A pan-cancer single-cell panorama of human natural killer cells.

Natural killer (NK) cells play indispensable roles in innate immune responses against tumor progression. To depict their phenotypic and functional diversities in the tumor microenvironment, we perform integrative single-cell RNA sequencing analyses on NK cells from 716 patients with cancer, covering 24 cancer types. We observed heterogeneity in NK cell composition in a tumor-type-specific manner. Notably, we have identified a group of tumor-associated NK cells that are enriched in tumors, show impaired anti-tumor functions, and are associated with unfavorable prognosis and resistance to immunotherapy. Specific myeloid cell subpopulations, in particular LAMP3+ dendritic cells, appear to mediate the regulation of NK cell anti-tumor immunity. Our study provides insights into NK-cell-based cancer immunity and highlights potential clinical utilities of NK cell subsets as therapeutic targets.

Landscape and Dynamics of Single Immune Cells in Hepatocellular Carcinoma.

The immune microenvironment of hepatocellular carcinoma (HCC) is poorly characterized. Combining two single-cell RNA sequencing technologies, we produced transcriptomes of CD45+ immune cells for HCC patients from five immune-relevant sites: tumor, adjacent liver, hepatic lymph node (LN), blood, and ascites. A cluster of LAMP3+ dendritic cells (DCs) appeared to be the mature form of conventional DCs and possessed the potential to migrate from tumors to LNs. LAMP3+ DCs also expressed diverse immune-relevant ligands and exhibited potential to regulate multiple subtypes of lymphocytes. Of the macrophages in tumors that exhibited distinct transcriptional states, tumor-associated macrophages (TAMs) were associated with poor prognosis, and we established the inflammatory role of SLC40A1 and GPNMB in these cells. Further, myeloid and lymphoid cells in ascites were predominantly linked to tumor and blood origins, respectively. The dynamic properties of diverse CD45+ cell types revealed by this study add new dimensions to the immune landscape of HCC.

Landscape of Infiltrating T Cells in Liver Cancer Revealed by Single-Cell Sequencing.

Systematic interrogation of tumor-infiltrating lymphocytes is key to the development of immunotherapies and the prediction of their clinical responses in cancers. Here, we perform deep single-cell RNA sequencing on 5,063 single T cells isolated from peripheral blood, tumor, and adjacent normal tissues from six hepatocellular carcinoma patients. The transcriptional profiles of these individual cells, coupled with assembled T cell receptor (TCR) sequences, enable us to identify 11 T cell subsets based on their molecular and functional properties and delineate their developmental trajectory. Specific subsets such as exhausted CD8+ T cells and Tregs are preferentially enriched and potentially clonally expanded in hepatocellular carcinoma (HCC), and we identified signature genes for each subset. One of the genes, layilin, is upregulated on activated CD8+ T cells and Tregs and represses the CD8+ T cell functions in vitro. This compendium of transcriptome data provides valuable insights and a rich resource for understanding the immune landscape in cancers.

Abdominal infectious complications associated with the dislocation of intraperitoneal part of drainage tube and poor drainage after major surgeries.

Abdominal drainage, serving as a diagnostic and therapeutic tool, has been widely applied to prevent complications after major abdominal surgical procedures. However, dislocation of intraperitoneal portion of drainage tube and poor drainage after major surgery has never been detailed. In this retrospective study, we determined whether postoperative abdominal infectious complications are attributed to dislocation of intraperitoneal portion of drainage tube. Patients were recruited from the Department of General Surgery at Beijing Shijitan Hospital, Capital Medical University, between June 2015 and June 2018. All of the enrolled patients had undergone different major abdominal surgical procedures with abdominal drainage. According to different fixation methods of the drainage tube, the patients were categorised as follows: group 1 as conventional extra-abdominal fixation where the tubes were fixed on abdominal wall; group 2 as double fixation where the tubes were fixed by both extra-abdominal and intra-abdominal fixation. Among 60 patients (40 in group 1 and 20 in group 2) with suspected postoperative abdominal infection, abdominal computed tomography (CT) was performed to determine the presence of abnormality. Dislocation of drainage tubes, morbidity, treatment, and prognosis were compared between the two groups. None of the patients showed slip knot or drainage tube slipping from the abdomen based on physical examination and CT imaging. Drainage tube was fixed firmly on the abdominal wall. In group 1, 18 (45%) patients developed postoperative complications resulting from abdominal infection where severe dislocation of intraperitoneal portion of drainage tubes was confirmed by CT. Drainage tubes of six cases were significantly dislocated to the anterior abdominal wall from the target area; 7 upper abdominal drainage tubes dislocated to the lower abdomen; and 5 lower abdominal drainage tubes dislocated to the upper abdomen. Common complications included localised peritonitis (n = 4), abdominal abscess (n = 8), and anastomotic leakage (n = 6). Among them, 8 patients were cured by abdominal puncture catheter drainage; 5 underwent secondary operation and 5 were cured by conservative treatment. In group 2, no tube dislocation was identified by CT. Five patients (25%) developed complications, including localised peritonitis (n = 1), abdominal abscess (n = 1), and anastomotic leakage (n = 3). All the five patients were cured by conservative treatment. Postoperative abdominal infection complications can stem from dislocation of intraperitoneal portion of drainage tube and poor drainage after major abdominal surgery. Maintaining the intraperitoneal portion of drainage tube at the proper location, for example, by applying intraabdominal fixation, is paramount to decrease the incidence and severity of postoperative complications.

HDM2 Promotes NEDDylation of Hepatitis B Virus HBx To Enhance Its Stability and Function.

Hepatitis B virus (HBV)-encoded X protein (HBx) plays a critical role in HBV-related hepatocarcinoma development. In this study, we demonstrate that HBx is specifically modified by NEDD8. We found that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability by preventing its ubiquitination-mediated degradation. Consistently, analysis of 160 hepatocellular carcinoma patient specimens indicated that the amount of HDM2 protein correlates with HBx protein level. We identified that HBx K91 and K95 as the key HBx NEDDylation sites and observed that the NEDDylation-deficient HBx has shorter half-life. We generated Huh7 cell lines which ectopically express wild-type and NEDDylation-deficient HBx and found that NEDDylation-deficient HBx showed less chromatin localization and less DDB1 binding. Consistently, the expression of HBx-regulated genes (IL-8, MMP9, and YAP) and HBV transcription (the activity of HBV enhancer and the amount of pgRNA transcribed from cccDNA) were significantly higher in cells expressing wild-type (WT) HBx than that in cells expressing mutant HBx. In addition, HBx-expressing cells proliferated faster than control and mutant HBx-expressing cells. We also showed that the ability of WT HBx-expressing cells to form tumors in nude mice was significantly higher than that of mutant HBx-expressing cells. In conclusion, we revealed that E3 ligase HDM2 promotes NEDDylation of HBx to enhance HBx stability and chromatin localization, which in turn favors HBx-dependent transcriptional regulation, cell proliferation, and HBV-driven tumor growth.IMPORTANCE Hepatitis B virus (HBV) HBx protein plays a critical role in viral replication and hepatocarcinogenesis. However, the regulation of HBx stability is not well understood. We found that HBx is modified by NEDD8 and that the HDM2 E3 ligase promotes HBx NEDDylation to enhance HBx stability by inhibiting its ubiquitination. We provide a new evidence to show the positive correlation between HDM2 and HBx in clinical hepatocellular carcinoma (HCC) samples. We also identified the major NEDDylation sites on HBx. Our studies indicate that the defective NEDDylation of HBx negatively affects its ability to activate the transcription of downstream genes and promote cell proliferation and tumor growth in vivo Taken together, our findings reveal a novel posttranslational modification of HBx by HDM2 which regulates its stability, subcellular localization, and functions. These findings indicate that HDM2 is an important regulator on HBx and a potential diagnosis/therapeutic marker for HBV-associated HCC.

Programmed Death Receptor 1 (PD1) Knockout and Human Telomerase Reverse Transcriptase (hTERT) Transduction Can Enhance Persistence and Antitumor Efficacy of Cytokine-Induced Killer Cells Against Hepatocellular Carcinoma.

BACKGROUND The weak antitumor efficacy and limited lifespan are the main obstacles that hinder the therapeutic effect of cytokine-induced killer (CIK) cell immunotherapy. In the study, we enhanced the persistence and the antitumor efficacy of CIK cell through PD-1 knockout and hTERT transduction. MATERIAL AND METHODS CIK cells were cultured from patients with hepatocellular carcinoma and PD-1 gene was knocked out through the Cas9 ribonucleoproteins (Cas9 RNPs) electroporation. TIDE assay, T7E1 mismatch cleavage assay, and clone Sanger sequencing were used to detect PD-1 knockout efficiency. The immunophenotype was analyzed by flow cytometry. After PD-1 knockout, the hTERT gene was transduced into PD-1 KO/CIK cells with lentiviral transduction. The hTERT expression and persistence of hTERT/PD-1 KO/CIK cells were evaluated by Western blotting and proliferation curve. The antitumor efficacy was detected by ELISPOT and cytotoxicity assay. The telomere length was measured by the Q-FISH and qPCR method. The karyotype assay was used to analyze the chromosome structural stability. RESULTS The optimal knockout efficiency of PD-1 gene in CIK cells could reach 41.23±0.52%. PD-1 knockout did not affect the immunophenotype of CIK cells. The hTERT transduction enhanced persistence and increased the telomere length. ELISPOT and cytotoxicity assay showed hTERT/PD-1 KO/CIK cells had an enhanced antitumor efficacy. Meanwhile, PD-1 KO/CIK cells transduced with hTERT showed a normal karyotype. CONCLUSIONS PD-1 knockout combined with hTERT transduction could prolong the lifespan and enhance antitumor efficacy of CIK cells against hepatocellular carcinoma cell line.

Gastric bypass procedure for type 2 diabetes patients with BMI <28 kg/m2.

OBJECTIVES: To evaluate the Roux-en-Y gastric bypass (GBP) procedure for patients suffering from type 2 diabetes mellitus (T2DM) with body mass index (BMI) <28 kg/m2. METHODS: Thirty-one patients suffering from T2DM were selected to undergo laparoscopic Roux-en-Y gastric bypass surgery and were enrolled at Beijing Shijitan Hospital between November 2012 and December 2014. The fasting plasma glucose (FPG), glycosylated hemoglobin (HbA1c), C-peptide, fasting insulin (FINS) and glucagon-like peptide-1 (GLP-1) of all patients were measured before and at 1, 3, 6 months after surgery. The results were compared and analyzed. RESULTS: Thirty-one patients suffering from T2DM successfully underwent GBP surgery (a mean age of 46 years), 14 were male and 17 were female. Among them, 7 patients had hypertriglyceridemia (HTG). The patients were followed up for 6 months. No major complications were found. The average BMI was 26.5 ± 1.4 kg/m2 before surgery. The average levels of FPG, HbA1c, C-peptide, FINS of all patients were statistically decreased after surgery, respectively, compared to those before surgery (P < 0.05). However, the mean GLP-1 of all patients was statistically increased after surgery compared to that before surgery (P < 0.05). At 6 months after surgery, 22 patients (71 %) achieved complete remission of T2DM with HbA1c < 6.5 %, 7 patients (23 %) gained partial remission of T2DM with 6.5 % ≤ HbA1c < 7.0 % and 2 patients (6 %) experienced no remission of T2DM. The mean serum triglyceride of 31 patients was statistically decreased after surgery compared to that before surgery (P < 0.05). CONCLUSIONS: This research shows that the GBP procedure is safe and effective for T2DM patients with BMI <28 kg/m2, and the condition of patients with HTG was greatly improved. However, further studies with larger samples and long-term follow-up are needed.

Hepatitis B virus inhibits apoptosis of hepatoma cells by sponging the MicroRNA 15a/16 cluster.

Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. In this study, we found that HBV inhibited the chemotherapy drug etoposide-induced apoptosis of hepatoma cells. Further analysis revealed that HBV mRNAs possess a microRNA 15a/16 (miR-15a/16)-complementary site (HBV nucleotides [nt] 1362 to 1383) that acts as a sponge to bind and sequester endogenous miR-15a/16. Consequently, Bcl-2, known as the target of miR-15a/16, was upregulated in HBV-infected cells. The data from HBV-transgenic mice further confirmed that HBV transcripts cause the reduction of miR-15a/16 and increase of Bcl-2. More importantly, we examined the levels of HBV transcripts and miR-15a/16 in HBV-infected HCC from patients and found that the amount of HBV mRNA and the level of miR-15a/16 were negatively correlated. Consistently, the level of Bcl-2 mRNA was upregulated in HBV-infected patients. In conclusion, we identified a novel HBV mRNA-miR-15a/16-Bcl-2 regulatory pathway that is involved in inhibiting etoposide-induced apoptosis of hepatoma cells, which may contribute to facilitating chronic HBV infection and hepatoma development.

Caffeic acid phenethyl ester derivative exerts remarkable anti-hepatocellular carcinoma effect, non-inferior to sorafenib, in vivo analysis.

Caffeic acid phenethyl ester (CAPE) and its derivatives exhibit considerable effects against hepatocellular carcinoma (HCC), with unquestioned safety. Here we investigated CAPE derivative 1' (CAPE 1') monotherapy to HCC, compared with sorafenib. HCC Bel-7402 cells were treated with CAPE 1', the IC50 was detected using CCK-8 analysis, and acute toxicity testing (5 g/kg) was performed to evaluate safety. In vivo, tumor growth after CAPE 1' treatment was evaluated using an subcutaneous tumor xenograft model. Five groups were examined, with group 1 given vehicle solution, groups 2, 3, and 4 given CAPE 1' (20, 50, and 100 mg/kg/day, respectively), and group 5 given sorafenib (30 mg/kg/day). Tumor volume growth and tumor volume-to-weight ratio were calculated and statistically analyzed. An estimated IC50 was 5.6 µM. Acute toxicity tests revealed no animal death or visible adverse effects with dosage up to 5 g/kg. Compared to negative controls, CAPE 1' treatment led to significantly slower increases of tumor volume and tumor volume-to-weight. CAPE 1' and sorafenib exerted similar inhibitory effects on HCC tumors. CAPE 1' was non-inferior to sorafenib for HCC treatment, both in vitro and in vivo. It has great potential as a promising drug for HCC, based on effectiveness and safety profile.

Multi-omics analysis reveals the landscape of tumor microenvironments in left-sided and right-sided colon cancer.

Background: Distinct clinical features and molecular characteristics of left-sided colon cancer (LCC) and right-sided colon cancer (RCC) suggest significant variations in their tumor microenvironments (TME). These differences can impact the efficacy of immunotherapy, making it essential to investigate and understand these disparities. Methods: We conducted a multi-omics analysis, including bulk RNA sequencing (bulk RNA-seq), single-cell RNA sequencing (scRNA-seq), and whole-exome sequencing (WES), to investigate the constituents and characteristic differences of the tumor microenvironment (TME) in left-sided colon cancer (LCC) and right-sided colon cancer (RCC). Result: Deconvolution algorithms revealed significant differences in infiltrated immune cells between left-sided colon cancer (LCC) and right-sided colon cancer (RCC), including dendritic cells, neutrophils, natural killer (NK) cells, CD4 and CD8 T cells, and M1 macrophages (P < 0.05). Notably, whole-exome sequencing (WES) data analysis showed a significantly higher mutation frequency in RCC compared to LCC (82,187/162 versus 18,726/115, P < 0.01). Single-cell analysis identified predominant tumor cell subclusters in RCC characterized by heightened proliferative potential and increased expression of major histocompatibility complex class I molecules. However, the main CD8 + T cell subpopulations in RCC exhibited a highly differentiated state, marked by T cell exhaustion and recent activation, defined as tumor-specific cytotoxic T lymphocytes (CTLs). Immunofluorescence and flow cytometry results confirmed this trend. Additionally, intercellular communication analysis demonstrated a greater quantity and intensity of interactions between tumor-specific CTLs and tumor cells in RCC. Conclusion: RCC patients with an abundance of tumor-specific cytotoxic T lymphocytes (CTLs) and increased immunogenicity of tumor cells in the TME may be better candidates for immune checkpoint inhibitor therapy.

Cross-tissue human fibroblast atlas reveals myofibroblast subtypes with distinct roles in immune modulation.

Fibroblasts, known for their functional diversity, play crucial roles in inflammation and cancer. In this study, we conduct comprehensive single-cell RNA sequencing analyses on fibroblast cells from 517 human samples, spanning 11 tissue types and diverse pathological states. We identify distinct fibroblast subpopulations with universal and tissue-specific characteristics. Pathological conditions lead to significant shifts in fibroblast compositions, including the expansion of immune-modulating fibroblasts during inflammation and tissue-remodeling myofibroblasts in cancer. Within the myofibroblast category, we identify four transcriptionally distinct subpopulations originating from different developmental origins, with LRRC15+ myofibroblasts displaying terminally differentiated features. Both LRRC15+ and MMP1+ myofibroblasts demonstrate pro-tumor potential that contribute to the immune-excluded and immune-suppressive tumor microenvironments (TMEs), whereas PI16+ fibroblasts show potential anti-tumor functions in adjacent non-cancerous regions. Fibroblast-subtype compositions define patient subtypes with distinct clinical outcomes. This study advances our understanding of fibroblast biology and suggests potential therapeutic strategies for targeting specific fibroblast subsets in cancer treatment.

Main Diagnostic Criteria Usually Does Not Work for Autoimmune Pancreatitis Wrongly Presuming Malignancy.

Background: Autoimmune pancreatitis (AIP) usually responds dramatically to steroid therapy. Occasionally, however, misdiagnosed patients have undergone pancreaticoduodenectomy. This study is aimed at providing useful information to improve the accuracy of diagnosis before surgery and thus avoid unnecessary resections in patients with AIP. Methods: From January 2015 to February 2020, a series of patients were enrolled, having undergone pancreaticoduodenectomy for presumed malignancy. AIP diagnoses were confirmed by postoperative pathology. The demographic and clinical data of the AIP patients were evaluated. The main diagnostic criteria (HISORt, Asian, and ICDC) for AIP were applied to assess whether and how unnecessary surgery could have been avoided. Results: A total of 124 cases of pancreaticoduodenectomy were performed for presumed malignancy. Six patients were diagnosed with benign disease and five with AIP. The prevalences of benign disease and AIP were 4.8% and 4%, respectively. Four patients were female and 1 male, with a mean age of 60.0 years old. Jaundice, pain, and weight loss were observed in 100%, 20%, and 40% of AIP patients, respectively. The radiologic features of the AIP patients were a diffusely enlarged gland (40.0%), a focally enlarged gland (40.0%), pancreatic ductal dilatation (60.0%), upstream parenchymal atrophy (20.0%), bile duct thickening (66.0%), and bile duct stricture (40.0%). Based on the diagnostic criteria for AIP, surgery could have been avoided in two cases. Conclusions: IgG4 measurement and integrated use of major diagnostic criteria should be emphasized in every patient eligible for pancreaticoduodenectomies.

Risk factors of secondary infection/recurrence after ablation for liver cancers: A systemic review and meta-analysis.

Background: This study aimed to systemically explore the risk factors of secondary infection/recurrence after ablation in patients with liver cancer. Methods: Relevant literature in PubMed, EMbase, and Cochrane Library databases were searched with keywords including "liver cancer or carcinoma," "ablation," "infectious or infection or recurrence," and "risk factor or relevant factor or correlative factor or influencing factor." Meta-analyses were performed and forest plots were drawn for risk factors, including the tumor size and location, number of tumor nodules, hepatitis B virus (HBV) DNA levels, serum alpha fetal protein (AFP) levels and serum albumin levels, Child-Pugh Class, and lack of antiviral therapy. A funnel plot was drawn to assess the publication bias. Results: A total of 23 studies were included from the initial 701 potentially relevant articles. Our meta-analyses showed that a large tumor size (odds ratio [OR] = 1.58; 95% confidence interval [CI]: 1.31-1.92); proximity to the colon, large vessels, and large hepatic vein (OR = 4.10; 95% CI: 2.26-7.43); multinodular tumor (OR = 2.10; 95% CI: 1.46-3.03), the higher HBV DNA levels (OR = 1.34; 95% CI: 1.09-0.64); higher serum AFP levels (OR = 1.56; 95% CI: 1.18-2.05), lower serum albumin levels (OR = 1.67; 95% CI: 1.06-2.65); Child-Pugh Class B and Class C (OR = 1.27; 95% CI: 1.05-1.54); and lack of antiviral therapy (OR = 1.75; 95% CI: 0.93-3.28) were associated with an increased risk of post-ablation infection/recurrence in patients with liver cancers. Conclusion: Our results indicated that the tumor size and location, number of tumor nodules, HBV DNA levels, serum AFP levels and serum albumin levels, Child-Pugh Class, and lack of antiviral therapy were the risk factors for post-ablation infection/recurrence in patients with liver cancer. Here, we have provided directions for the clinical prevention of secondary infection/recurrence in patients with liver cancer who underwent ablation therapy.

Immune phenotypic linkage between colorectal cancer and liver metastasis.

The tumor microenvironment (TME) is connected to immunotherapy responses, but it remains unclear how cancer cells and host tissues differentially influence the immune composition within TME. Here, we performed single-cell analyses for autologous samples from liver metastasized colorectal cancer to disentangle factors shaping TME. By aligning CD45+ cells across different tissues, we classified exhausted CD8+ T cells (Texs) and activated regulatory T cells as M-type, whose phenotypes were associated with the malignancy, while natural killer and mucosal-associated invariant T cells were defined as N-type, whose phenotypes were associated with the niche. T cell receptor sharing between Texs in primary and metastatic tumors implicated the presence of common peripheral non-exhausted precursors. For myeloid cells, a subset of dendritic cells (DC3s) and SPP1+ macrophages were M-type, and the latter were predominant in liver metastasis, indicating its pro-metastasis role. Our analyses bridge immune phenotypes of primary and metastatic tumors, thereby helping to understand the tumor-specific contexture and identify the pro-metastasis components.

Dual selective sensor for exosomes in serum using magnetic imprinted polymer isolation sandwiched with aptamer/graphene oxide based FRET fluorescent ignition.

The selective and sensitive detection of cancerous exosomes in serum is critical for early disease diagnosis and improved prognosis. Previous exosome-related research has been limited by a lack of well-understanding in exosomes as well as the challenging background interference of body fluid. Molecularly imprinted polymers (MIPs) and nucleic acid aptamers can be regarded as the two alternatives to antibodies. When using imprinted polymer technology, comprehensive and precise information about the target constituents is not required. In this study, a novel kind of dual selective fluorescent nanosensor for the poorly characterized exosomes was constructed by integrating magnetic MIP selective exosome capture sandwiched with an aptamer/graphene oxide fluorescence resonance energy transfer system (FRET) based selective 'turn-on' exosome labeling heterogeneously. The overall strategy performance was successively evaluated using lysozyme and exosomes as targets. Good linearity and high sensitivity achieved were demonstrated. The LOD of exosomal detection in serum was 2.43 × 106 particles/mL, lower than other immunology based detection methods. The discrimination between serum from breast cancer patients and healthy people was also primarily studied. In conclusion, the developed sensor with outstanding selectivity, high detection sensitivity, simplicity, low cost, and wide applicability for known or unknown targets present significant potential in challenging clinical diagnosis.

Identification of promiscuous HLA-DR-restricted CD4⁺ T-cell epitopes on the cancer-testis antigen HCA587.

The cancer testis antigen HCA587 is an attractive candidate for T cell-based immunotherapy because it is overexpressed in a wide spectrum of malignant tumors but not normal tissues, except testis. Several CTL epitopes derived from HCA587 have been described. Our aim was to identify helper T lymphocyte epitopes of HCA587 for the optimization of T cell-based immunotherapies against HCA587-expressing tumors. Candidate helper T lymphocyte epitopes for HCA587 were predicted using the SYFPEITHI algorithm and were tested for their ability to induce helper T lymphocyte responses by in vitro peptide vaccination of CD4(+) T lymphocytes from healthy individuals and hepatocellular carcinoma patients. Four CD4(+) T-cell epitopes for HCA587 (p43-57, p145-159, p186-200 and p249-263) were identified. Among them, the p43-57 epitope was shown to be naturally processed and presented by HCA587-expressing tumor cells as well as autologous dendritic cells pulsed with whole-protein HCA587. Notably, this epitope behaved as a promiscuous T-cell epitope as it stimulated T cells in the context of more than one HLA class II allele. Thus, p43-57 is the first HCA587-derived major histocompatibility complex class II-restricted epitope to fulfil all prerequisites for use as a peptide vaccine in patients with HCA587-expressing tumors.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro.

Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

Simultaneous measurements of serum AFP, GPC-3 and HCCR for diagnosing hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a prevalent malignant tumor. Tumor markers are very useful in early diagnosis; however a single marker is rather limited. We launched a test to increase the diagnostic sensitivity through the combined detection. METHODOLOGY: Serum concentration of three tumor-markers, Glypican-3 (GPC-3), Human-Cervical-Cancer-Oncogene (HCCR) and a-fetoprotein (AFP), were determined in 189 samples: 101 cases of HCC, 40 cases of cirrhosis, 18 cases of hepatitis and 30 cases of control healthy donors. Every marker was evaluated for its diagnostic value by one-way-analysis-of-variance and receiver-operating-characteristics analysis. RESULTS: GPC-3 was the best marker with an area under the curve (AUC) of 0.892; using 26.8ng/mL as the cut-off for HCC diagnosis, GPC-3 has a sensitivity of 51.5% and maintains a specificity of 92.8%. HCCR, with an AUC of 0.831, can reach a sensitivity of 22.8% and maintain a specificity of 90.9% if the cut-off is set as 58.8mAU/mL. With an AUC of 0.827, the efficacy and sensitivity of AFP were 36.6% and 98.5% when using 199.3ng/mL as the cut-off. No significant correlation was found between these three markers. Simultaneously detecting three markers can significantly increases the sensitivity to 80.2%, much higher than AFP alone. CONCLUSIONS: GPC-3 and HCCR are useful tumor markers complementary to AFP for clinical diagnosis of HCC.

In vitro and in vivo studies of biodegradable Zn-Li-Mn alloy staples designed for gastrointestinal anastomosis.

Zn-0.8 wt.% Li-0.1 wt.% Mn wire with the diameter of 0.3 mm was fabricated and further processed into gastrointestinal staple, and its in vitro and in vivo biodegradation behavior and biocompatibility were studied systematically. The experimental Zn-Li-Mn alloy staple could deform from the original U-shape to B-shape without fracture, indicating its good mechanical property. Due to the residual stress concentration caused by anastomosis deformation, the feet and leg arc part of the staple were more prone to degradation. The Zn-Li-Mn alloy staple sustained integrity after immersion in Hanks' solution and simulated gastric fluid (SGF) for 28 days, and the degradation rate in SGF was about 4 times of that in Hanks' solution. Furthermore, Zn-Li-Mn alloy staples were utilized for gastrointestinal anastomosis in pig models, with clinically-used titanium alloy staples as a comparison. No anastomotic leakage and severe inflammation were observed after operation. The Zn-Li-Mn alloy staple maintained mechanical integrity within 8 weeks' implantation. The gastrointestinal tissue healed after 12 weeks, and no obvious side effects were detected during the whole implantation period, demonstrating the good biocompatibility of Zn-Li-Mn alloy staple. Thus, Zn-Li-Mn alloy staple fabricated in this work displayed the promising potential in the gastrointestinal anastomosis.

DNASE1L3 as a Prognostic Biomarker Associated with Immune Cell Infiltration in Cancer.

OBJECTIVES: Deoxyribonuclease 1 like 3 (DNASE1L3) is critically involved in apoptosis and immune response, however, its role in cancer has yet to be deciphered. We aimed to explore the prognostic value of DNASE1L3 across a series of malignancies. METHODS: Based on Oncomine database and Tumor Immune Estimation Resource (TIMER), expression profiling of DNASE1L3 was detailed in malignancies. Using PrognoScan, Kaplan-Meier Plotter, GEPIA2, and bc-GenEcMiner v4.5, prognostic value of DNASE1L3 was estimated in diverse cancers. Based on TIMER, association between DNASEL13 expression and immune infiltration was examined in various cancers. Then, mRNA level of DNASE1L3 in hepatocellular carcinoma (HCC) samples (n=22) and stomach adenocarcinoma (STAD) samples (n=17) was measured with qRT-PCR. Immunohistochemistry was performed to confirm expression of DNASE1L3 in paraffin-embedded tissues of HCC (n=9) and lung adenocarcinoma (n=20). RESULTS: DNASE1L3 was downregulated in multiple cancers, including breast invasive carcinoma (BRCA), cholangiocarcinoma (CHOL), liver hepatocellular carcinoma (LIHC), and lung adenocarcinoma (LUAD). A lower level of DNASE1L3 correlated with poorer prognosis in various cancers, especially in breast, liver, kidney, stomach, lung adenocarcinoma and sarcoma (SARC). Moreover, DNASE1L3 was positively related to immune cell infiltration in many cancers, including BRCA, LIHC, STAD, LUAD, and SARC. DNASE1L3 was significantly associated with CCR7/CCL19 in cancers. DNASE1L3 was downregulated in HCC and STAD tissues as demonstrated by qRT-PCR, as well as in HCC and LUAD samples, as shown by immunohistochemistry. CONCLUSION: DNASE1L3 has potential to serve as a prognostic biomarker in cancer of the breast, kidney, liver, stomach, lung adenocarcinoma and sarcoma. Down-regulation of DNASE1L3 may participate in immune escape via CCR7/CCL19 axis.