TET2 (Ten Eleven Translocation 2) is a dioxygenase that converts methylcytosine (mC) to hydroxymethylcytosine (hmC). TET2 loss-of-function mutations are highly frequent in subtypes of T-cell lymphoma that harbor follicular helper T (Tfh)-cell-like features, such as angioimmunoblastic T-cell lymphoma (30–83%) or peripheral T-cell lymphoma, not otherwise specified (10–49%), as well as myeloid malignancies. Here, we show that middle-aged Tet2 knockdown (Tet2gt/gt) mice exhibit Tfh-like cell overproduction in the spleen compared with control mice. The Tet2 knockdown mice eventually develop T-cell lymphoma with Tfh-like features after a long latency (median 67 weeks). Transcriptome analysis revealed that these lymphoma cells had Tfh-like gene expression patterns when compared with splenic CD4-positive cells of wild-type mice. The lymphoma cells showed lower hmC densities around the transcription start site (TSS) and higher mC densities at the regions of the TSS, gene body and CpG islands. These epigenetic changes, seen in Tet2 insufficiency-triggered lymphoma, possibly contributed to predated outgrowth of Tfh-like cells and subsequent lymphomagenesis. The mouse model described here suggests that TET2 mutations play a major role in the development of T-cell lymphoma with Tfh-like features in humans.
The tumor suppressor gene p53 regulates apoptosis in response to DNA damage. Promoter selectivity of p53 depends on mainly its phosphorylation. Particularly, the phosphorylation at serine-46 of p53 is indispensable in promoting pro-apoptotic genes that are, however, poorly determined. In the current study, we identified palmdelphin as a pro-apoptotic gene induced by p53 in a phosphorylated serine-46-specific manner. Upregulation of palmdelphin was observed in wild-type p53-transfected cells, but not in serine-46-mutated cells. Expression of palmdelphin was induced by p53 in response to DNA damage. In turn, palmdelphin induced apoptosis. Intriguingly, downregulation of palmdelphin resulted in necroptosis-like cell death via ATP depletion. Upon DNA damage, palmdelphin dominantly accumulated in the nucleus to induce apoptosis. These findings define palmdelphin as a target of serine-46-phosphorylated p53 that controls cell death in response to DNA damage.
palmdelphin; p53; DNA damage; apoptosis
We examined nitrate-dependent Fe2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “Candidatus Brocadia sinica” anaerobically oxidized Fe2+ and reduced NO3− to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein−1 min−1, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “Ca. Brocadia sinica” (10 to 75 nmol NH4+ mg protein−1 min−1). A 15N tracer experiment demonstrated that coupling of nitrate-dependent Fe2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO3− by “Ca. Brocadia sinica.” The activities of nitrate-dependent Fe2+ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO3− ± SD of “Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe2+ oxidation was further demonstrated by another anammox bacterium, “Candidatus Scalindua sp.,” whose rates of Fe2+ oxidation and NO3− reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein−1 min−1, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO2− to consumed NH4+ (ΔNO2−/ΔNH4+) and produced NO3− to consumed NH4+ (ΔNO3−/ΔNH4+). These reactions are preferable to the application of anammox processes for wastewater treatment.
High-resolution CT angiography (CTA) is currently available using multidetector row CT (MDCT); however, its use for small artery visualisation has been limited. To evaluate its capability, we investigated CTA visualisation for difference in number of the lenticulostriate artery (LSA) branches between normotensive and hypertensive patients, because hypertension is a major cause of LSA damage.
This was a retrospective study evaluating cerebrovascular CTA at our hospital conducted from February 2008 to June 2009 under approval of the institutional review board. 117 patients (39 males and 78 females, 19–88 years old) were included. CTA was conducted using a 64 channel MDCT. Total numbers of LSA branches were examined for differences by age with regression analysis and the presence or absence of hypertension and/or aneurysm using two-sample t-tests. A p-value <0.016 was considered statistically significant after correction for multiple comparisons. A multiple variable analysis of three factors was also conducted.
The average number of LSA branches was 3.6 [95% confidence interval (CI) 3.0–4.1] and 4.4 (95% CI 4.1–4.7), respectively, for a patient with and without history of hypertension, and the difference was statistically significant (p=0.013). The difference was approximately one branch in the multiple variable analysis. No significant correlation was observed for age and no significant difference was observed for the presence or absence of aneurysms.
Contrast-enhanced CTA can visualise significant differences in the number of LSA branches among patients with and without hypertension.
Advances in knowledge
Current high-resolution CTA can visualise LSA well, which enables finding a difference in the LSA between normotensive subjects and hypertensive patients.
Tenascin-C (TNC), a large hexameric extracellular glycoprotein, is a pleiotropic molecule with multiple domains binding to a variety of receptors mediating a wide range of cellular functions. We earlier reported that TNC induces epithelial–mesenchymal transition (EMT)-like change in breast cancer cells. In the present study, we clarified TNC receptor involvement in this process. Among integrins previously reported as TNC receptors, substantial expression of αv, α2, β1 and β6 subunits was detected by quantitative PCR and immunoblotting in MCF-7 cells. Integrin β6 mRNA was remarkably upregulated by transforming growth factor (TGF)-β1 treatment, and protein expression was prominently increased by additional exposure to TNC. Immunofluorescent labeling demonstrated integrin αvβ6 accumulation in focal adhesions after TNC treatment, especially in combination with TGF-β1. The α2 and β1 subunits were mainly localized at cell–cell contacts, αv being found near cell cluster surfaces. Immunoprecipitation showed increase in αvβ1 heterodimers, but not α2β1, after TNC treatment. Activated β1 subunits detected by an antibody against the Ca2+-dependent epitope colocalized with αv in focal adhesion complexes, associated with FAK phosphorylation at tyrosine 925. Neutralizing antibodies against αv and β1 blocked EMT-like change caused by TNC alone. In addition, anti-αv and combined treatment with anti-β1 and anti-αvβ6 inhibited TGF-β1/TNC-induced EMT, whereas either of these alone did not. Integrin subunits αv, β1 and β6, but not α2, bound to TNC immobilized on agarose beads in a divalent cation-dependent manner. Treatments with neutralizing antibodies against β1 and αvβ6 reduced αv subunit bound to the beads. Immunohistochemistry of these receptors in human breast cancer tissues demonstrated frequent expression of β6 subunits in cancer cells forming scattered nests localized in TNC-rich stroma. These findings provide direct evidence that binding of αvβ6 and αvβ1 integrins to TNC as their essential ligand induces EMT-like change in breast cancer cells.
breast cancer cells; epithelial–mesenchymal transition; integrin αvβ1; integrin αvβ6; tenascin-c; transforming growth factor β
Molecular characterisation using gene-expression profiling will undoubtedly improve the prediction of treatment responses, and ultimately, the clinical outcome of cancer patients.
To establish the procedures to identify responders to FOLFOX therapy, 83 colorectal cancer (CRC) patients including 42 responders and 41 non-responders were divided into training (54 patients) and test (29 patients) sets. Using Random Forests (RF) algorithm in the training set, predictor genes for FOLFOX therapy were identified, which were applied to test samples and sensitivity, specificity, and out-of-bag classification accuracy were calculated.
In the training set, 22 of 27 responders (81.4% sensitivity) and 23 of 27 non-responders (85.1% specificity) were correctly classified. To improve the prediction model, we removed the outliers determined by RF, and the model could correctly classify 21 of 23 responders (91.3%) and 22 of 23 non-responders (95.6%) in the training set, and 80.0% sensitivity and 92.8% specificity, with an accuracy of 69.2% in 29 independent test samples.
Random Forests on gene-expression data for CRC patients was effectively able to stratify responders to FOLFOX therapy with high accuracy, and use of pharmacogenomics in anticancer therapy is the first step in planning personalised therapy.
colorectal cancer; FOLFOX therapy; machine learning algorithm; class predictor; personalised therapy
We previously reported that a faecal cyclooxygenase-2 (COX-2) mRNA assay was useful for identifying colorectal cancer (CRC). This study sought to investigate the factors that contribute to faecal COX-2 mRNA expression in subjects with CRC.
The study cohort comprised 78 patients with CRC and 36 control subjects. The expressions of COX-2, β-2-microglobulin (B2M), carcinoembryonic antigen (CEA), E-cadherin (E-cad), and CD45 mRNA in faeces and COX-2 mRNA expression in tissue were determined by quantitative real-time RT–PCR.
The level of faecal expression of COX-2 mRNA in CRC was significantly higher than that in controls. A significant correlation was found between faecal COX-2 mRNA expression and faecal B2M, CEA, E-cad, or CD45 mRNAs, markers of exfoliated total cells, colonocytes, and leukocytes, respectively. A significant correlation was found between the expression of COX-2 mRNA in faeces and tumour surface area, COX-2 mRNA expression in primary tumour. There was no difference in faecal COX-2 mRNA expression between proximal CRC and distal CRC.
COX-2 mRNA expression in faeces seems to originate from tumour lesion and to be affected by factors such as the number of exfoliated cells, exfoliation of inflammatory cells, COX-2 mRNA expression in tumour, and tumour size.
colorectal cancer; faeces; RNA; COX-2; exfoliated cell
In patients with stage IV gastric cancer, systemic chemotherapy is the key treatment. Combination chemotherapy (cis-diamminedichloride platinum plus S-1 and docetaxel plus S-1) results in long-term survival in clinical practice. In selected cases, additional (adjuvant) surgery may result in further long-term survival. This study aimed to evaluate the efficacy of adjuvant surgery following the response to chemotherapy for advanced gastric cancer. Based on response to chemotherapy, the indications for adjuvant surgery (surgery after the response to chemotherapy) are that resection is expected to be curative rather than palliative, provided that no other distant metastases occur. The study included 20 advanced gastric cancer patients who had undergone gastrectomies after the response to the combination chemotherapy of docetaxel and S-1, between September 2003 and December 2008 at Hiroshima University Hospital. At a median follow-up of 980 days, the median overall survival was 855 days. A 2- and 3-year survival was observed in 80 and 54.9% of patients, respectively, following macroscopic curative surgery. In the palliative group, the median overall survival was 510 days, but a 3-year survival was not observed. In the partial response group, the median overall survival was 865 days and a 3-year survival was observed in 37% of patients. One-year survival was not observed in the stable disease group. The patient survival in the partial response group was statistically more prolonged than in the stable disease group. The median overall survival in patients with liver metastasis was 865 days, while that in patients with peritoneal dissemination was 510 days. In conclusion, adjuvant surgery may be effective in gastric cancer patients diagnosed as stage IV by means of liver or distant lymph node metastasis, except in cases of peritoneal dissemination.
gastric cancer; adjuvant surgery; S-1; docetaxel
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Background: Epidermal growth factor receptor (EGFR) inhibitors are effective in a subset of patients with non-small-cell lung cancer (NSCLC). We previously showed that E-cadherin expression associates with gefitinib activity. Here, we correlated the expressions of ErbB-3 and E-cadherin in NSCLC tumors and cell lines, their effect on response to gefitinib, and induction of both by the histone deacetylase (HDAC) inhibitors vorinostat and SNDX-275.
Methods: Real-time RT-PCR was carried out on RNA isolated from 91 fresh-frozen NSCLC samples and from 21 NSCLC lines. Protein expression was evaluated with western blot and flow cytometry. Apoptosis was assessed using vibrant apoptosis assay.
Results: Expressions of E-cadherin and ErbB-3 correlated significantly in primary tumors (r = 0.38, P < 0.001) and in cell lines (r = 0.88, P < 0.001). Cotransfection of ErbB-3 and E-cadherin in a gefitinib-resistant cell line showed enhanced apoptotic response to gefitinib. vorinostat and SNDX-275 induced ErbB-3 and E-cadherin in gefitinib-resistant cell lines. When gefitinib-resistant lines were treated with vorinostat and gefitinib, synergistic effects were detected in four of the five lines tested.
Conclusion: ErbB-3 and E-cadherin are coexpressed and induced by HDAC inhibitors. For tumors with low ErbB-3 and E-cadherin expressions, the combination of HDAC and EGFR-tyrosine kinase inhibitors increased expression of both genes and produced more than additive apoptotic effect.
E-cadherin; EGFR; ErbB3; HDAC; NSCLC; TKI
A 60-year old man with a 10-year history of multiple system atrophy (MSA) was found in respiratory arrest. After 4 months of respiratory support with two episodes of septic shock, he died. Autopsy disclosed severe atrophy of the mesencephalon, brainstem, medulla oblongata and cerebellum. Gallyas–Braak, α-synuclein and ubiquitin-positive inclusions in the cytoplasm of glial cells were evident, despite the severe ischaemic damage due to respiratory arrest and subsequent respiratory support for 4 months. The cause of respiratory arrest was not identified, but could be explained by the natural history of MSA. The bereaved family, who had suspected malpractice, was satisfied with the explanation based on the investigation performed by eight expert doctors, one expert nurse, two coordinator nurses and two lawyers in the model project promoted by the Japanese government.
To investigate whether the ratio of early transmitral flow velocity (E) to early diastolic mitral annular velocity (E′) predict prognosis in patients with non‐valvular atrial fibrillation.
230 patients with non‐valvular atrial fibrillation were enrolled and studied. According to E/E′ value, patients were divided into groups with lower (group A with E/E′ ⩽ 15) and higher (group B with E/E′ > 15) E/E′.
During follow up (average 245 days), 21 (9.1%) deaths were documented. All cause death (15/90 (16.7%) v 6/140 (4.3%)), cardiac death (10 (11.1%) v 2 (1.4%)) and congestive heart failure (16 (17.8%) v 8 (5.7%)) were more common in group B than in group A (all p < 0.01). A Kaplan–Meier survival curve showed that the cumulative survival rate was significantly lower in group B than in group A (log rank p = 0.0013). By multivariate logistic regression analysis, E/E′ (χ2 = 4.47, odds ratio (OR) 1.05, 95% confidence interval (CI) 1.01 to 1.11, p = 0.03) and age (χ2 = 6.45, OR 1.06, 95% CI 1.01 to 1.11, p = 0.02) were independent predictors of mortality.
The Doppler‐derived index of left ventricular filling pressure, E/E′, is a powerful predictor of the clinical outcome of patients with non‐valvular atrial fibrillation.
conjunctiva; immunohistochemistry; mucinous glycoprotein; pterygium
The Japanese Medical Act section 21 states that doctors must report unnatural deaths to the police, even though the term “unnatural death” is not defined by law. However, many doctors are reluctant to report potential therapeutic deaths (PTDs). The Japanese Society of Legal Medicine has submitted guidelines for unnatural death, including PTD. These define a PTD as an unexpected death, the cause of which is unknown, but which is potentially related to medical practice. Such deaths are “reportable” to the coroner in the UK. In this study, we addressed the question of whether physicians would report each of eight hypothetical PTDs. Although the clinical societies (the Japan Society of Internal Medicine and the Japan Surgical Society) declare that doctors must report deaths due to gross negligence, 60% of the participants said that they would not report gross negligence involving an overdose in cases where they had obtained informed consent or had provided an explanation after the death occurred. This can be accounted for by the mistaken belief on the part of the participants that obtaining informed consent exempts Japanese physicians from the duty of reporting PTDs. The attitude of Japanese physicians is caused by the death investigation system, which is designed to discover whether a crime has been committed rather than focusing on the cause of death. Accordingly, the Japanese Government has decided to commission a pilot study from an independent organisation in which medical specialists will investigate PTDs in order to prevent deaths occurring as a result of gross negligence.
coroner's inquest; informed consent; medical malpractice; unnatural death
The mechanisms of the cellular origin and cell proliferation in the idiopathic epiretinal membrane (ERM) are unsolved. The aim of this study was to examine the expression of cell cycle related molecules and glutamine synthetase (GS), which is expressed in Müller cells and their processes, in ERM tissues.
The ERMs were surgically removed using pars plana vitrectomy. Formalin fixed, paraffin embedded ERM tissues were analysed by immunohistochemistry with anti‐cyclin D1, p27 (KIP1), proliferating cell nuclear antigen (PCNA), and GS antibodies.
The histopathological findings showed that all the ERMs consisted of oval or spindle mononuclear cells with thin collagen‐like tissues. Immunoreactivity for GS was detected in collagen‐like tissues of ERM, presenting a continuous, isodense pattern. GS immunopositive cells in all cases expressed PCNA in their nuclei. Nuclear immunoreactivity for cyclin D1 was noted in the ERM constituent cells, whereas p27 (KIP1) positive nuclei were not detected.
Cyclin D1 and PCNA were expressed in the idiopathic ERM, which was mainly derived from Müller cells and extensions of their processes.
cyclin; epiretinal membrane; glutamine synthetase; proliferating cell nuclear antigen
coronary disease; diabetes mellitus; microcirculation; nitric oxide synthase
segmented myocardial perfusion; selective intracoronary injection; 256 slice cone beam computed tomography
Objective: To determine the effect of one or multiple co-morbid conditions on the diagnostic accuracy of coronary flow velocity reserve (CFVR) in a heterogeneous patient population.
Methods: CFVR was measured in the left anterior descending coronary artery (LAD) by transthoracic Doppler echocardiography (TTDE) in 318 consecutive patients before elective coronary angiography. CFVR was calculated as the average peak diastolic velocity during intravenous ATP infusion divided by baseline flow velocity. All patients underwent coronary angiography within 48 hours. Significant LAD stenosis was defined as > 50% luminal narrowing. Diagnostic accuracy of CFVR was analysed according to the type and number of risk factors that may adversely affect microvascular function.
Results: CFVR was measured in 309 patients, of whom 105 were found to have significant LAD stenosis based on coronary angiography. CFVR < 2.0 had a sensitivity of 86% and a specificity of 77% for predicting significant LAD stenosis. Left ventricular hypertrophy (LVH) was the only factor that significantly lowered diagnostic accuracy (61% with LVH v 84% without LVH, p < 0.001). Diagnostic accuracy was not affected by increasing number of risk factors.
Conclusions: The diagnostic accuracy of CFVR by TTDE for detecting significant LAD stenosis remains high in a more clinically relevant population with multiple cardiovascular co-morbidities. Only the presence of LVH adversely affected diagnostic accuracy.
coronary flow velocity reserve; microvascular dysfunction; transthoracic Doppler echocardiography
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Aims: The perforin mediated pathway is the major pathway of cytotoxicity induced by activated T cells and natural killer cells, and may be involved in the development of pulmonary fibrosis.
Methods: Perforin and granzyme B expression were examined in idiopathic pulmonary fibrosis by means of immunohistochemistry, and perforin knockout mice were used to examine whether or not perforin mediated cytotoxicity participates in the pathophysiology of bleomycin induced pneumopathy.
Results: Perforin and granzyme B expression were upregulated in infiltrating lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis compared with normal lung parenchyma. Perforin and granzyme B expression were upregulated predominantly in infiltrating mononuclear cells after bleomycin instillation in wild-type mice. Although the development of bleomycin induced pneumopathy was not completely prevented, the pathological grade of inflammation and fibrosis, and the number of apoptotic cells in lung tissue, were significantly decreased in perforin knockout mice compared with wild-type mice.
Conclusions: These results suggest that perforin mediated apoptosis may be associated with the pathophysiology of lung injury and fibrosis.
perforin; granzyme B; lung injury; fibrosis; apoptosis
Aim: To examine the expression of p65, one of nuclear factor-kappa B (NF-κB), in the conjunctival epithelium of the C57Bl6 mouse and a patient with epidemic keratoconjunctivitis (EKC).
Methods: Normal and epithelial scraped cornea obtained 6 hours after the injury were processed for paraffin section. Samples of a normal and an EKC conjunctival epithelium were obtained using impression cytology. Both samples were analysed by immunocytochemistry using anti-p65 antibody.
Results: Immunocytochemistry with the anti-NF-κB p65 antibody revealed that p65 was localised in the cytoplasm of the conjunctival epithelium in the C57Bl6 mouse without the treatment. Six hours after the scraping of the cornea, p65 protein was expressed in the nuclei of the conjunctival epithelium. p65 was localised in the cytoplasm of the conjunctival epithelium in the human normal eye. p65 protein was expressed in the nuclei of the conjunctival epithelial cells in the EKC patient.
Conclusion: These findings suggest that NF-κB was activated in the conjunctiva in the epithelial scraping of the mouse cornea and in human EKC.
adenovirus; corneal ulcer; keratoconjunctivitis; NF-κB
A 33 year old man was admitted to hospital six days after the onset of abdominal pain. There was hypereosinophilia, but the cause could not be identified (primary hypereosinophilia). The hypereosinophilia, high C reactive protein concentration, and gastrointestinal symptoms were alleviated by corticosteroid treatment. Unexpectedly, after this apparent recovery, he was found dead on the 27th day after admission. Necropsy disclosed two solid tumours primarily composed of eosinophils in the ascending and transverse colon. The cause of the sudden death was pulmonary artery emboli, derived from a thrombus in the left iliac vein.
hypereosinophilia syndrome; solid tumour; sudden death; pulmonary artery emboli; major basic protein
Background: A recent study using a Doppler guide wire showed that coronary flow velocity measurements immediately after coronary reperfusion were useful in predicting recovery of regional left ventricular function. The value of coronary flow velocity analyses during follow up after reperfusion has not been established in the clinical setting.
Objective: To evaluate coronary flow velocity measurements in predicting recovery of regional left ventricular function during short term follow up after acute anterior myocardial infarction, using transthoracic Doppler echocardiography (TTDE).
Methods: 30 consecutive patients with anterior acute myocardial infarction were studied. They all underwent successful coronary angioplasty for lesions in the left anterior descending coronary artery (LAD). Using TTDE, coronary flow velocity in the LAD was recorded on days 1 and 3, and at one and two weeks after reperfusion. Regional wall motion was analysed by the wall motion score index (WMSI), calculated as an average of segmental scores in the LAD territory before reperfusion and one month after the infarction.
Results: Deceleration time of diastolic flow velocity (DDT) in patients with viable myocardium (WMSI in LAD territory at one month, < 2.0) was significantly longer after recanalisation than in patients without viable myocardium (WMSI in LAD territory at one month, > 2.0): 657 (226) v 271 (117) ms on day 1, p < 0.001; 732 (219) v 373 (217) ms on day 3, p < 0.01; and 903 (107) v 577 (300) ms at one week, p < 0.01. However, the difference at two weeks (991 (75) v 795 (281) ms) was not significant. For the prediction of viable myocardium, DDT > 600 ms had a sensitivity of 78% and a specificity of 92% on day 1, and a sensitivity of 78% and a specificity of 84% on day 3. At one and two weeks, DDT > 600 ms was sensitive (100% and 100%, respectively) but less specific (46% and 26%, respectively) for predicting viable myocardium.
Conclusions: Non-invasive assessment of coronary flow velocity using TTDE within three days of successful coronary angioplasty in patients with anterior acute myocardial infarction is useful in predicting recovery of regional left ventricular function.
echocardiography; myocardial blood flow; coronary artery disease