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Abstract In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.

The relationship between energy production and cancer is attracting attention. This study aimed to investigate the clinicopathological significance of fumarate hydratase (FH), a tricarboxylic acid cycle enzyme, in gastric cancer using autoantibodies as biomarkers. The study analyzed 116 patients who underwent gastric cancer surgery and 96 healthy controls. Preoperative serum FH autoantibody (s-FH-Ab) titers were analyzed using an immunosorbent assay with an amplified luminescent proximity homogeneous assay. Receiver operating characteristic analysis was used to determine the cutoff s-FH-Ab titer. Clinicopathological factors and prognosis were compared between the high and low s-FH-Ab groups. The s-FH-Ab levels were significantly higher in the gastric cancer group than in the control group (p = 0.01). Levels were elevated even in patients with stage I gastric cancer compared with healthy controls (p = 0.02). A low s-FH-Ab level was significantly associated with distant metastasis (p = 0.01), peritoneal dissemination (p < 0.05), and poor overall survival (p < 0.01). Multivariate analysis revealed that low s-FH-Ab levels were an independent risk factor for poor prognosis (p < 0.01). Therefore, s-FH-Ab levels may be a useful biomarker for early diagnosis and the prediction of prognosis in patients with gastric cancer.

Inflammation is closely associated with cerebrovascular diseases, cardiovascular diseases, diabetes, and cancers, and it is accompanied by the development of autoantibodies in the early stage of inflammation-related diseases. Hence, it is meaningful to discover novel antibody biomarkers targeting inflammation-related diseases. In this study, Jumonji C-domain-containing 6 (JMJD6) was identified by the serological identification of antigens through recombinant cDNA expression cloning. In particular, JMJD6 is an antigen recognized in serum IgG from patients with unstable angina pectoris (a cardiovascular disease). Then, the serum antibody levels were examined using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay and a purified recombinant JMJD6 protein as an antigen. We observed elevated levels of serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with inflammation-related diseases such as ischemic stroke, acute myocardial infarction (AMI), diabetes mellitus (DM), and cancers (including esophageal cancer, EC; gastric cancer; lung cancer; and mammary cancer), compared with the levels in healthy donors. The s-JMJD6-Ab levels were closely associated with some inflammation indicators, such as C-reactive protein and intima–media thickness (an atherosclerosis index). A better postoperative survival status of patients with EC was observed in the JMJD6-Ab-positive group than in the negative group. An immunohistochemical analysis showed that JMJD6 was highly expressed in the inflamed mucosa of esophageal tissues, esophageal carcinoma tissues, and atherosclerotic plaques. Hence, JMJD6 autoantibodies may reflect inflammation, thereby serving as a potential biomarker for diagnosing specific inflammation-related diseases, including stroke, AMI, DM, and cancers, and for prediction of the prognosis in patients with EC.

Objectives: We evaluated the prognostic impact of a novel C-reactive protein (CRP) cut-off value (0.6 mg/dl) and carcinoembryonic antigen (CEA)/carbohydrate antigen 19-9 (CA19-9) in stage II/III colorectal cancer. Methods: Four hundred ninety-eight patients with stage II (n = 275) or stage III (n = 223) colorectal cancer, surgically treated between January 2010 and December 2016, were analyzed. The optimal CRP cut-off value was fixed at 0.6 mg/dl to predict recurrence based on the receiver operating characteristic curve. Prognostic factors, including CRP/CEA/CA19-9 status, for relapse-free survival (RFS) were evaluated by multivariate analysis. Results: Recurrent rates were 15% and 32% in stages II and III, respectively. In stage II, CRP, CEA, and CA19-9 were not significant prognostic factors for RFS. In stage III, the RFS of the low CRP group was significantly better than that of the high CRP group (p = 0.002). In stage III, the RFS of CRP(−)/CEA(−) or CRP(−)/CA19-9(−) was significantly better than the other group, as opposed to the RFS of the CEA(−)/CA19-9(−) group that was not. The CRP(−)/CEA(−)/CA19-9(−) group recurrence rate in stage III was significantly better than the CRP(+)/CEA(−)/CA19-9(−) group (20% vs. 50%, p = 0.006). Multivariate analysis revealed that CRP(−)/CEA(−)/CA19-9(−) (p = 0.04) and non-T4 (p < 0.001) were good independent prognostic factors in stage III. The CRP(−)/CEA(−)/CA19-9(−)/non-T4 group recurrence rate in stage III was 11% (8 out of 73). Conclusions: In stage III, the CRP(−)/CEA(−)/CA19-9(−)/non-T4 group is favorable risk for recurrence.

Abstract This study aimed to evaluate the uptake of the clinical effectiveness of [S-methyl-11C]-L-methionine positron emission tomography/computed tomography (MET PET/CT) in patients with esophageal cancer and to investigate MET PET/CT imaging parameters to assess early response for esophageal cancer with neoadjuvant carbon ion radiotherapy (CIRT). MET PET/CT scans were performed in nineteen patients before and 3 weeks after completion of CIRT. After Surgery, the effect of neoadjuvant CIRT was investigated by examining the relationship between each parameter of MET uptake and the histological assessment (grade and tumor residual ratio). Four parameters of MET uptake were the maximum and minimum standardized uptake values of pre and post CIRT (pre-SUVmax, pre-SUVmean, post-SUVmax, and post-SUVmean). MET PET/CT imaging of esophageal cancer was clearly demonstrated. The post-SUVmax was the most suitable parameter. When the cutoff value was set as post-SUVmax = 6.21, the sensitivity, the specificity, and the accuracy of Grades 3 were 100.0%, 63.6%, and 78.9%, respectively. And there was a positive relationship between the tumor residual ratio and post-SUVmax (R 2 = 0.38, p

IntroductionAutoantibodies against inflammatory cytokines may be used for the prevention of atherosclerosis. Preclinical studies consider colony-stimulating factor 2 (CSF2) as an essential cytokine with a causal relationship to atherosclerosis and cancer. We examined the serum anti-CSF2 antibody levels in patients with atherosclerosis or solid cancer.MethodsWe measured the serum anti-CSF2 antibody levels via amplified luminescent proximity homogeneous assay-linked immunosorbent assay based on the recognition of recombinant glutathione S-transferase-fused CSF2 protein or a CSF2-derived peptide as the antigen.ResultsThe serum anti-CSF2 antibody (s-CSF2-Ab) levels were significantly higher in patients with acute ischemic stroke (AIS), acute myocardial infarction (AMI), diabetes mellitus (DM), and chronic kidney disease (CKD) compared with healthy donors (HDs). In addition, the s-CSF2-Ab levels were associated with intima-media thickness and hypertension. The analyzes of samples obtained from a Japan Public Health Center-based prospective study suggested the utility of s-CSF2-Ab as a risk factor for AIS. Furthermore, the s-CSF2-Ab levels were higher in patients with esophageal, colorectal, gastric, and lung cancer than in HDs but not in those with mammary cancer. In addition, the s-CSF2-Ab levels were associated with unfavorable postoperative prognosis in colorectal cancer (CRC). In CRC, the s-CSF2-Ab levels were more closely associated with poor prognosis in patients with p53-Ab-negative CRC despite the lack of significant association of the anti-p53 antibody (p53-Ab) levels with the overall survival.ConclusionS-CSF2-Ab was useful for the diagnosis of atherosclerosis-related AIS, AMI, DM, and CKD and could discriminate poor prognosis, especially in p53-Ab-negative CRC.

The emergence of escape variants of SARS-CoV-2 carrying mutations in the spike protein poses a challenge for therapeutic antibodies. Here, we show that through the comprehensive engineering of the variable region of the neutralizing monoclonal antibody 5A6, the engineered antibody, 5A6CCS1, is able to neutralize SARS-CoV-2 variants that escaped neutralization by the original 5A6 antibody. In addition to the improved affinity against variants, 5A6CCS1 was also optimized to achieve high solubility and low viscosity, enabling a high concentration formulation for subcutaneous injection. In cynomolgus monkeys, 5A6CCS1 showed a long plasma half-life and good subcutaneous bioavailability through engineering of the variable and constant region. These data demonstrate that 5A6CCS1 is a promising antibody for development against SARS-CoV-2 and highlight the importance of antibody engineering as a potential method to counteract escape variants.

Abstract Background Real‐world outcomes of nivolumab treatment for gastric cancer and associated prognostic factors remain unclear; the present study aimed to evaluate both items. Methods A total of 278 consecutive patients treated with nivolumab for gastric cancer during 2017‐2019 were enrolled in this multi‐institutional retrospective cohort study. The impact of laboratory findings, immune‐related adverse events (irAEs), and clinicopathological factors on long‐term survival was evaluated using the Cox proportional hazards model. Results The response rate was 11.7% in patients with measurable lesions. The overall and progression‐free survival estimates were 6.77 and 2.53 months, respectively. The incidence of irAEs was 30.6% (6.8% for grade ≥3). There were no treatment‐related deaths. Multivariate analysis revealed that C‐reactive protein level of ≤0.5 mg/dL (hazard ratio = 0.476, P 3.5 g/dL (hazard ratio = 0.688, P = .045), performance status 0 (hazard ratio = 0.711, P = .028), lymphocyte count >1000/μL (hazard ratio = 0.686, P = .027), and differentiated histological type (hazard ratio = 0.740, P = .046) were independently associated with improved survival. The median survival of patients with four or more good prognostic factors was 18.3 months. Conclusion Nivolumab showed safety and survival benefits in patients with previously treated unresectable or recurrent gastric cancer. Low C‐reactive protein level, irAE occurrence, high albumin level, high lymphocyte count, and differentiated histological type may affect outcomes. The presence of four or more good prognostic factors may help identify likely long‐term survivors.

Abstract Aim Clinical staging is vital for selecting appropriate candidates and designing neoadjuvant treatment strategies for advanced tumors. The aim of this review was to evaluate diagnostic abilities of clinical TNM staging for gastrointestinal, gastrointestinal cancers. Methods We conducted a systematic review of recent publications to evaluate the accuracy of diagnostic modalities on gastrointestinal cancers. A systematic literature search was performed in PubMed/MEDLINE using the keywords “TNM staging,” “T4 staging,” “distant metastases,” “esophageal cancer,” “gastric cancer,” and “colorectal cancer,” and the search terms used in Cochrane Reviews between January 2005 to July 2020. Articles focusing on preoperative diagnosis of: (a) depth of invasion; (b) lymph node metastases; and (c) distant metastases were selected. Results After a full‐text search, a final set of 55 studies (17 esophageal cancer studies, 26 gastric cancer studies, and 12 colorectal cancer studies) were used to evaluate the accuracy of clinical TNM staging. Positron emission tomography–computed tomography (PET‐CT) and/or magnetic resonance imaging (MRI) were the best modalities to assess distant metastases. Fat and fiber mode of CT may be useful for T4 staging of esophageal cancer, CT was a partially reliable modality for lymph node staging in gastric cancer, and CT combined with MRI was the most reliable modality for liver metastases from colorectal cancer. Conclusion The most reliable diagnostic modality differed among gastrointestinal cancers depending on the type of cancer. Therefore, we propose diagnostic algorithms for clinical staging for each type of cancer.

Abstract A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.

Abstract Background Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS. Methods Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay. Results The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297–311 of DIDO1, 426–440 of FOXJ2, and 607–621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case–control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS. Conclusions Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.

Abstract Atherosclerosis has been considered as the main cause of morbidity, mortality, and disability worldwide. The first screening for antigen markers was conducted using the serological identification of antigens by recombinant cDNA expression cloning, which has identified adaptor-related protein complex 3 subunit delta 1 (AP3D1) as an antigen recognized by serum IgG antibodies of patients with atherosclerosis. Serum antibody levels were examined using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using a recombinant protein as an antigen. It was determined that the serum antibody levels against AP3D1 were higher in patients with acute ischemic stroke (AIS), transient ischemic attack, diabetes mellitus (DM), cardiovascular disease, chronic kidney disease (CKD), esophageal squamous cell carcinoma (ESCC), and colorectal carcinoma than those in the healthy donors. The area under the curve values of DM, nephrosclerosis type of CKD, and ESCC calculated using receiver operating characteristic curve analysis were higher than those of other diseases. Correlation analysis showed that the anti-AP3D1 antibody levels were highly associated with maximum intima-media thickness, which indicates that this marker reflected the development of atherosclerosis. The results of the Japan Public Health Center-based Prospective Study indicated that this antibody marker is deemed useful as risk factors for AIS.

Atherosclerosis (AS) and cancers are major global causes of mortality and morbidity. They also share common modifiable pathogenesis risk factors. As the same strategies used to predict AS could also detect certain cancers, we sought novel serum antibody biomarkers of cancers in atherosclerotic sera sampled by liquid biopsy. Using serological antigen identification by cDNA expression cloning (SEREX) and western blot, we screened and detected the antigens BRCA1-Associated ATM Activator 1 (BRAT1) and WD Repeat Domain 1 (WDR1) in the sera of patients with transient ischemic attacks (TIA). Amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) established the upregulation of serum BRAT1 antibody (BRAT1-Abs) and WDR1 antibody (WDR1-Abs) in patients with AS-related diseases compared with healthy subjects. ROC and Spearman’s correlation analyses showed that BRAT1-Abs and WDR1-Abs could detect AS-related diseases. Thus, serum BRAT1-Abs and WDR1-Abs are potential AS biomarkers. We used online databases and AlphaLISA detection to compare relative antigen and serum antibody expression and found high BRAT1 and BRAT1-Abs expression in patients with GI cancers. Significant increases (> 0.6) in the AUC for BRAT1-Ab vs. esophageal squamous cell carcinoma (ESCC), gastric cancer, and colorectal cancer suggested that BRAT1-Ab exhibited better predictive potential for GI cancers than WDR1-Ab. There was no significant difference in overall survival (OS) between BRAT1-Ab groups (P = 0.12). Nevertheless, a log-rank test disclosed that the highest serum BRAT1-Ab levels were associated with poor ESCC prognosis at 5–60 weeks post-surgery. We validated the foregoing conclusions by comparing serum BRAT1-Ab and WDR1-Ab levels based on the clinicopathological characteristics of the patients with ESCC. Multiple statistical approaches established a correlation between serum BRAT1-Ab levels and platelet counts. BRAT1-Ab upregulation may enable early detection of AS and GI cancers and facilitate the delay of disease progression. Thus, BRAT1-Ab is a potential antibody biomarker for the diagnosis of AS and GI cancers and strongly supports the routine clinical application of liquid biopsy in chronic disease detection and diagnosis.

Abstract Background The positive response and the clinical usefulness of 14 serum antibodies in patients with esophageal squamous cell carcinoma (ESCC) were examined in this study. The Cancer Genome Atlas (TCGA) was used to investigate the frequency of gene expressions, mutations, and amplification of these 14 antigens and also the possible effects of antibody induction. Methods Blood serum derived from 85 patients with ESCC was collected and analyzed for the 14 antibodies using ELISA. The prognosis between positive and negative antibodies were then compared. The antibody panel included LGALS1, HCA25a, HCC-22-5, and HSP70. Results Patient serum was positive for all antibodies, except VEGF, with the positive rates ranging from 1.18 to 10.59%. Positive rates for LGALS1, HCA25a, HCC-22-5, and HSP70 were > 10%. TCGA data revealed that all antigen-related genes had little or no mutation or amplification, and hence an increase in gene expression affected antibody induction. The positive results from the panel accounted for the positive rate comparable to the combination of CEA and SCC. No significant association was observed between the presence of antibodies and disease prognosis. Conclusions The detection rates of LGALS1, HCA25a, HCC-22-5, and HSP70 were 10% higher in patients with ESCC. Gene overexpression may be involved in such antibody production. These four antibodies were applied as a panel in comparison with conventional tumor markers. Moreover, it was confirmed that the combination of this panel and the conventional tumor markers significantly improved the positive rate.

Abstract Background Although the first-line therapy for early-stage gastric mucosa-associated lymphoid tissue lymphoma is the eradication of Helicobacter pylori, the effect of eradication in Helicobacter pylori-negative cases is unclear. In this case report, we describe a surgical option for a case of Barrett’s esophageal cancer with concurrent gastric mucosa-associated lymphoid tissue lymphoma. Case presentation A 79-year-old man was admitted to our hospital with Barrett’s esophageal cancer and gastric mucosa-associated lymphoid tissue lymphoma. Initially, we performed endoscopic submucosal dissection for Barrett’s esophageal cancer. Since residual tumor was observed after the endoscopic submucosal dissection, we performed an esophagectomy with two-field lymph node dissection, which was followed by placement of a gastric conduit via the posterior mediastinal route. He was discharged 14 days after surgery. Although no additional treatment exists for mucosa-associated lymphoid tissue lymphoma, no recurrent disease has been detected to date. Conclusion An option to use a portion of the stomach with low-grade malignant mucosa-associated lymphoid tissue lymphoma as a conduit after esophagectomy was suggested.

Abstract Background and Aim We evaluated the clinicopathological and prognostic significance of serum p53 (s‐p53‐Abs) and serum NY‐ESO‐1 autoantibodies (s‐NY‐ESO‐1‐Abs) in esophageal squamous cell carcinoma (ESCC), gastric cancer and hepatocellular carcinoma (HCC). Patients and Methods A total of 377 patients, 85 patients with ESCC, 248 patients with gastric cancer, and 44 patients with HCC were enrolled to measure s‐p53‐Abs and s‐NY‐ESO‐1‐Abs titers by the enzyme‐linked immunosorbent assay before treatment. The clinicopathological significance and prognostic impact of the presence of autoantibodies were evaluated. Expression data based on the Cancer Genome Atlas and the prognostic impact of gene expression was also examined for discussion. Results The positive rates of s‐p53‐Abs were 32.9% in ESCC, 15% in gastric cancer, and 4.5% in HCC. The positive rates of s‐NY‐ESO‐1‐Abs were 29.4% in ESCC, 9.7% in gastric cancer, and 13.6% in HCC. The presence of s‐p53‐Abs was not associated with tumor progression in these three cancer types. On the other hand, the presence of s‐NY‐ESO‐1‐Abs was significantly associated with tumor progression in ESCC and gastric cancer. The presence of s‐p53‐Abs and/or s‐NY‐ESO‐1‐Abs was significantly associated with poor prognosis in gastric cancer but not in ESCC nor HCC. Conclusions The presence of s‐p53‐Abs and/or s‐NY‐ESO‐1‐Abs was associated with tumor progression in ESCC and gastric cancer. These autoantibodies might have poor prognostic impacts on gastric cancer (UMIN000014530).

Abstract Background Programmed death ligand 1 (PD‐L1) inhibitor has been approved as one of the standard therapies for various cancers. Some reports have shown that serum PD‐L1 level is associated with advanced tumor stages and poor prognosis; however, corresponding pathological information in esophageal cancer patients is lacking. Therefore, we evaluated the clinicopathological and prognostic impact of serum PD‐L1 levels in surgically treated esophageal cancer. Methods A total of 150 patients who underwent radical resection for esophageal cancer were included in the study. Preoperative serum PD‐L1 levels were analyzed using the enzyme‐linked immunosorbent assay kit. A cutoff level of 65.6 pg/mL was used to divide the patients into two groups, and univariate and multivariate analyses were conducted to compare the clinicopathological characteristics and prognoses between these two groups. Results Although significant associations between serum PD‐L1 levels and clinicopathological variables were observed, serum PD‐L1 level was significantly associated with high neutrophil counts, high CRP levels, low albumin levels, and high squamous cell carcinoma antigen levels. Furthermore, serum PD‐L1 level was associated with poor overall survival independent to TNM factors. Conclusions High preoperative level of serum PD‐L1 is a prognostic factor for poor overall survival in patients with surgically treated esophageal cancer.

BackgroundEsophageal cancer often appears as postoperative metastasis or recurrence after radical surgery. Although we had previously reported that serum programmed cell death ligand 1 (PD-L1) level correlated with the prognosis of esophageal cancer, further novel biomarkers are required for more precise prediction of the prognosis. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with the cholesterol metabolism. But there was no report of relationship between serum PCSK9 antibody and cancer. Therefore, we investigated whether anti-PCSK9 antibodies could be a novel biomarker for solid cancer.MethodsSerum levels of anti-PCSK9 antibodies and antigens in patients with solid cancer were analyzed using amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA). The reactivity of serum antibodies against recombinant PCSK9 protein was investigated by Western blotting, and the expression of PCSK9 antigens in esophageal cancer tissues was examined by immunohistochemical staining.ResultsAlphaLISA showed that serum anti-PCSK9 antibody (s-PCSK9-Ab) levels were significantly higher in patients with esophageal cancer, gastric cancer, colorectal cancer, lung cancer, and breast cancer than in healthy donors, and patients with esophageal cancer had the highest levels. The presence of serum antibody in patients was confirmed by Western blotting. There was no apparent correlation between s-PCSK9-Ab and PCSK9 antigen levels. Immunohistochemical staining demonstrated the expression of PCSK9 antigen in both the cytoplasm and nuclear compartments of esophageal squamous cell carcinoma tissue but not in normal tissue. Compared with patients with low s-PCSK9-Ab levels, those with high s-PCSK9-Ab levels had a favorable postoperative prognosis after radical surgery for esophageal cancer. In the multivariate analysis, tumor depth and s-PCSK9-Ab level were identified as independent prognostic factors. In the univariate analysis of clinicopathological features, high PCSK9 antibody levels were not associated with sex, age, location, tumor depth, lymph node status, squamous cell carcinoma antigen, or p53-Ab, whereas they correlated significantly with PD-L1 levels, which were associated with unfavorable prognosis. Correlation between s-PCSK9-Ab and PD-L1 levels was also confirmed in the logistic regression analysis; therefore, low s-PCSK9-Ab levels could discriminate another poor prognosis group other than high-PD-L1 group.ConclusionsPatients with solid cancer had higher s-PCSK9-Ab levels than healthy donors. High s-PCSK9-Ab levels indicated better prognosis for overall survival after surgery in patients with esophageal cancer.

The differential diagnosis of reactive mesothelial hyperplasia and mesothelioma is difficult. We present a rare case of diffuse pleural thickening with thoracic contraction that was indistinguishable from mesothelioma. A 66-year-old woman with no history of asbestos exposure visited our hospital with a complaint of dyspnea. The clinical findings included circumferential pleural thickening on chest computed tomography image and a high concentration of hyaluronic acid in the pleural fluid. Pleural biopsies obtained by thoracoscopy under local anesthesia were pathologically consistent with mesothelioma, but the patient refused to take any kind of mesothelioma treatments. Four months later, she consented to a surgical pleural biopsy under general anesthesia to obtain larger tissue samples, which included typical proliferating polygonal cells positive for CAM5.2, calretinin, WT-1, D2-40, CK5/6, epithelial membrane antigen, and glucose transporter-1 and negative for carcinoembryonic antigen, BerEP4, and MOC31. The analysis was consistent with diagnosis of epithelioid mesothelioma. Fluorescence in situ hybridization, however, showed the presence of p16 gene, and the expression of BRCA1-associated protein-1 was detected by immunohistochemistry. Our final diagnosis was diffuse pleural thickening unrelated to asbestos exposure. Differential diagnosis of diffuse pleural thickening and malignant mesothelioma is thus difficult and routine immunohistochemical examinations are often insufficient for accurate diagnosis. Multiple diagnostic methods are required for correct diagnosis in a clinically marginal case.

Abstract Background Discoidin domain receptor 1 (DDR1) is a collagen-activated receptor tyrosine kinase extensively implicated in diseases such as cancer, atherosclerosis and fibrosis. Multiple preclinical studies, performed using either a gene deletion or a gene silencing approaches, have shown this receptor being a major driver target of fibrosis and glomerulosclerosis. Methods The present study investigated the role and relevance of DDR1 in human crescentic glomerulonephritis (GN). Detailed DDR1 expression was first characterized in detail in human GN biopsies using a novel selective anti-DDR1 antibody using immunohistochemistry. Subsequently the protective role of DDR1 was investigated using a highly selective, novel, small molecule inhibitor in a nephrotoxic serum (NTS) GN model in a prophylactic regime and in the NEP25 GN mouse model using a therapeutic intervention regime. Results DDR1 expression was shown to be mainly limited to renal epithelium. In humans, DDR1 is highly induced in injured podocytes, in bridging cells expressing both parietal epithelial cell (PEC) and podocyte markers and in a subset of PECs forming the cellular crescents in human GN. Pharmacological inhibition of DDR1 in NTS improved both renal function and histological parameters. These results, obtained using a prophylactic regime, were confirmed in the NEP25 GN mouse model using a therapeutic intervention regime. Gene expression analysis of NTS showed that pharmacological blockade of DDR1 specifically reverted fibrotic and inflammatory gene networks and modulated expression of the glomerular cell gene signature, further validating DDR1 as a major mediator of cell fate in podocytes and PECs. Conclusions Together, these results suggest that DDR1 inhibition might be an attractive and promising pharmacological intervention for the treatment of GN, predominantly by targeting the renal epithelium.

Abstract Background Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. Methods We used type 5 Ad in which the expression of E1A gene was activated by 5′-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. Results The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. Conclusions Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

Abstract Background Replication-competent adenoviruses (Ad) produced cytotoxic effects on infected tumors and have been examined for the clinical applicability. A biomarkers to predict the cytotoxicity is valuable in a clinical setting. Methods We constructed type 5 Ad (Ad5) of which the expression of E1A gene was activated by a 5′ regulatory sequences of survivin, midkine or cyclooxygenase-2, which were highly expressed in human tumors. We also produced the same replication-competent Ad of which the fiber-knob region was replaced by that of Ad35 (AdF35). The cytotoxicity was examined by a colorimetric assay with human tumor cell lines, 4 kinds of pancreatic, 9 esophageal carcinoma and 5 mesothelioma. Ad infectivity and Ad-mediated gene expression were examined with replication-incompetent Ad5 and AdF35 which expressed the green fluorescence protein gene. Expression of cellular receptors for Ad5 and AdF35 was also examined with flow cytometry. A transcriptional activity of the regulatory sequences was investigated with a luciferase assay in the tumor cells. We then investigated a possible correlation between Ad-mediated cytotoxicity and the infectivity/gene expression, the transcriptional activity or the p53 genotype. Results We found that the cytotoxicity was greater with AdF35 than with Ad5 vectors, but was not correlated with the Ad infectivity/gene expression irrespective of the fiber-knob region or the E1A-activating transcriptional activity. In contrast, replication-competent Ad produced greater cytotoxicity in p53 mutated than in wild-type esophageal carcinoma cells, suggesting a possible association between the cytotoxicity and the p53 genotype. Conclusions Sensitivity to Ad-mediated cytotoxic activity was linked with the p53 genotype but was not lineally correlated with the infectivity/gene expression or the E1A expression.

Abstract Background Mesothelioma is resistant to conventional treatments and is often defective in p53 pathways. We then examined anti-tumor effects of metformin, an agent for type 2 diabetes, and combinatory effects of metformin and nutlin-3a, an inhibitor for ubiquitin-mediated p53 degradation, on human mesothelioma. Methods We examined the effects with a colorimetric assay and cell cycle analyses, and investigated molecular events in cells treated with metformin and/or nutlin-3a with Western blot analyses. An involvement of p53 was tested with siRNA for p53. Results Metformin suppressed cell growth of 9 kinds of mesothelioma including immortalized cells of mesothelium origin irrespective of the p53 functional status, whereas susceptibility to nutlin-3a was partly dependent on the p53 genotype. We investigated combinatory effects of metformin and nutlin-3a on, nutlin-3a sensitive MSTO-211H and NCI-H28 cells and insensitive EHMES-10 cells, all of which had the wild-type p53 gene. Knockdown of p53 expression with the siRNA demonstrated that susceptibility of MSTO-211H and NCI-H28 cells to nutlin-3a was p53-dependent, whereas that of EHMES-10 cells was not. Nevertheless, all the cells treated with both agents produced additive or synergistic growth inhibitory effects. Cell cycle analyses also showed that the combination increased sub-G1 fractions greater than metformin or nutlin-3a alone in MSTO-211H and EHMES-10 cells. Western blot analyses showed that metformin inhibited downstream pathways of the mammalian target of rapamycin (mTOR) but did not activate the p53 pathways, whereas nutlin-3a phosphorylated p53 and suppressed mTOR pathways. Cleaved caspase-3 and conversion of LC3A/B were also detected but it was dependent on cells and treatments. The combination of both agents in MSTO-211H cells rather suppressed the p53 pathways that were activated by nutrin-3a treatments, whereas the combination rather augmented the p53 actions in NCI-H28 and EHMES-10 cells. Conclusion These data collectively indicated a possible interactions between mTOR and p53 pathways, and the combinatory effects were attributable to differential mechanisms induced by a cross-talk between the pathways.

Abstract.: We examined the involvement of the Na+/Ca2+ exchanger in the automaticity of the pulmonary vein myocardium with a specific inhibitor, SEA0400. Action potentials were recorded from the myocardial layer of isolated guinea-pig pulmonary vein preparations, and Ca2+ transients were recorded from the cardiomyocytes. Spontaneous electrical activity was observed in 17.7% of the preparations, which was inhibited by either SEA0400 or ryanodine. In quiescent preparations, ouabain induced electrical activity and spontaneous Ca2+ transients, which were inhibited by SEA0400, as well as ryanodine. These results provide pharmacological evidence that the Na+/Ca2+ exchanger underlies the automaticity of the pulmonary vein myocardium. Keywords:: pulmonary vein myocardium, Na+/Ca2+ exchanger, automaticity

The chronic atrioventricular block dog is a useful model for predicting the future onset of drug-induced long QT syndrome in clinical practice. To better understand the arrhythmogenic profile of this model, we recorded the action potentials of the isolated ventricular tissues in the presence and absence of the class III antiarrhythmic drug nifekalant. The action potential durations of the Purkinje fiber and free wall of the right ventricle were longer in the chronic atrioventricular block dogs than in the dogs with normal sinus rhythm. Nifekalant in concentrations of 1 and 10 µM prolonged the action potential durations of Purkinje fiber and the free wall in a concentration-dependent manner. The extent of prolongation was greater in the chronic atrioventricular block dogs than in the normal dogs. However, increase of temporal dispersion of ventricular repolarization including early afterdepolarization was not detected by nifekalant in either group of dogs, indicating lack of potential to trigger arrhythmias in vitro. These results suggest that the ventricular repolarization delay in the chronic atrioventricular block model by nifekalant may largely depend on the decreased myocardial repolarization reserve, whereas the trigger for lethal arrhythmia was not generated in the in vitro condition in contrast to the in vivo experiment. Keywords:: action potential, chronic atrioventricular block dog, nifekalant

Involvement of the Na+/Ca2+ exchanger in ouabain-induced inotropy and arrhythmogenesis was examined with a specific inhibitor, SEA0400. In right ventricular papillary muscle isolated from guinea-pig ventricle, 1 µM SEA0400, which specifically inhibits the Na+/Ca2+ exchanger by 80%, reduced the ouabain (1 µM)-induced positive inotropy by 40%, but had no effect on the inotropy induced by 100 µM isobutyl methylxantine. SEA0400 significantly inhibited the contracture induced by low Na+ solution. In HEK293 cells expressing the Na+/Ca2+ exchanger, 1 µM ouabain induced an increase in intracellular Ca2+, which was inhibited by SEA0400. The arrhythmic contractions induced by 3 µM ouabain were significantly reduced by SEA0400. These results provide pharmacological evidence that the Na+/Ca2+ exchanger is involved in ouabain-induced inotropy and arrhythmogenesis. Keywords:: ouabain, Na+/Ca2+ exchanger, SEA0400, inotropy, arrhythmia

Interaction of Fas and Fas ligand (FasL) plays an important role in the regulation of immune responses by inducing apoptosis of activated cells; however, a possible role of FasL in DNA vaccination has not been well understood. We examined whether administration of DNA encoding FasL gene enhanced antitumor effects in mice that were vaccinated with DNA expressing a putative tumor antigen gene, β-galactosidase (β-gal). Growth of β-gal-positive Colon 26 tumors was retarded in the syngeneic mice immunized with β-gal and FasL DNA compared with those vaccinated with β-gal or FasL DNA. We did not detect increased numbers of β-gal-specific CD8+ T cells in lymph node of mice that received combination of β-gal and FasL DNA, but amounts of anti-β-gal antibody increased with the combination but not with β-gal or FasL DNA injection alone. Subtype analysis of anti-β-gal antibody produced by the combination of β-gal and FasL DNA or β-gal DNA injection showed that IgG2a amounts were greater in mice injected with both DNA than those with β-gal DNA alone, but IgG2b amounts were lower in both DNA-injected than β-gal DNA-injected mice. These data suggest that FasL is involved in boosting humoral immunity against a gene product encoded by coinjected DNA and enhances the vaccination effects.

Acquired tracheal stenosis remains a challenging problem for otolaryngologists. The objective of this study was to determine whether the Sendai virus (SeV)-mediated c-myc suppressor, a far upstream element (FUSE)-binding protein (FBP)-interacting repressor (FIR), modulates wound healing of the airway mucosa, and whether it prevents tracheal stenosis in an animal model of induced mucosal injury. A fusion gene-deleted, non-transmissible SeV vector encoding FIR (FIR-SeV/ΔF) was prepared. Rats with scraped airway mucosae were administered FIR-SeV/ΔF through the tracheostoma. The pathological changes in the airway mucosa and in the tracheal lumen were assessed five days after scraping. Untreated animals showed hyperplasia of the airway epithelium and a thickened submucosal layer with extensive fibrosis, angiogenesis, and collagen deposition causing lumen stenosis. By contrast, the administration of FIR-SeV/ΔF decreased the degree of tracheal stenosis (P < 0.05) and improved the survival rate (P < 0.05). Immunohistochemical staining showed that c-Myc expression was downregulated in the tracheal basal cells of the FIR-SeV/ΔF-treated animals, suggesting that c-myc was suppressed by FIR-SeV/ΔF in the regenerating airway epithelium of the injured tracheal mucosa. The airway-targeted gene therapy of the c-myc suppressor FIR, using a recombinant SeV vector, prevented tracheal stenosis in a rat model of airway mucosal injury.

Interferons (IFNs) have been tested for the therapeutic effects in various types of malignancy, but mechanisms of the anti-tumors effects and the differential biological activities among IFN members are dependent on respective cell types. In this study, we examined growth inhibitory activities of type I and III IFNs on 5 kinds of human mesothelioma cells bearing wild-type p53 gene, and showed that type I IFNs but not type III IFNs decreased the cell viabilities. Moreover, growth inhibitory activities and up-regulated expression levels of the major histocompatibility complexes class I antigens were greater with IFN-β than with IFN-α treatments. Cell cycle analyses demonstrated that type I IFNs increased S- and G2/M-phase populations, and subsequently sub-G1-phase fractions. The cell cycle changes were also greater with IFN-β than IFN-α treatments, and these data collectively showed that IFN-β had stronger biological activities than IFN-α in mesothelioma. Type I IFNs-treated cells increased p53 expression and the phosphorylation levels, and activated apoptotic pathways. A combinatory use of IFN-β and cisplatin or pemetrexed, both of which are the current first-line chemotherapeutic agents for mesothelioma, produced synergistic anti-tumor effects, which were also evidenced by increased sub-G1-phase fractions. These data demonstrated firstly to our knowledge that IFN-β produced synergistic anti-tumor effects with cisplatin or pemetrexed on mesothelioma through up-regulated p53 expression.

Although excessive fructose intake is epidemiologically linked with dyslipidemia, obesity, and diabetes, the mechanisms regulating plasma fructose are not well known. Cells transfected with sodium/glucose cotransporter 5 (SGLT5), which is expressed exclusively in the kidney, transport fructose in vitro; however, the physiological role of this transporter in fructose metabolism remains unclear. To determine whether SGLT5 functions as a fructose transporter in vivo, we established a line of mice lacking the gene encoding SGLT5. Sodium-dependent fructose uptake disappeared in renal brush border membrane vesicles from SGLT5-deficient mice, and the increased urinary fructose in SGLT5-deficient mice indicated that SGLT5 was the major fructose reabsorption transporter in the kidney. From this, we hypothesized that urinary fructose excretion induced by SGLT5 deficiency would ameliorate fructose-induced hepatic steatosis. To test this hypothesis we compared SGLT5-deficient mice with wild-type mice under conditions of long-term fructose consumption. Paradoxically, however, fructose-induced hepatic steatosis was exacerbated in the SGLT5-deficient mice, and the massive urinary fructose excretion was accompanied by reduced levels of plasma triglycerides and epididymal fat but fasting hyperinsulinemia compared with fructose-fed wild-type mice. There was no difference in food consumption, water intake, or plasma fructose between the two types of mice. No compensatory effect by other transporters reportedly involved in fructose uptake in the liver and kidney were indicated at the mRNA level. These surprising findings indicated a previously unrecognized link through SGLT5 between renal fructose reabsorption and hepatic lipid metabolism.

We examined anti-tumor effects of zoledronic acid (ZOL), one of the bisphosphonates agents clinically used for preventing loss of bone mass, on human mesothelioma cells bearing the wild-type p53 gene. ZOL-treated cells showed activation of caspase-3/7, -8 and -9, and increased sub-G1 phase fractions. A combinatory use of ZOL and cisplatin (CDDP), one of the first-line anti-cancer agents for mesothelioma, synergistically or additively produced the cytotoxicity on mesothelioma cells. Moreover, the combination achieved greater anti-tumor effects on mesothelioma developed in the pleural cavity than administration of either ZOL or CDDP alone. ZOL-treated cells as well as CDDP-treated cells induced p53 phosphorylation at Ser 15, a marker of p53 activation, and up-regulated p53 protein expression levels. Down-regulation of p53 levels with siRNA however did not influence the ZOL-mediated cytotoxicity but negated the combinatory effects by ZOL and CDDP. In addition, ZOL treatments augmented cytotoxicity of adenoviruses expressing the p53 gene on mesothelioma. These data demonstrated that ZOL-mediated augmentation of p53, which was not linked with ZOL-induced cytotoxicity, played a role in the combinatory effects with a p53 up-regulating agent, and suggests a possible clinical use of ZOL to mesothelioma with anti-cancer agents.

In guinea pig ventricular cardiomyocytes, the R(−)-enantiomer of efonidipine concentration-dependently blocked T-type Ca2+ current with 85% inhibition at 1 μM. In contrast, R(−)-efonidipine (1 μM) had no effect on the L-type Ca2+ current and Ca2+ transient in cardiomyocytes and contractile force in papillary muscles. Thus, R(−)-efonidipine is a highly selective blocker of the T-type Ca2+ current in native myocardia. Keywords:: R(−)-efonidipine, T-type Ca2+ channel, cardiomyocyte

Subtotal esophagectomy with extended lymph node dissection is a standard procedure for thoracic esophageal squamous cell carcinoma. Three-field lymphadenectomy, including neck dissection, is a standard type of lymph node dissection for complete clearance of tumor cells. Based on various series of analyses for lymph node metastases, the appropriate indication for neck dissection has been clarified. Herein, we describe the established techniques of neck dissection and review recent topics of three-field lymph node dissection for thoracic esophageal squamous cell carcinoma.

Recently identified interleukin-28 and -29 belong to a novel type III interferon (IFN) family, which could have distinct biological properties from type I and II IFNs. Type I IFNs, IFN-α/β, have been clinically applied for treating a certain kind of malignancies for over 30 years, but a wide range of the adverse effects hampered the further clinical applications. Type III IFNs, IFN-λs, have similar signaling pathways as IFN-α/β and inhibits proliferation of tumor cells through cell cycle arrest or apoptosis. Restricted patterns of type III IFN receptor expression in contrast to ubiquitously expressed IFN-α/β receptors suggest that type III IFNs have limited cytotoxicity to normal cells and can be a possible anticancer agent. In this paper, we summarize the current knowledge on the IFN-λs-mediated tumor cell death and discuss the functional difference between type I and III IFNs.

Objective: Unusual clinical course. Background: Although recurrence after surgery for esophagogastric junction (EGJ) adenocarcinoma frequently develops in the mediastinal and para-aortic lymph nodes (LN), distant LN recurrence in the mesocolon is rare. We report a rare case of ileocecal LN metastasis in the ascending mesocolon after radical surgery for an EGJ adenocarcinoma. Case Report: We performed subtotal esophagectomy with mediastinal and para-gastric LN dissection in a patient with an advanced EGJ adenocarcinoma. Clinicopathologically, the patient was diagnosed with type I EGJ adenocarcinoma based on Siewert's classification (pathological T3N1M0). One year after surgery, computed tomography showed enlarged lymph nodes around the ileocolic artery, and further examination was performed. Although positron emission tomography-computed tomography showed that the lesion had moderate uptake of fluorodeoxyglucose, we did not find the reason for the enlarged lymph nodes. Finally, laparoscopic ileocecal resection was performed for diagnostic and therapeutic purposes. Clinicopathological tests revealed that the specimen was a moderately differentiated adenocarcinoma, which was strongly suspected to be a metastasis of the EGJ adenocarcinoma. Conclusions: We encountered a rare case of EGJ adenocarcinoma that spread to the ileocecal LN in the ascending mesocolon. To the best of our knowledge, this is the first such report in the literature to date. Laparoscopic ileocecal resection for metastasis to the ascending mesocolon seems reasonable as a local control. [ABSTRACT FROM AUTHOR]