Diphenyl-tetrazol-propanamide Derivatives Act as Dual-Specific Antagonists of Platelet CLEC-2 and Glycoprotein VI.

BACKGROUND: Platelet C-type lectin-like receptor 2 (CLEC-2) induces platelet activation and aggregation after clustering by its ligand podoplanin (PDPN). PDPN, which is not normally expressed in cells in contact with blood flow, is induced in inflammatory immune cells and some malignant tumor cells, thereby increasing the risk of venous thromboembolism (VTE) and tumor metastasis. Therefore, small-molecule compounds that can interfere with the PDPN-CLEC-2 axis have the potential to become selective antiplatelet agents. METHODS AND RESULTS: Using molecular docking analysis of CLEC-2 and a PDPN-CLEC-2 binding-inhibition assay, we identified a group of diphenyl-tetrazol-propanamide derivatives as novel CLEC-2 inhibitors. A total of 12 hit compounds also inhibited PDPN-induced platelet aggregation in humans and mice. Unexpectedly, these compounds also fit the collagen-binding pocket of the glycoprotein VI molecule, thereby inhibiting collagen interaction. These compounds also inhibited collagen-induced platelet aggregation, and one compound ameliorated collagen-induced thrombocytopenia in mice. For clinical use, these compounds will require a degree of chemical modification to decrease albumin binding. CONCLUSION: Nonetheless, as dual activation of platelets by collagen and PDPN-positive cells is expected to occur after the rupture of atherosclerotic plaques, these dual antagonists could represent a promising pharmacophore, particularly for arterial thrombosis, in addition to VTE and metastasis.

Contributions of histone tail clipping and acetylation in nucleosome transcription by RNA polymerase II.

The N-terminal tails of histones protrude from the nucleosome core and are target sites for histone modifications, such as acetylation and methylation. Histone acetylation is considered to enhance transcription in chromatin. However, the contribution of the histone N-terminal tail to the nucleosome transcription by RNA polymerase II (RNAPII) has not been clarified. In the present study, we reconstituted nucleosomes lacking the N-terminal tail of each histone, H2A, H2B, H3 or H4, and performed RNAPII transcription assays. We found that the N-terminal tail of H3, but not H2A, H2B and H4, functions in RNAPII pausing at the SHL(-5) position of the nucleosome. Consistently, the RNAPII transcription assay also revealed that the nucleosome containing N-terminally acetylated H3 drastically alleviates RNAPII pausing at the SHL(-5) position. In addition, the H3 acetylated nucleosome produced increased amounts of the run-off transcript. These results provide important evidence that the H3 N-terminal tail plays a role in RNAPII pausing at the SHL(-5) position of the nucleosome, and its acetylation directly alleviates this nucleosome barrier.

The Limited Effect of a History of COVID-19 on Antibody Titers and Adverse Reactions Following BNT162b2 Vaccination: A Single-Center Prospective Study.

Background and Objectives: The need for, and ideal frequency of, the vaccination against coronavirus disease 2019 (COVID-19) of previously infected individuals have not yet been sufficiently evaluated. The aim of this study was to examine the anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody status and adverse reactions after vaccination among medical staff with or without a history of COVID-19. Materials and Methods: A single-center prospective study was performed at Fukuoka University Chikushi Hospital. We investigated the presence of the anti-SARS-CoV-2 antibody titer among medical staff before and after mRNA vaccination with the BNT162b2. The levels of immunoglobulin G antibody were quantitatively measured at six points-before vaccination, after the first vaccination, at three points after the second vaccination, and finally, after the third vaccination-and the levels were then compared based on the COVID-19 infection history. Results: The previously infected (before the first vaccination) subjects (n = 17) showed a marked increase in antibody titers two weeks after the first vaccination and four weeks after the second vaccination. Although they were able to maintain a certain level of antibody titers until 30 weeks after the second vaccination, the titers fell in the same way as observed in the non-infected subjects. The subjects who did not receive the third vaccination due to adverse reactions to previous vaccines (n = 1) or who were positive for COVID-19 prior to the third vaccination (n = 2) were excluded from the subsequent analyses. Among non-infected subjects (n = 36), smokers had lower peak antibody titers than the others. The previously infected subjects had a significantly higher incidence of adverse reactions after the first vaccination but had a similar incidence of adverse reactions after the second and third vaccinations compared to the non-infected subjects. Conclusions: A history of COVID-19 may influence only the initial increase in anti-SARS-CoV-2 antibody titers and the occurrence of adverse reactions after the first vaccination.

Effective connectivity and criminal sentencing decisions: dynamic causal models in laypersons and legal experts.

This magnetic resonance imaging study is designed to obtain relevant implications for criminal justice and explores the effective connectivity underlying expertise. Laypersons and experts considered sentences for remorseful and remorseless defendants, respectively, with and without mitigation, in hypothetical murder cases. Two groups revealed no differential activation. However, dynamic causal modeling analysis found distinct patterns of connectivity associated with subjects' expertise and mitigating factors. In sentencing for remorseful defendants, laypersons showed increased strength in all bidirectional connections among activated regions of Brodmann area (BA) 32, BA23, the right posterior insula, and the precuneus. In contrast, legal experts sentenced based on mitigation reasoning, showed increased strength only in the bidirectional connection between the insula and the precuneus. When sentencing for remorseless ones without mitigation, both laypersons and experts increased the connection strength, but with reverse directionality, between regions; legal experts strengthened connectivity from BA10 to other regions, that is, the right anterior insula and BA23, but the directionality was reversed in laypersons. In addition, the strength of connection to BA32 and BA10 was correlated with changes in punishments by mitigating factors. This is a crucial result that establishes the validity of the connectivity estimates, which were uninformed by the independent (behavioral) differences in the severity of punishment.

Pelvic organ prolapse and Japanese lifestyle: prevalence and risk factors in Japan.

INTRODUCTION AND HYPOTHESIS: Little is known about the prevalence of pelvic organ prolapse (POP). We aimed to evaluate the prevalence of POP and identify its risk factors in Japan. METHODS: This was a single-centre, cross-sectional study. We recruited Japanese women seen for a Pap smear from July 2018 through May 2019. After providing their informed consent, subjects were asked to complete questionnaires. Pelvic organ support was assessed using the POP quantification (POP-Q) system by an examiner. Logistic regression analyses were conducted to identify risk factors for POP. RESULTS: There were 1032 women aged 21 to 84 years. The distribution of POP-Q stage was stage 0, 38.0%; stage I, 45.0%; stage II, 16.4%; stage III, 0.6%; and stage IV, 0%. Rates (95% confidence interval [CI]) of stage II or greater in each age group were 6.6% (2.4-10.8) in 20 s-30 s; 17.6% (13.3-21.9) in 40 s; 17.1% (12.9-21.3) in 50 s; 18.0% (12.6-23.4) in 60 s; and 28.7% (19.6-37.9) in 70 s and over. Multivariate analysis revealed the following risk factors for POP, with odds ratio (95% CI): body mass index [BMI] ≥ 25 kg/m2, 1.63 (1.05-2.51); BMI < 18.5 kg/m2, 0.40 (0.17-0.94); hysterectomy, 4.09 (1.55-10.80); ≥ 3 vaginal deliveries, 2.26 (1.19-4.28); and ≥ 1 cup of coffee per day, 0.63 (0.43-0.92). CONCLUSION: Among Japanese women undergoing routine gynaecological examinations, 17.1% (14.7-19.5) had POP-Q stage II or greater. Overweight, hysterectomy and ≥ 3 vaginal deliveries increased the risk for POP, whereas underweight and daily coffee consumption decreased it.

Individual feedback and monitoring of endoscopist performance improves the adenoma detection rate in screening colonoscopy: a prospective case-control study.

BACKGROUND AND PURPOSE: Previous reports have suggested that a longer withdrawal time (WT) during colonoscopy led to an improved adenoma detection rate (ADR); however, there are few controlled studies that substantiated monitoring WT as an educational method. We aimed to validate a feedback and monitoring system to improve the ADR in screening colonoscopy in a prospective case-control setting. METHODS: After collecting data in the pre-feedback period (3.5 months), the individual performance and the average ADR and WT values of the facility were provided to 6 endoscopists in the intervention group, while 3 endoscopists were isolated as the control group during the feedback period (2 weeks). The intervention group consisted of two subgroups, the Fast and Slow WT groups, according to the results from the pre-feedback period. The endoscopists in the intervention group were instructed to be aware of their own WT in each examination during the post-feedback period (4 months). The performances of all endoscopists in the post-feedback period were analyzed and compared with those in the pre-feedback period. RESULTS: Among the initial analyses, the correlation analysis and multivariate analysis revealed that WT was an independent predictor for the ADR (P = 0.0101). After providing individual performance feedback and instruction regarding real-time WT monitoring, the WT was significantly prolonged in the Fast WT group (P = 0.0346) but did not change in the Slow WT and control groups. In addition, the ADR of the Fast WT group significantly improved after the intervention (P = 0.024), whereas the ADR of the Slow WT and control groups did not change. CONCLUSION: Providing individual feedback on ADR and WT and monitoring WT helped improve the endoscopists' ADRs.

Acetylation-modulated communication between the H3 N-terminal tail domain and the intrinsically disordered H1 C-terminal domain.

Linker histones (H1s) are key structural components of the chromatin of higher eukaryotes. However, the mechanisms by which the intrinsically disordered linker histone carboxy-terminal domain (H1 CTD) influences chromatin structure and gene regulation remain unclear. We previously demonstrated that the CTD of H1.0 undergoes a significant condensation (reduction of end-to-end distance) upon binding to nucleosomes, consistent with a transition to an ordered structure or ensemble of structures. Here, we show that deletion of the H3 N-terminal tail or the installation of acetylation mimics or bona fide acetylation within H3 N-terminal tail alters the condensation of the nucleosome-bound H1 CTD. Additionally, we present evidence that the H3 N-tail influences H1 CTD condensation through direct protein-protein interaction, rather than alterations in linker DNA trajectory. These results support an emerging hypothesis wherein the H1 CTD serves as a nexus for signaling in the nucleosome.

A randomized, double-blind, placebo-controlled, phase 3 study of tivantinib in Japanese patients with MET-high hepatocellular carcinoma.

A previous randomized phase 2 study of hepatocellular carcinoma revealed that the c-Met inhibitor tivantinib as second-line treatment significantly prolonged progression-free survival in a subpopulation whose tumor samples highly expressed c-Met (MET-high). Accordingly, this phase 3 study was conducted to evaluate the efficacy of tivantinib as a second-line treatment for Japanese patients with MET-high hepatocellular carcinoma. This randomized, double-blind, placebo-controlled study was conducted at 60 centers in Japan. Hepatocellular carcinoma patients with one prior sorafenib treatment and those with MET-high tumor samples were eligible for inclusion. Registered patients were randomly assigned to either the tivantinib or placebo group at a 2:1 ratio and were treated with twice-a-day oral tivantinib (120 mg bid) or placebo until the discontinuation criteria were met. The primary endpoint was progression-free survival while the secondary endpoints included overall survival and safety. Between January 2014 and June 2016, 386 patients provided consent, and 195 patients were randomized to the tivantinib (n = 134) or placebo (n = 61) group. Median progression-free survival was 2.8 (95% confidence interval: 2.7-2.9) and 2.3 (1.5-2.8) mo in the tivantinib and placebo groups, respectively (hazard ratio = 0.74, 95% confidence interval: 0.52-1.04, P = .082). Median overall survival was 10.3 (95% confidence interval: 8.1-11.6) and 8.5 (6.2-11.4) mo in the tivantinib and placebo group, respectively (hazard ratio = 0.82, 95% confidence interval: 0.58-1.15). The most common tivantinib-related grade ≥3 adverse events were neutropenia (31.6%), leukocytopenia (24.8%), and anemia (12.0%). This study did not confirm the significant efficacy of tivantinib as a second-line treatment for Japanese patients with MET-high hepatocellular carcinoma. (NCT02029157).

Quality Indicators for the Detection of Helicobacter pylori-Negative Early Gastric Cancer: A Retrospective Observational Study.

BACKGROUND/AIMS: While Helicobacter pylori (HP)-negative gastric cancer is frequently reported, little is known about the predictors for detecting HP-negative early gastric cancer (EGC). We aimed to evaluate the predictors for the detection of HP-negative EGC. METHODS: We retrospectively reviewed 13,477 consecutive asymptomatic cases where upper endoscopy was performed by nine physicians from April 2017 to March 2019 and analyzed the detection rate of high-risk lesions (HRLs), including EGC, tubular adenoma, and lymphoma, according to the status of HP infection. The observation time was corrected for multiple regression analyses. RESULTS: For all physicians, the average observation time for screening HP-eradicated and -naïve patients was shorter than that for screening HP-positive patients (p<0.05). Multiple regression analyses revealed that the observation time in the three groups was an independent predictor for detecting HRLs in HP-eradicated patients (p=0.03106, 0.01263, and 0.02485, respectively), while experience of endoscopy was an independent predictor for detecting HRLs in HP-naïve patients (p=0.02638). CONCLUSION: While observation time during screening endoscopy was a quality indicator for detecting HRLs in HP-eradicated patients, experience of endoscopy was a quality indicator for detecting HRLs in HP-naïve patients.

Human pursuance of equality hinges on mental processes of projecting oneself into the perspectives of others and into future situations.

In the pursuance of equality, behavioural scientists disagree about distinct motivators, that is, consideration of others and prospective calculation for oneself. However, accumulating data suggest that these motivators may share a common process in the brain whereby perspectives and events that did not arise in the immediate environment are conceived. To examine this, we devised a game imitating a real decision-making situation regarding redistribution among income classes in a welfare state. The neural correlates of redistributive decisions were examined under contrasting conditions, with and without uncertainty, which affects support for equality in society. The dorsal anterior cingulate cortex (dACC) and the caudate nucleus were activated by equality decisions with uncertainty but by selfless decisions without uncertainty. Activation was also correlated with subjective values. Activation in both the dACC and the caudate nucleus was associated with the attitude to prefer accordance with others, whereas activation in the caudate nucleus reflected that the expected reward involved the prospective calculation of relative income. The neural correlates suggest that consideration of others and prospective calculation for oneself may underlie the support for equality. Projecting oneself into the perspective of others and into prospective future situations may underpin the pursuance of equality.

Combination therapy with zoledronic acid and cetuximab effectively suppresses growth of colorectal cancer cells regardless of KRAS status.

Targeted molecular therapy is an effective anticancer strategy. Anti-EGFR monoclonal antibodies such as cetuximab (CTX) have been approved for the treatment of various malignancies, including colorectal cancer (CRC) with wild-type KRAS. However, their efficacy in patients with KRAS mutations has not been established. Therefore, we investigated whether CTX treatment was effective as a single agent or in combination with zoledronic acid (ZOL) in human CRC cell lines with different KRAS status. CRC cell lines SW48 (wild-type KRAS) and LS174T (mutant KRAS) were treated with ZOL, CTX and a combination of both drugs. Cytotoxicity was measured using the MTT assay. Changes in the levels of intracellular signaling proteins were evaluated using western blot analysis. Finally, we evaluated the efficacy of the combination treatment in an in vivo xenograft model. We observed that ZOL apparently inhibited growth in both cell lines, whereas CTX showed little effect. ZOL also increased the levels of unprenylated RAS. Combined ZOL and CTX treatment was synergistic in both cell lines and was associated with inhibition of the RAS-MAPK and AKT-mTOR signaling pathways. Furthermore, the combination treatment was more effective in suppressing the growth of xenografts derived from both SW48 and LS174T cells; this effect was associated with increased apoptosis. These results demonstrate that ZOL inhibits the growth of colon cancer cells regardless of KRAS status, and combination therapy using ZOL and CTX enhances this growth suppression. These findings suggest a novel strategy for the treatment of CRC independent of KRAS mutational status.

Computed tomograpy evaluation of morphological changes, clinical response and survival in colorectal cancer liver metastasis treated by regorafenib: A case report.

Pathological response to preoperative chemotherapy was recently found to be correlated with improved survival and has been proposed as a new outcome end-point following resection of liver metastases from colorectal cancer (CRC). It was demonstrated that, particularly after therapy with bevacizumab, CRC liver metastases decreased in size and underwent distinct morphological changes on computed tomography (CT). However, morphological changes in response to treatment with regorafenib have not yet been reported. A 73-year-old male patient with synchronous multiple liver and lung metastases from colon cancer was treated with regorafenib as a fifth-line therapy. CT imaging revealed a decrease in the tumor size and distinct morphological changes, namely homogeneous attenuation and sharp tumor-liver interface. The patient continued to take regorafenib for 8 months. Thus, regorafenib appeared to be effective as a last-line chemotherapy. In particular, the unique morphological changes of the metastases on CT imaging of may represent a method for evaluating the effects of CRC cancer therapy.

Efficacy of recombinant human soluble thrombomodulin in patients with sepsis and disseminated intravascular coagulation in the gastroenterology field.

Recombinant human soluble thrombomodulin (rTM) was approved recently and has been used for treatment of disseminated intravascular coagulation (DIC). The aim of the present study was to evaluate the efficacy of rTM for DIC. The data of 53 inpatients with sepsis-induced DIC were retrospectively analyzed. Patients were classified into the rTM treatment group (n=25) and conventional treatment group (rTM not used) was the control group (n=28). Diagnosis of DIC was made according to the criteria for acute DIC of the Japan Association of Acute Medicine. Platelet count, prothrombin time-international normalized ratio, levels of fibrin/fibrinogen degradation products (FDP), C-reactive protein and DIC scores were measured on days 0, 3 and 7. Furthermore, the DIC resolution rate was assessed on days 3 and 7. Prior to treatment, DIC scores were 5.0±1.0 in the rTM group and 5.9±1.3 in the control group (P<0.05). Significant intra-group improvements were observed in all the parameters, except for FDP in the two groups. Significant improvements were observed in the DIC scores in the rTM group (Δ2.0±1.9 vs. Δ1.5±1.9, P=0.001). Therefore, the results suggest that rTM would be a useful medicine for treatment of DIC in the gastroenterology field.

Metastatic seminoma in the duodenum: diagnosis with endoscopy followed by successful treatment.

Seminomas rarely metastasize to the gastrointestinal tract. In general, these lesions metastasize to the lungs or retroperitoneal lymph nodes. A 34-year-old Japanese man who had undergone orchiectomy for seminoma two years earlier experienced shortness of breath and tarry stools. The patient presented at our hospital and was diagnosed with metastatic seminoma to the third portion of the duodenum on double balloon endoscopy. He was effectively treated with chemotherapy and continues to progress well, with no episodes of recurrence.

Phase I clinical trial of peptide vaccination with URLC10 and VEGFR1 epitope peptides in patients with advanced gastric cancer.

Peptide vaccine treatment has attracted attention in recent years as a new therapy option for chemotherapy­resistant, advanced, unresectable cancer. The safety of peptide vaccination with HLA-A*2402-restricted URLC10-A24-177 and VEGFR1-A12-9 1084 epitope peptides (fixed 2-mg dose) was investigated in a phase I clinical trial of patients with advanced gastric cancer who were refractory to chemotherapy. We determined the HLA genotype of the subjects after enrollment, results of which were held by the evaluation committee and kept from both patients and investigators until completion of the study. The primary end­point was safety of the peptide vaccination. The secondary end­points were immunological responses and clinical outcome, which were compared between the HLA-A*2402-positive and HLA-A*2402-negative groups. The peptides were subcutaneously administered on day 1, 8, 15 and 22 within a 28-day treatment cycle. A total of 14 patients was enrolled in this study; 12 of the 14 patients received 4 or more vaccinations (at least 1 course). No patient had a severe treatment-related adverse event. Findings from evaluation of clinical responses after a single course showed that 4 cases had stable disease and 8 cases had progressive disease. The median overall survival time (MST) for the 12 patients was 3.9 months. The MSTs in the HLA-A*2402­positive and HLA-A*2402­negative groups were, 4.2 and 3.6 months (p=0.9164), respectively. The results of this study showed that vaccination with URLC10 and VEGFR1 peptides was a safe treatment for advanced gastric cancer. This trial was registered with University Hospital Medical Information Network (UMIN, no. 000002409).

Varicella-zoster virus ORF49 functions in the efficient production of progeny virus through its interaction with essential tegument protein ORF44.

The ORF49 tegument protein of varicella-zoster virus (VZV) is one of the core gene products that is conserved among herpesvirus family members. Although ORF49 is known to be a cell-tropic factor, its detailed functions remain elusive. ORF44 is another core gene product reported to be essential, although its characterization and detailed functional analysis have not been reported. These two core gene products form a complex in other herpesviruses beyond the host species and herpesvirus subfamilies. Here, we show that complex formation between ORF44 and ORF49 is conserved in VZV. We serendipitously found that binding is eliminated by an amino acid substitution at position 129 (phenylalanine 129), and four amino acids in the carboxyl-terminal half of the acidic cluster in ORF49 (i.e., aspartate-phenylalanine-aspartate-glutamate from positions 41 to 44 [41DFDE44]) were identified as its binding motif. Alanine substitutions in each domain rendered the ORF44F129A mutation lethal for VZV, similar to deletion of the entire ORF44. The phenotype of the ORF49-41AAAA44 mutation was comparable to that of the ORF49-defective virus, including small-plaque formation, impaired growth, and low infectious virus production. These results suggest that the interaction between ORF44 and ORF49 is essential for their role in VZV infection and that ORF49 is required for the efficient production of infectious progeny virus mediated by the conserved interaction between the two proteins.

Repression of Factor VIII Inhibitor Development with Apoptotic Factor VIII-expressing Embryonic Stem Cells.

Development of factor VIII (fVIII)-neutralizing antibodies, called inhibitors, is a challenging problem in the management of hemophilia A patients. We explored the possibility of pretreatment with apoptotic fVIII-expressing embryonic stem (ES) cells to prevent the development of fVIII inhibitors. Murine ES cells integrated with the human F8 gene were differentiated into embryoid bodies, dissociated to a single cell suspension, subjected to hypo-osmotic shock to induce apoptosis, and intraperitoneally injected into hemophilia A mice. Inhibitors were induced by periodic intraperitoneal injections of recombinant human fVIII (rhfVIII). In the groups in which intraperitoneal injections of rhfVIII began at 1-3 weeks after pretreatment, the titers of inhibitors were significantly lower after the third administration of rhfVIII compared with that in the control group in which apoptotic Ainv18 ES cells (without the human F8 gene) were used for pretreatment, and continued to show lower levels until the sixth administration of rhfVIII. These results suggest that pretreatment with apoptotic hfVIII-expressing ES cells might be promising for the prevention of fVIII inhibitor development in hemophilia A patients.

Decreased FANCJ caused by 5FU contributes to the increased sensitivity to oxaliplatin in gastric cancer cells.

BACKGROUND: Oxaliplatin is effective against many types of cancer, and the combination of 5-fluorouracil (5FU) and oxaliplatin is synergistically effective against gastric cancer, as well as colon cancer. The FANCJ protein is one of the Fanconi anemia (FA) gene products, and its interaction with the tumor suppressor BRCA1 is required for DNA double-strand break (DSB) repair. FANCJ also functions in interstrand crosslinks (ICLs) repair by linking to mismatch repair protein complex MLH1-PMS2 (MutLα). While oxaliplatin causes ICLs, 5FU is considered to cause DSBs. Therefore, we investigated the importance of FANCJ in the synergistic effects of oxaliplatin and 5FU in MKN45 gastric cancer cells and the derived 5FU-resistant cell line, MKN45/F2R. METHODS: MKN1, TMK1, MKN45, and MKN45/F2R (5FU-resistant) gastric cancer cells were treated with 5FU and/or oxaliplatin. The signaling pathway was evaluated by a western blotting analysis and reverse transcription polymerase chain reaction (RT-PCR). Drug resistance was evaluated by the 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. RESULTS: In MKN45 cells, the combination of 5FU and oxaliplatin had synergistic effects. DSBs appeared when the cells were treated with 5FU. FANCJ was down-regulated, and BRCA1 was induced in a dose- and time-dependent manner. MKN45 cells showed increased sensitivity to oxaliplatin when FANCJ was knocked down by short interfering (si) RNA. However, these findings were not observed in MKN45/F2R 5FU-resistant cells. CONCLUSION: These results strongly suggest that the decrease in FANCJ caused by 5FU treatment leads to an increase in sensitivity to oxaliplatin, thus indicating that the FANCJ protein plays an important role in the synergism of the combination of 5FU and oxaliplatin.

Extracellular signal-regulated kinase and Akt activation play a critical role in the process of hepatocyte growth factor-induced epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) has recently been studied to elucidate mechanisms of the liver metastatic process. We investigated EMT in the process of liver metastasis and the effects of chemotherapy on EMT cells as therapeutic strategy for colorectal liver metastasis. We used the CT26 murine colorectal carcinoma cell line to create an in vivo mouse liver metastasis model. Liver tumors were stained immuno-histochemically. Expression of proteins associated with TGF-ß/Smad and hepatocyte growth factor (HGF)/c-Met pathways were investigated by western blotting. Cells with c-Met mRNA knockdown by siRNA techniques showed clearly reduced liver metastases compared with regular cells at 21 days. TGF-ß and HGF induced EMT expression, but signal transduction was quite different. TGF-ß induced ERK, but not Akt phosphory-lation. HGF mediated both ERK and Akt phosphorylation. Akt inhibitor blocked Akt phosphorylation but did not affect TGF-ß-induced activation of ERK, Snail and Slug. U-0126 did not reduce Snail activity by TGF-ß at a concentration to block ERK phosphorylation. However, Akt inhibitor and U-0126 completely inhibited HGF-induced Slug activation. 5-FU mediated cell death in the EMT process induced by TGF-ß more effectively than HGF. ERK/Akt signaling, but not the Smad pathway, may be one of the main processes in HGF-induced EMT, despite the Smad pathway, but not ERK/Akt, being critical for TGF-ß-induced EMT. The MAPK/Akt pathway is indispensable in HGF/c-Met signaling. The ERK/Akt pathway particularly may be critical in the HGF-induced EMT process. However, long-term use of chemotherapeutic agents may induce drug resistance and distant metastases through EMT-related signaling pathway activation.

Interferon-gamma-mediated tissue factor expression contributes to T-cell-mediated hepatitis through induction of hypercoagulation in mice.

UNLABELLED: Concanavalin A (Con A) treatment induces severe hepatitis in mice in a manner dependent on T cells, interferon (IFN)-gamma, and tumor necrosis factor (TNF). Treatment with the anticoagulant heparin protects against hepatitis, despite healthy production of IFN-γ and TNF. Here, we investigated molecular and cellular mechanisms for hypercoagulation-mediated hepatitis. After Con A challenge, liver of wild-type (WT) mice showed prompt induction of Ifnγ and Tnf, followed by messenger RNA expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), which initiate blood coagulation and inhibit clot lysis, respectively. Mice developed dense intrahepatic fibrin deposition and massive liver necrosis. In contrast, Ifnγ(-/-) mice and Ifnγ(-/-) Tnf(-/-) mice neither induced Pai1 or Tf nor developed hepatitis. In WT mice TF blockade with an anti-TF monoclonal antibody protected against Con A-induced hepatitis, whereas Pai1(-/-) mice were not protected. Both hepatic macrophages and sinusoidal endothelial cells (ECs) expressed Tf after Con A challenge. Macrophage-depleted WT mice reconstituted with hematopoietic cells, including macrophages deficient in signal transducer and activator of transcription-1 (STAT1) essential for IFN-γ signaling, exhibited substantial reduction of hepatic Tf and of liver injuries. This was also true for macrophage-depleted Stat1(-/-) mice reconstituted with WT macrophages. Exogenous IFN-γ and TNF rendered T-cell-null, Con A-resistant mice deficient in recombination-activating gene 2, highly susceptible to Con A-induced liver injury involving TF. CONCLUSIONS: Collectively, these results strongly suggest that proinflammatory signals elicited by IFN-γ, TNF, and Con A in both hepatic macrophages and sinusoidal ECs are necessary and sufficient for the development of hypercoagulation-mediated hepatitis.