Nicotinamide N -methyltransferase promotes M2 macrophage polarization by IL6 and MDSC conversion by GM-CSF in gallbladder carcinoma.

BACKGROUND AND AIMS: Nicotinamide N -methyltransferase (NNMT), an enzyme responsible for the methylation of nicotinamide, is involved in many metabolic pathways in adipose tissue and the liver. However, the role of NNMT in editing the tumor immune microenvironment is not well understood. APPROACH AND RESULTS: Here, we identified that NNMT can promote IL6 and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by decreasing the tri-methyl-histone H3 levels on the promoters of IL6 and CSF2 (encoding GM-CSF) and CCAAT/Enhancer Binding Protein, an essential transcription factor for IL6 expression, thus promoting differentiation of macrophages into M2 type tumor-associated macrophages and generation of myeloid-derived suppressor cells from peripheral blood mononuclear cells. Treatment of xenografted tumor models overexpressing NNMT gallbladder carcinoma (GBC) cells with the NNMT inhibitor JBSNF-000088 resulted in compromised tumor development and decreased expression levels of IL6, GM-CSF, tumor-associated macrophage marker CD206, and myeloid-derived suppressor cell marker CD33 but increased expression levels of CD8. In addition, elevated expression of NNMT in tumors of patients with GBC was correlated with increased expression levels of CD206 and CD33 but with decreased levels of CD8 and survival of patients. CONCLUSIONS: These data highlight the critical role of NNMT in GBC progression. Inhibition of NNMT by JBSNF-000088 is a potential molecular target for GBC immunotherapy.

CD73 and PD-L1 as Potential Therapeutic Targets in Gallbladder Cancer.

Gallbladder cancer (GBC) is one of the most common and aggressive biliary tract cancers with a dismal prognosis. Ongoing clinical trials are evaluating a few selected immune checkpoint inhibitors (ICIs) as monotherapy for the treatment of GBC patients. However, only a subset of patients benefits from these treatments. To improve ICI therapy response, molecular mechanisms that confer resistance to immune checkpoint (IC) blockade needs to be explored. Epithelial-to-mesenchymal transition (EMT) program and cancer stem cells (CSCs) have been implicated as key processes that confer ICI treatment resistance. However, in GBC the EMT-CSC-IC axis has not yet been clearly elucidated. This study aims to examine the aberrant expression of ICs associated with CSC and EMT. We successfully enriched CSCs by utilizing a 3-dimensional culture system and established a reversible EMT model with human GBC NOZ cell line. Notably, ICs CD73 and PD-L1 were closely associated with both CSC and EMT phenotypes. Knockdown of CD73 or PD-L1 reduced the proliferative and motile abilities of both adherent monolayers and anchorage-free spheroids. In conclusion, blocking CD73 and PD-L1 offer a promising therapeutic strategy for targeting highly aggressive populations with CSC and EMT phenotype to improve GBC patient prognosis.

Neutrophilic inflammation in gallbladder carcinoma correlates with patient survival: A case-control study.

Gallbladder carcinoma is an uncommon malignancy with an overall 5-year survival of less than 5%. Gallbladder carcinoma has been strongly linked with cholelithiasis and chronic inflammation. Case reports and series have described cholecystitis with acute (neutrophilic) inflammation in association with gallbladder carcinoma, although a clear relationship to patient outcome has not been established. Our series included 8 cases of gallbladder carcinoma with high tumor-associated neutrophils (>25 per high power field) that were associated with shorter patient survival (Cox regression coefficient 6.2, p = 0.004) than age- and stage-matched controls. High tumor-associated neutrophils were not associated with gallbladder rupture/perforation or increased bacterial load measured by 16S PCR. Neutrophilic inflammation with gallbladder carcinoma correlates to shorter survival, independent of patient age and stage of carcinoma. The findings suggest that the degree of neutrophilic inflammation may have prognostic significance in specimens from patients with gallbladder carcinoma after cholecystectomy. Further studies with larger case numbers are needed to confirm and generalize these findings.

Clinical epidemiology of gallbladder cancer in North-Central India and association of immunological markers, NLR, MLR and PLR in the diagnostic/prognostic prediction of GBC.

INTRODUCTION: The incidence of Gallbladder Cancer (GBC) is found to be increasing in the rural populations of north-central India. Role of multiple demographic factors, including poor socio-economic conditions, illiteracy and miserable primary healthcare services appear to be significant factors for this increase. Here, we aim to assess the present status of GBC in north-central India and evaluate the role of immunological markers in its management. METHODS: A total of 1845 cases of different Gallbladder diseases, including GBC, from rural and urban areas both, registered at CHRI, Gwalior during 2009-2014 and 2018 were included in this study. The demographic and clinical information of the patients were analysed using various statistical tests. RESULTS: Of all the cases (1845) included in this study, 1125 (60.97%) were diagnosed with GBC, of which, 707 (62.84%) were from rural background and 418 (37.15%) from urban settings. Mean age for GBC cases for both male and female was about 53.49 years. Females were more affected, being 70.37%, while male patients were only 29.63%. The pathological investigations showed elevated levels of total bilirubin and liver function enzymes both. The NLR, PLR and MLR were found to be significantly associated with different clinical parameters as well as OS. CONCLUSION: We infer that the growing trend of GBC, particularly in rural areas, in north-central India is primarily associated with the lack of awareness, inadequate medical support and poor socio-economic conditions. Evaluation of haematological markers may help in the predictive diagnosis/ prognosis and or management of GBC cases in the studied population.

Single-cell RNA-sequencing atlas reveals an MDK-dependent immunosuppressive environment in ErbB pathway-mutated gallbladder cancer.

BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.

The predictive value of systemic immune inflammation index for postoperative survival of gallbladder carcinoma patients.

BACKGROUND: Growing evidence indicates that systemic immune inflammation index (SII) can predict the prognosis of various solid tumors. The objective of this study aimed to investigate the efficacy of SII in predicting the prognosis of gallbladder carcinoma (GBC) patients after radical surgery. METHODS: A consecutive series of 93 patients with GBC who underwent radical resection were enrolled in the retrospective study. The cutoff value for the SII was calculated using the time-dependent receiver operating characteristic (ROC) curve analysis by overall survival (OS) prediction. The associations between the SII and the clinicopathologic characteristics were analyzed using Pearson's χ2 test and Fisher's exact test. Survival curves were calculated using the Kaplan-Meier method. Univariate analysis was performed to evaluate the prognostic relevance of preoperative parameters. The multivariate Cox regression proportional hazard model was used to assess variables significant on univariate analysis. RESULTS: The Kaplan-Meier survival analysis and the multivariate analysis of patients with GBC who received radical resection showed SII independently predicted OS. The univariate analysis showed that the TNM stage, SII, CA19-9, ALP, prealbumin, NLR, MLR, lymph node metastasis, and histopathological type were all associated with overall survival. In time-dependent ROC analysis, the area of the SII-CA19-9 under the ROC curve (AUC) was higher than that of the preoperative SII or CA19-9 levels for the prediction of OS. CONCLUSION: Our results demonstrate that high SII was a predictor of poor long-term outcomes among patients with GBC undergoing curative surgery. SII-CA19-9 classification may be more effective in predicting the postoperative prognosis of GBC patients.

Immune Microenvironment in Gallbladder Adenocarcinomas.

Programmed death-1 (PD1) expression has not been reported in gallbladder adenocarcinoma. In this study we examined PD1 expression in gallbladder cancer to explore the correlation between PD1 expression and the clinicopathologic parameters. We found that 98% (46/47) cases expressed programmed death-ligand 1 (PD-L1) with 85% cases being PD-L1 3+. PD1+ tumor-infiltrating lymphocytes (TILs) were present in 78.7% cases (37/47). The tumor size was significantly smaller and the stromal CD3+ TILs were significantly higher in tumors with PD1+ TILs than those with PD1- TILs. In the tumors with size of <3 cm, stromal CD3+ TILs >115/HPF or stromal CD8+ TILs >45/HPF were associated with much better survival than those with stromal CD3+ TILs ≤115/HPF or stromal CD8+ TILs ≤45/HPF. In tumors with the size of 3 cm or larger, PD1+ TILs or stromal CD8+ TILs >45/HPF carried a significantly poorer survival than PD1- tumors or stromal CD8+ TILs <=45/HPF. No correlation was identified between PD1 expression and lymphovascular invasion, distant metastasis, pathologic tumor stage or prognostic stage. Multivariate survival analysis showed that tumor TNM stage and age were independent prognostic factors in gallbladder adenocarcinomas. We conclude that gallbladder adenocarcinomas may have high PD-L1 expression and PD1+ TILs. Smaller tumor size and greater amount of stromal CD3+ T cells were found in tumors with PD1+ TILs. In small tumors (<3 cm), high stromal CD3+ TILs or high stromal CD8+ TILs were associated with better survival. However, in large tumors (≥3 cm), PD1+ TILs or high stromal CD8+ TILs carried a poorer survival. Our study implied that immune-based therapy including PD1/PD-L1 checkpoint blockade might be useful in gallbladder adenocarcinomas.

GLI2 but not GLI1/GLI3 plays a central role in the induction of malignant phenotype of gallbladder cancer.

We previously reported that Hedgehog (Hh) signal was enhanced in gallbladder cancer (GBC) and was involved in the induction of malignant phenotype of GBC. In recent years, therapeutics that target Hh signaling have focused on molecules downstream of smoothened (SMO). The three transcription factors in the Hh signal pathway, glioma­associated oncogene homolog 1 (GLI1), GLI2, and GLI3, function downstream of SMO, but their biological role in GBC remains unclear. In the present study, the biological significance of GLI1, GLI2, and GLI3 were analyzed with the aim of developing novel treatments for GBC. It was revealed that GLI2, but not GLI1 or GLI3, was involved in the cell cycle­mediated proliferative capacity in GBC and that GLI2, but not GLI1 or GLI3, was involved in the enhanced invasive capacity through epithelial­mesenchymal transition. Further analyses revealed that GLI2 may function in mediating gemcitabine sensitivity and that GLI2 was involved in the promotion of fibrosis in a mouse xenograft model. Immunohistochemical staining of 66 surgically resected GBC tissues revealed that GLI2­high expression patients had fewer numbers of CD3+ and CD8+ tumor­infiltrating lymphocytes (TILs) and increased programmed cell death ligand 1 (PD­L1) expression in cancer cells. These results suggest that GLI2, but not GLI1 or GLI3, is involved in proliferation, invasion, fibrosis, PD­L1 expression, and TILs in GBC and could be a novel therapeutic target. The results of this study provide a significant contribution to the development of a new treatment for refractory GBC, which has few therapeutic options.

The immunological characteristics of gallbladder carcinoma and advances in immunotherapy practices.

Gallbladder carcinoma (GBC) is one of the most common malignant tumors in the biliary system, ranking sixth among gastrointestinal malignancies. In addition, the incidence of GBC has recently increased in China. GBC metastasizes early and invades adjacent organs such as the liver, making patients with GBC ineligible for radical surgery and giving them a poor prognosis. What is more, GBC is more inclined to develop chemo-resistance, which requires new strategies for clinical intervention. Cancer immunotherapy has made great advances over the past few years, with improved clinical efficacy against multiple malignancies, including GBC. This review summarizes the immunological characteristics of GBC as well as current advances in immunotherapies for GBC in order to provide new insights into future treatment and prevention of GBC.

High Systemic Inflammation Response Index (SIRI) Indicates Poor Outcome in Gallbladder Cancer Patients with Surgical Resection: A Single Institution Experience in China.

PURPOSE: The systemic inflammation response index (SIRI) has been reported to have prognostic ability in various solid tumors but has not been studied in gallbladder cancer (GBC). We aimed to determine its prognostic value in GBC. MATERIALS AND METHODS: From 2003 to 2017, patients with confirmed GBC were recruited. To determine the SIRI's optimal cutoff value, a time-dependent receiver operating characteristic curve was applied. Univariate and multivariate Cox analyses were performed for the recognition of significant factors. Then the cohort was randomly divided into the training and the validation set. A nomogram was constructed using the SIRI and other selected indicators in the training set, and compared with the TNM staging system. C-index, calibration plots, and decision curve analysis were performed to assess the nomogram's clinical utility. RESULTS: One hundred twenty-four patients were included. The SIRI's optimal cutoff value divided patients into high (≥ 0.89) and low SIRI (< 0.89) groups. Kaplan-Meier curves according to SIRI levels were significantly different (p < 0.001). The high SIRI group tended to stay longer in hospital and lost more blood during surgery. SIRI, body mass index, weight loss, carbohydrate antigen 19-9, radical surgery, and TNM stage were combined to generate a nomogram (C-index, 0.821 in the training cohort, 0.828 in the validation cohort) that was significantly superior to the TNM staging system both in the training (C-index, 0.655) and validation cohort (C-index, 0.649). CONCLUSION: The SIRI is an independent predictor of prognosis in GBC. A nomogram based on the SIRI may help physicians to precisely stratify patients and implement individualized treatment.

Low immune index correlates with favorable prognosis but with reduced benefit from chemotherapy in gallbladder cancer.

Use of immune index is a new potential approach for cancer classification and prediction. To investigate the status and clinical effect of immune index in gallbladder cancer (GBC), 238 GBC patients from Zhongshan Hospital affiliated to Fudan University were involved in the present study, including 113 patients in a training set and 125 patients in a validation set. Five immune cells (macrophages, neutrophils, regulatory T cells, cytotoxic T cells and mast cells) were selected based on a literature review and the immune index for each patient was calculated using the LASSO regression. A low immune index (<1) was defined as immunotype A and a high immune index (≥1) was defined as immunotype B. The 5-year overall survival rate for immunotype A was higher than that for immunotype B in the training set and the validation set (70.0% vs 37.0%, P < 0.001; 68.9% vs 47.5%, P = 0.002; respectively). Moreover, the immune index showed higher prediction efficiency compared with all the single immune cells which we selected. When combined with the immune index, the areas under the curve (AUC) of the TNM staging system in both sets were elevated from 0.677 to 0.787 and from 0.631 to 0.694, respectively. Interestingly, gemcitabine-based chemotherapy only benefits stage II patients of immunotype B and stage III patients of both immunotype A and immunotype B (P = 0.015, P = 0.030, P = 0.011, respectively) but does not work in stage II patients of immunotype A (P = .307). Taken together, the immune index could effectively predict prognosis and the benefits of gemcitabine-based chemotherapy and might improve on the TNM staging system.

Genomic ERBB2/ERBB3 mutations promote PD-L1-mediated immune escape in gallbladder cancer: a whole-exome sequencing analysis.

OBJECTIVES: Patients with gallbladder carcinoma (GBC) lack effective treatment methods largely due to the inadequacy of both molecular characterisation and potential therapeutic targets. We previously uncovered a spectrum of genomic alterations and identified recurrent mutations in the ErbB pathway in GBC. Here, we aimed to study recurrent mutations of genes and pathways in a larger cohort of patients with GBC and investigate the potential mechanisms and clinical significance of these mutations. DESIGN: We performed whole-exome sequencing (WES) in 157 patients with GBC. Functional experiments were applied in GBC cell lines to explore the oncogenic roles of ERBB2/ERBB3 hotspot mutations, their correlation with PD-L1 expression and the underlying mechanisms. ERBB inhibitors and a PD-L1 blocker were used to evaluate the anticancer activities in co-culture systems in vitro and in vivo. RESULTS: WES identified ERBB2 and ERBB3 mutations at a frequency of 7%-8% in the expanded cohort, and patients with ERBB2/ERBB3 mutations exhibited poorer prognoses. A set of in vitro and in vivo experiments revealed increased proliferation/migration on ERBB2/ERBB3 mutation. Ectopic expression of ERBB2/ERBB3 mutants upregulated PD-L1 expression in GBC cells, effectively suppressed normal T-cell-mediated cytotoxicity in vitro through activation of the PI3K/Akt signalling pathway and contributed to the growth and progression of GBC in vivo. Treatment with an ERBB2/ERBB3 inhibitor or a PD-L1 monoclonal antibody reversed these immunosuppressive effects, and combined therapy revealed promising therapeutic activities. CONCLUSIONS: ERBB2/ERBB3 mutations may serve as useful biomarkers in identifying patients who are sensitive to ERBB2/ERBB3 inhibitors and PD-L1 monoclonal antibody treatment. TRIAL REGISTRATION NUMBER: NCT02442414;Pre-results.

Tumor lysate-based vaccines: on the road to immunotherapy for gallbladder cancer.

Immunotherapy based on checkpoint blockers has proven survival benefits in patients with melanoma and other malignancies. Nevertheless, a significant proportion of treated patients remains refractory, suggesting that in combination with active immunizations, such as cancer vaccines, they could be helpful to improve response rates. During the last decade, we have used dendritic cell (DC) based vaccines where DCs loaded with an allogeneic heat-conditioned melanoma cell lysate were tested in a series of clinical trials. In these studies, 60% of stage IV melanoma DC-treated patients showed immunological responses correlating with improved survival. Further studies showed that an essential part of the clinical efficacy was associated with the use of conditioned lysates. Gallbladder cancer (GBC) is a high-incidence malignancy in South America. Here, we evaluated the feasibility of producing effective DCs using heat-conditioned cell lysates derived from gallbladder cancer cell lines (GBCCL). By characterizing nine different GBCCLs and several fresh tumor tissues, we found that they expressed some tumor-associated antigens such as CEA, MUC-1, CA19-9, Erb2, Survivin, and several carcinoembryonic antigens. Moreover, heat-shock treatment of GBCCLs induced calreticulin translocation and release of HMGB1 and ATP, both known to act as danger signals. Monocytes stimulated with combinations of conditioned lysates exhibited a potent increase of DC-maturation markers. Furthermore, conditioned lysate-matured DCs were capable of strongly inducing CD4+ and CD8+ T cell activation, in both allogeneic and autologous cell co-cultures. Finally, in vitro stimulated CD8+ T cells recognize HLA-matched GBCCLs. In summary, GBC cell lysate-loaded DCs may be considered for future immunotherapy approaches.

High CD8+ and absence of Foxp3+ T lymphocytes infiltration in gallbladder tumors correlate with prolonged patients survival.

BACKGROUND: Gallbladder cancer (GBC), although infrequent in industrialized countries, has high incidence rates in certain world regions, being a leading cause of death among elderly Chilean women. Surgery is the only effective treatment, and a five-year survival rate of advanced-stage patients is less than 10%. Hence, exploring immunotherapy is relevant, although GBC immunogenicity is poorly understood. This study examined the relationship between the host immune response and GBC patient survival based on the presence of tumor-infiltrating lymphocytes at different disease stages. METHODS: Tumor tissues from 80 GBC patients were analyzed by immunohistochemistry for the presence of CD3+, CD4+, CD8+, and Foxp3+ T cell populations, and the results were associated with clinical stage and patient survival. RESULTS: The majority of tumor samples showed CD3+ T cell infiltration, which correlated with better prognosis, particularly in advanced disease stages. CD8+, but not CD4+, T cell infiltration correlated with improved survival, particularly in advanced disease stages. Interestingly, a < 1 CD4+/CD8+ T cell ratio was related with increased survival. Additionally, the presence of Foxp3+ T cells correlated with decreased patient survival, whereas a ≤ 1 Foxp3+/CD8+ T cell ratio was associated with improved patient survival. CONCLUSIONS: Depending on the disease stage, the presence of CD8+ and absence of Foxp3+ T cell populations in tumor tissues correlated with improved GBC patient survival, and thus represent potential markers for prognosis and management of advanced disease, and supports testing of immunotherapy.

Long-lasting complete response status of advanced stage IV gall bladder cancer and colon cancer after combined treatment including autologous formalin-fixed tumor vaccine: two case reports.

BACKGROUND: The prognosis of advanced (stage IV) cancer of the digestive organs is very poor. We have previously reported a case of advanced breast cancer with bone metastasis that was successfully treated with combined treatments including autologous formalin-fixed tumor vaccine (AFTV). Herein, we report the success of this approach in advanced stage IV (heavily metastasized) cases of gall bladder cancer and colon cancer. CASE PRESENTATION: Case 1: A 61-year-old woman with stage IV gall bladder cancer (liver metastasis and lymph node metastasis) underwent surgery in May 2011, including partial resection of the liver. She was treated with AFTV as the first-line adjuvant therapy, followed by conventional chemotherapy. This patient is still alive without any recurrence, as confirmed with computed tomography, for more than 5 years. Case 2: A 64-year-old man with stage IV colon cancer (multiple para-aortic lymph node metastases and direct abdominal wall invasion) underwent non-curative surgery in May 2006. Following conventional chemotherapy, two courses of AFTV and radiation therapy were administered sequentially. This patient has had no recurrence for more than 5 years. CONCLUSION: We report the success of combination therapy including AFTV in cases of liver-metastasized gall bladder cancer and abdominal wall-metastasized colon cancer. Both patients experienced long-lasting, complete remission. Therefore, combination therapies including AFTV should be considered in patients with advanced cancer of the digestive organs.

Therapeutic effects of long-circulating miR-135a-containing cationic immunoliposomes against gallbladder carcinoma.

Gallbladder carcinoma (GBC) is the most common malignant tumour in the biliary tract, but effective therapeutics are lacking. Based on our previous studies, miR-135a is a potential tool to inhibit GBC proliferation. In this study, we constructed miR-135a-loaded DSPE-PEG2000 liposomes modified with Anti-EGFR antibodies (Anti-EGFR-CIL-miR-135a). The results of an analysis of their physicochemical properties indicated the particle size of it was 222.0 ± 2.1 nm in diameter with an uptake efficiency of 86.5%. Next, the post-treatment biological behaviours of GBC, specifically, invasion, metastasis and apoptosis, were evaluated. miR-135a inhibited GBC invasion and metastasis and promoted apoptosis compared to controls. Additionally, miR-135a targeted and regulated the expression of ROCK1, HOXA10 and BCL-2. Due to the targeted effects of Anti-EGFR-CIL-miR-135a, the GBC tumour growth rate was 60% lower in an in vivo xenograft-bearing mouse model compared to controls. Thus, Anti-EGFR-CIL-miR-135a is a promising therapeutic strategy to combat GBC.

Effects of combined epidural and general anesthesia on intraoperative hemodynamic responses, postoperative cellular immunity, and prognosis in patients with gallbladder cancer: A randomized controlled trial.

BACKGROUND: This study is supposed to investigate the effects of combined epidural and general anesthesia on intraoperative hemodynamic responses, postoperative cellular immunity, and prognosis in patients with gallbladder cancer (GBC). METHODS: One hundred forty-four GBC patients were selected and randomly divided into the general anesthesia (GA) group and the combined epidural-general anesthesia (CEGA) group. Before anesthesia induction (t0), at intubation (t1), at the beginning of surgery (t2), 5 minutes after pneumoperitoneum (t3), at the end of surgery (t4), after recovery of spontaneous breathing (t5), after regaining consciousness (t6), and after extubation (t7), the heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and the depth of anesthesia (bispectral index [BIS]) were detected. Blood samples were separately collected 30 minutes before anesthesia induction (T1), 2 hours after the beginning of surgery (T2), at the end of surgery (T3), 1 day after surgery (T4), 3 days after surgery (T5). The survival rates of T cell subsets (CD3+, CD4+, CD8+, CD4+/CD8+) and natural killer (NK) cells were determined by flow cytometry. Postoperative nausea and vomiting (PONV), visual analog scale (VAS), and sedation-agitation scale (SAS) were performed to assess postoperative adverse reactions. A 3-year follow-up was conducted. RESULTS: Compared with the GA group, the CEGA group had significant lower SBP values at t5 and t6, lower DBP values at t1, t3, t4, and t5, lower HR values at t1 and t5, and higher BIS values at t4, t5, t6, and t7. No PONV was observed in the CEGA group. In comparison to the GA group, the VAS was markedly increased and survival rates of CD3+, CD4+, and CD4+/CD8+ cells were increased at T2, T3, T4, and T5 in the CEGA group. The 1-year, 2-year, and 3-year survival rates were not evidently different between the CEGA group and the GA group. CONCLUSION: Our study provides evidence that the combined epidural-general anesthesia might attenuate intraoperative hemodynamic responses and improve postoperative cellular immunity, so that it might be a more available anesthesia method for GBC patients.

Prognostic significance of immune cells in the tumor microenvironment and peripheral blood of gallbladder carcinoma patients.

BACKGROUND: The role of the interaction between tumor cells and inflammatory cells in gallbladder carcinoma (GBC) is unclear. Inflammatory cells exist in both the tumor immune microenvironment and the host peripheral blood circulatory system. In the current study, we examined the prognostic value of inflammatory cells in the tumor microenvironment and peripheral blood in patients with GBC. METHODS: 98 patients with GBC were recruited in this retrospective study. Using immunohistochemistry, we examined tumor-infiltrating CD3+ generic T-cells, CD8+ cytotoxic T-cells, CD45RO+ memory T-cells, and CD15+ neutrophils. Peripheral venous blood samples were also collected, and absolute neutrophil count (ANC), absolute lymphocyte count (ALC) and neutrophil/lymphocyte ratio (NLR) were measured. The relationships between these variables and patient outcome were evaluated. RESULTS: Survival analysis revealed that the density of CD3+ cell infiltrates in the tumor microenvironment was positively correlated with overall survival (OS) and the density of CD15+ cell infiltrates was negatively correlated with the OS. The combined analysis showed that a high density of CD3+ cell infiltrates combined with a low density of CD15+ cell infiltrates was an independent prognostic factor for GBC. In peripheral blood, survival analysis suggested that ANC and NLR were negatively correlated, while ALC was positively correlated with OS. Multivariate survival analysis showed that NLR was an independent prognostic factor for gallbladder cancer prognosis. CONCLUSIONS: The results indicate that the combination of high density of CD3+ cell infiltrates combined with a low density of CD15+ cell infiltrates in tumor samples and pretreatment peripheral blood NLR were independent prognostic factors in patients with GBC.

Amorphous aggregate adducts of linker histone H1 turn highly immunologic in the cancers of oesophagus, stomach, gall bladder and ovary.

Hyperglycaemic influence on carcinogenesis and tumour progression is emerging as a link between diabetes and cancer. This work establishes the disturbed structural integrity of nucleosomal linker histone H1 by methyglyoxal (MG) and then correlates the role of modified H1 in the auto-immunopathogenesis of multiple cancers. MG modification caused a loss of free ε-amino groups in H1 and raised its ß-sheet structural component with a consequence of non amyloid aggregation. It changed the folding-unfolding denaturation pattern of H1 and attached itself to the lysine residues of the protein eventually making up Nε-(carboxyethyl) lysine. The structural variations act as extra antigenic determinants on H1 that yield aggressive antibody response, when immunised in rabbits. The ELISA tests proved the immunoglobulin response very specific and gel based studies established the preferential binding of antibodies generated against MG-H1 with the modified protein. Cross reaction analysis inferred the multiple specific natures of immunoglobulins with binding tendencies against different inhibitors. The immunoglobulin content in blood sera derived from human subjects with tumours of oesophagus, stomach, gall bladder and ovary confirmed the antibody presence against MG-H1 and competitive ELISA showed their high specificity. This may suggest a link between nucleosomal linker H1, hyperglycaemia, glycoxidation and cancer.