence showed a close sequence homology with Ixodes persulcatus lipocalin. The HlLIP gene was constitutively detected in all developmental stages and in all tissues of the unfed female tick. The ORF of the HlLIP gene was sub-cloned into pET-32a (+) to obtain the recombinant protein (rHlLIP) and its immunogenicity was comfirmed by western blot. A vaccination trial on rabbits against H. longicornis infestation demonstrated that the rHlLIP protein could significantly prolong the period of tick blood feeding, and reduce tick engorged weight, oviposition and egg hatching rate. The vaccination efficacy of the rHlLIP protein was 60.17 % based on engorged weight, oviposition and egg hatching rate of ticks. The results obtained in this study demonstrate that rHlLIP protein is a promising antigen that could potentially be developed as a vaccine against H. longicornis infestation.

Angiopoietin-2 induces angiogenesis via exosomes in human hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common primary liver cancer and is a highly vascularized solid tumor. Angiopoietin-2 (ANGPT2) has been described as an attractive target for antiangiogenic therapy. Exosomes are small extracellular vesicles secreted by most cell types and contribute to cell-to-cell communication by delivering functional cargo to recipient cells. The expression of ANGPT2 in tumor-derived exosomes remains unknown. METHODS: We detected the ANGPT2 expression in HCC-derived exosomes by immunoblotting, enzyme-linked immunosorbent assay and immunogold labeling, then observed exosomal ANGPT2 internalization and recycling by confocal laser scanning microscopy, co-immunoprecipitation and immunoblotting. We used two HCC cell lines (Hep3B and MHCC97H) to overexpress ANGPT2 by lentivirus infection or knockdown ANGPT2 by the CRISPR/Cas system, then isolated exosomes to coculture with human umbilical vein endothelial cells (HUVECs) and observed the angiogenesis by Matrigel microtubule formation assay, transwell migration assay, wound healing assay, cell counting kit-8 assay, immunoblotting and in vivo tumorigenesis assay. RESULTS: We found that HCC-derived exosomes carried ANGPT2 and delivered it into HUVECs by exosome endocytosis, this delivery led to a notable increase in angiogenesis by a Tie2-independent pathway. Concomitantly, we observed that HCC cell-secreted exosomal ANGPT2 was recycled by recipient HUVECs and might be reused. In addition, the CRISPR-Cas systems to knock down ANGPT2 significantly inhibited the angiogenesis induced by HCC cell-secreted exosomal ANGPT2, and obviously suppressed the epithelial-mesenchymal transition activation in HCC. CONCLUSIONS: Taken together, these results reveal a novel pathway of tumor angiogenesis induced by HCC cell-secreted exosomal ANGPT2 that is different from the classic ANGPT2/Tie2 pathway. This way may be a potential therapeutic target for antiangiogenic therapy. Video Abstract.

Vps4A mediates the localization and exosome release of ß-catenin to inhibit epithelial-mesenchymal transition in hepatocellular carcinoma.

We previously reported that Vps4A acted as a tumor suppressor by influencing the microRNA profiles of exosomes and their parental cells in hepatocellular carcinoma (HCC). However, the underlying mechanism and if Vps4A contributes to sorting proteins into exosomes are not well known. Here, we performed mass spectrometry analysis of the immunoprecipitated Vps4A complex and confirmed that Vps4A was associated with ß-catenin and CHMP4B. Through this interaction, Vps4A promoted the plasma membrane (PM) localization and exosome release of ß-catenin. Silencing Vps4A or CHMP4B decreased the PM localization and exosome sorting of ß-catenin. Vps4A overexpression decreased ß-catenin signaling pathway and inhibited epithelial-mesenchymal transition (EMT) and motility of HCC cells. And, silencing Vps4A or CHMP4B promoted EMT in HCC. Furthermore, the expression of Vps4A was significantly related to that of several EMT markers in HCC tissues and the level of exosomal ß-catenin in patients with metastatic HCC was significantly lower compared to that of control patients. In conclusion, through the interaction with CHMP4B and ß-catenin, Vps4A regulates the PM localization and exosome sorting of ß-catenin, consequently decreases ß-catenin signaling, and thereby inhibits EMT and metastasis in HCC.

Medical expenditure for esophageal cancer in China: a 10-year multicenter retrospective survey (2002-2011).

BACKGROUND: Esophageal cancer is associated with substantial disease burden in China, and data on the economic burden are fundamental for setting priorities in cancer interventions. The medical expenditure for the diagnosis and treatment of esophageal cancer in China has not been fully quantified. This study aimed to examine the medical expenditure of Chinese patients with esophageal cancer and the associated trends. METHODS: From 2012 to 2014, a hospital-based multicenter retrospective survey was conducted in 37 hospitals in 13 provinces/municipalities across China as a part of the Cancer Screening Program of Urban China. For each esophageal cancer patient diagnosed between 2002 and 2011, clinical information and expense data were extracted by using structured questionnaires. All expense data were reported in Chinese Yuan (CNY; 1 CNY = 0.155 USD) based on the 2011 value and inflated using the year-specific health care consumer price index for China. RESULTS: A total of 14,967 esophageal cancer patients were included in the analysis. It was estimated that the overall average expenditure per patient was 38,666 CNY, and an average annual increase of 6.27% was observed from 2002 (25,111 CNY) to 2011 (46,124 CNY). The average expenditures were 34,460 CNY for stage I, 39,302 CNY for stage II, 40,353 CNY for stage III, and 37,432 CNY for stage IV diseases (P < 0.01). The expenditure also differed by the therapy type, which was 38,492 CNY for surgery, 27,933 CNY for radiotherapy, and 27,805 CNY for chemotherapy (P < 0.05). Drugs contributed to 45.02% of the overall expenditure. CONCLUSIONS: These conservative estimates suggested that medical expenditures for esophageal cancer in China substantially increased in the last 10 years, treatment for early-stage esophageal cancer costs less than that for advanced cases, and spending on drugs continued to account for a considerable proportion of the overall expenditure.

Effect of propofol on generation of inflammatory mediator of monocytes.

OBJECTIVE: To evaluate the effect of propofol with different concentrations on the expression of inflammatory mediators of interleukin and tumor-necrosis factor-α (TNF-α) by stimulating the mouse primary monocytes and human monocytic cell line with lipopolysaccharide (LPS) and also discuss the effect of propofol on the secretion of inflammatory mediator and its possible molecular mechanism. METHODS: The mononuclear cells of mouse spleen were separated and then purified to obtain the primary monocytes. The dose-effect relationship of production of pro-inflammatory cytokines by monocytes which were stimulated by LPS, namely the monocytes were stimulated by the dose of 0-500 ng/mL for 24 h. ELISA was employed to detect the concentration of IL-6, IL-8 and TNF-α. The effect of propofol on the secretion of above pro-inflammatory cytokines by the monocytes was observed. Cells were divided into the control group, the 0.1% DMSO group, the LPS group and the treatment group with LPS + different dose of propofol (propofol 1-100 µg/mL). ELISA was employed to detect the concentration of IL-6, IL-8 and TNF-α. The change in the expression of important signaling molecules in Toll-like receptor and NF-κB signaling pathway was detected after THP-1 cells were treated with propofol. RESULTS: The concentration of TNF-α was (3863 ± 153) pg/mL after 12 h of stimulation by LPS and then its concentration was decreased gradually. But the concentration of IL-6 and IL-8 was relatively high after 24 h of stimulation by LPS, (5627 ± 330) pg/mL and (1626 ± 200) pg/mL, respectively. The propofol could inhibit the expression of IL-6, IL-8 and TNF-α caused by LPS. After the intervention treatment of 50 µg/mL propofol, the concentration of IL-6, IL-8 and TNF-α was significantly decreased (P < 0.01). CONCLUSIONS: The propofol can inhibit the expression of TLR-4 and NF-κB to inhibit the activation of p38 and the expression of pro-inflammatory cytokines.

Vps4A functions as a tumor suppressor by regulating the secretion and uptake of exosomal microRNAs in human hepatoma cells.

UNLABELLED: The deregulation of microRNAs (miRNAs) plays an important role in human hepatocarcinogenesis. In this study, we highlight exosomes as mediators involved in modulating miRNA profiles in hepatocellular carcinoma (HCC) cells. First, we examined the different miRNA expression profiles in HCC cells and HCC cell-derived exosomes. Next, coculture experiments indicated that HCC cell-derived exosomes promoted the cell growth, migration, and invasion of HCC cells and had the ability to shuttle miRNAs to recipient cells. Further, our data showed that Vps4A, a key regulator of exosome biogenesis, was frequently down-regulated in HCC tissues. The reduction of Vps4A in HCC tissues was associated with tumor progression and metastasis. In vitro studies revealed that Vps4A repressed the growth, colony formation, migration, and invasion of HCC cells. We further investigated the role and involvement of Vps4A in suppressing the bioactivity of exosomes and characterized its ability to weaken the cell response to exosomes. By small RNA sequencing, we demonstrated that Vps4A facilitated the secretion of oncogenic miRNAs in exosomes as well as accumulation and uptake of tumor suppressor miRNAs in cells. A subset of Vps4A-associated miRNAs was identified. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the phosphatidylinositol-3-kinase/Akt signaling pathway was the most likely candidate pathway for modulation by these miRNAs. Indeed, we proved that the phosphatidylinositol-3-kinase/Akt pathway was inactivated by Vps4A overexpression. CONCLUSION: Exosome-mediated miRNA transfer is an important mechanism of self-modulation of the miRNA expression profiles in HCC cells, and Vps4A may function as a tumor suppressor, which utilizes exosomes as mediators to regulate the secretion and uptake of miRNAs in hepatoma cells; these observations provide new insights into the development of HCC.

Decision tree analysis of traditional risk factors of carotid atherosclerosis and a cutpoint-based prevention strategy.

BACKGROUND: Reducing the exposure to risk factors for the prevention of cardio-cerebral vascular disease is a crucial issue. Few reports have described practical interventions for preventing cardiovascular disease in different genders and age groups, particularly detailed and specific cutpoint-based prevention strategies. METHODS: We collected the health examination data of 5822 subjects between 20 and 80 years of age. The administration of medical questionnaires and physical examinations and the measurement of blood pressure, fasting plasma glucose (FPG) and blood lipids [total cholesterol (TC), triglycerides (TG), high density lipoprotein-cholesterol (HDL-C), and low density lipoprotein-cholesterol (LDL-C)] were performed by physicians. Carotid ultrasound was performed to examine the carotid intima-media thickness (CIMT), which was defined as carotid atherosclerosis when CIMT ≥0.9 mm. Decision tree analysis was used to screen for the most important risk factors for carotid atherosclerosis and to identify the relevant cutpoints. RESULTS: In the study population, the incidence of carotid atherosclerosis was 12.20% (men: 14.10%, women: 9.20%). The statistical analysis showed significant differences in carotid atherosclerosis incidence between different genders (P<0.0001) and age groups (P<0.001). The decision tree analysis showed that in men, the most important traditional risk factors for carotid atherosclerosis were TC (cutpoint [CP]: 6.31 mmol/L) between the ages of 20-40 and FPG (CP: 5.79 mmol/L) between the ages of 41-59. By comparison, LDL-C (CP: 4.27 mmol/L) became the major risk factor when FPG ≤5.79 mmol/L. FPG (CP: 5.52 mmol/L) and TG (CP: 1.51 mmol/L) were the most important traditional risk factors for women between 20-40 and 41-59 years of age, respectively. CONCLUSION: Traditional risk factors and relevant cutpoints were not identical in different genders and age groups. A specific gender and age group-based cutpoint strategy might contribute to preventing cardiovascular disease.

Functional distinction of rat liver natural killer cells from spleen natural killer cells under normal and acidic conditions in vitro.

BACKGROUND: The microenvironment within solid tumors has often been shown to exhibit an acidic extracellular pH. Although the morphologic and functional differences in natural killer (NK) cells of the liver and spleen have been reported previously under physiological conditions, the difference under acidic conditions is still unclear. This study was to investigate the differences in the morphological and functional characteristics between rat liver and spleen NK cells under normal and acidic conditions in vitro. METHODS: Liver and spleen NK cells were isolated and purified from Sprague-Dawley rats by density gradient centrifugation and the Dynabeads(®) FlowComp(TM) Flexi system, and stimulated for 4 days with or without IL-2 or treated with low pH or control for different times. Morphology was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cell death and proliferation assays were performed by flow cytometry, IFN-gamma production was tested by ELISA, and cytotoxic activity was evaluated by lactate dehydrogenase (LDH) release assay. RESULTS: Liver NK cells had significantly higher levels of cytotoxic activity than spleen NK cells under normal and acidic conditions, and the maximum difference was observed at pH 5.6. Further analysis revealed that the cytotoxic activity of NK cells was correlated with morphology, cell death, proliferative activity and IFN-gamma production. By TEM, liver NK cells contained a greater number of electron-dense granules per cell at pH 5.6. Moreover, a modest elevation of cell death and reduction of proliferation of liver NK cells occurred within a range of 5.6-7.2. Interestingly, an acidic extracellular pH only marginally, and not significantly, suppressed IFN-gamma production by liver NK cells. CONCLUSION: The sharp morphological and functional differences shown by the two types of NK cells in vitro indicate that liver NK cells are unexpectedly resistant to pH shock.

Anticancer drugs cause release of exosomes with heat shock proteins from human hepatocellular carcinoma cells that elicit effective natural killer cell antitumor responses in vitro.

Failure of immune surveillance related to inadequate host antitumor immune responses has been suggested as a possible cause of the high incidence of recurrence and poor overall survival outcome of hepatocellular carcinoma. The stress-induced heat shock proteins (HSPs) are known to act as endogenous "danger signals" that can improve tumor immunogenicity and induce natural killer (NK) cell responses. Exosome is a novel secretory pathway for HSPs. In our experiments, the immune regulatory effect of the HSP-bearing exosomes secreted by human hepatocellular carcinoma cells under stress conditions on NK cells was studied. ELISA results showed that the production of HSP60, HSP70, and HSP90 was up-regulated in both cell lines in a stress-specific manner. After exposure to hepatocellular carcinoma cell-resistant or sensitive anticancer drugs (hereafter referred to as "resistant" or "sensitive" anticancer drug), the membrane microvesicles were actively released by hepatocellular carcinoma cells, differing in their ability to present HSPs on the cell surface, which were characterized as exosomes. Acting as a decoy, the HSP-bearing exosomes efficiently stimulated NK cell cytotoxicity and granzyme B production, up-regulated the expression of inhibitory receptor CD94, and down-regulated the expression of activating receptors CD69, NKG2D, and NKp44. Notably, resistant anticancer drugs enhanced exosome release and generated more exosome-carried HSPs, which augmented the activation of the cytotoxic response. In summary, our findings demonstrated that exosomes derived from resistant anticancer drug-treated HepG2 cells conferred superior immunogenicity in inducing HSP-specific NK cell responses, which provided a clue for finding an efficient vaccine for hepatocellular carcinoma immunotherapy.

High incidence of biliary complications in rat liver transplantation: can we avoid it?

AIM: To investigate how to reduce the incidence of biliary complications in rat orthotopic liver transplantation. METHODS: A total of 165 male Wistar rats were randomly divided into three groups: Group A, orthotropic liver transplantation with modified "two-cuff" technique; Group B, bile duct was cut and reconstructed without transplantation; and Group C, only laparotomy was performed. Based on the approaches used for biliary reconstruction, Group A was divided into two sub-groups:A1 (n = 30), duct-duct reconstruction, and A2 (n = 30), duct-duodenum reconstruction. To study the influence of artery reconstruction on bile duct complication, Group B was divided into four sub-groups: B1 (n = 10), duct-duct reconstruction with hepatic artery ligation, B2 (n = 10), duct-duct reconstruction without hepatic artery ligation, B3 (n = 10), duct-duodenum reconstruction with hepatic artery ligation, and B4 (n = 10), duct-duodenum reconstruction without hepatic artery ligation. The samples were harvested 14 d after operation or at the time when significant biliary complication was found. RESULTS: In Group A, the anhepatic phase was 13.7 ± 1.06 min, and cold ischemia time was 50.5 ± 8.6 min. There was no significant difference between A1 and A2 in the operation duration. The time for biliary reconstruction was almost the same among all groups. The success rate for transplantation was 98.3% (59/60). Significant differences were found in the incidence of biliary complications in Groups A (41.7%), B (27.5%) and C (0%). A2 was more likely to have biliary complications than A1 (50% vs 33.3%). B3 had the highest incidence of biliary complications in Group B. CONCLUSION: Biliary complications are almost inevitable using the classical "two cuff" techniques, and duct-duodenum reconstruction is not an ideal option in rat orthotopic liver transplantation.

Donor liver natural killer cells alleviate liver allograft acute rejection in rats.

BACKGROUND: Liver enriched natural killer (NK) cells are of high immune activity. However, the function of donor liver NK cells in allogeneic liver transplantation (LTx) remains unclear. METHODS: Ten Gy of whole body gamma-irradiation (WBI) from a 60Co source at 0.6 Gy/min was used for depleting donor-derived leukocytes, and transfusion of purified liver NK cells isolated from the same type rat as donor (donor type liver NK cells, dtlNKs) through portal vein was performed immediately after grafting the irradiated liver. Post-transplant survival observation on recipients and histopathological detection of liver grafts were adoptive to evaluate the biological impact of donor liver NK cells on recipients' survival in rat LTx. RESULTS: Transfusion of dtlNKs did not shorten the survival time among the recipients of spontaneous tolerance model (BN to LEW rat) after rat LTx, but prolonged the liver graft survival among the recipients depleted of donor-derived leukocytes in the acute rejection model (LEW to BN rat). Compared to the recipients in the groups which received the graft depleted of donor-derived leukocytes, better survival and less damage in the allografts were also found among the recipients in the two different strain combinations of liver allograft due to transfusion of dtlNKs. CONCLUSIONS: Donor liver NK cells alone do not exacerbate liver allograft acute rejection. Conversely, they can alleviate it, and improve the recipients' survival.