Activin/Nodal/TGF-ß Pathway Inhibitor Accelerates BMP4-Induced Cochlear Gap Junction Formation During in vitro Differentiation of Embryonic Stem Cells.

Mutations in gap junction beta-2 (GJB2), the gene that encodes connexin 26 (CX26), are the most frequent cause of hereditary deafness worldwide. We recently developed an in vitro model of GJB2-related deafness (induced CX26 gap junction-forming cells; iCX26GJCs) from mouse induced pluripotent stem cells (iPSCs) by using Bone morphogenetic protein 4 (BMP4) signaling-based floating cultures (serum-free culture of embryoid body-like aggregates with quick aggregation cultures; hereafter, SFEBq cultures) and adherent cultures. However, to use these cells as a disease model platform for high-throughput drug screening or regenerative therapy, cell yields must be substantially increased. In addition to BMP4, other factors may also induce CX26 gap junction formation. In the SFEBq cultures, the combination of BMP4 and the Activin/Nodal/TGF-ß pathway inhibitor SB431542 (SB) resulted in greater production of isolatable CX26-expressing cell mass (CX26+ vesicles) and higher Gjb2 mRNA levels than BMP4 treatment alone, suggesting that SB may promote BMP4-mediated production of CX26+ vesicles in a dose-dependent manner, thereby increasing the yield of highly purified iCX26GJCs. This is the first study to demonstrate that SB accelerates BMP4-induced iCX26GJC differentiation during stem cell floating culture. By controlling the concentration of SB supplementation in combination with CX26+ vesicle purification, large-scale production of highly purified iCX26GJCs suitable for high-throughput drug screening or regenerative therapy for GJB2-related deafness may be possible.

Modeling gap junction beta 2 gene-related deafness with human iPSC.

There are >120 forms of non-syndromic deafness associated with identified genetic loci. In particular, mutation of the gap junction beta 2 gene (GJB2), which encodes connexin (CX)26 protein, is the most frequent cause of hereditary deafness worldwide. We previously described an induction method to develop functional CX26 gap junction-forming cells from mouse-induced pluripotent stem cells (iPSCs) and generated in vitro models for GJB2-related deafness. However, functional CX26 gap junction-forming cells derived from human iPSCs or embryonic stem cells (ESCs) have not yet been reported. In this study, we generated human iPSC-derived functional CX26 gap junction-forming cells (iCX26GJCs), which have the characteristics of cochlear supporting cells. These iCX26GJCs had gap junction plaque-like formations at cell-cell borders and co-expressed several markers that are expressed in cochlear supporting cells. Furthermore, we generated iCX26GJCs derived from iPSCs from two patients with the most common GJB2 mutation in Asia, and these cells reproduced the pathology of GJB2-related deafness. These in vitro models may be useful for establishing optimal therapies and drug screening for various mutations in GJB2-related deafness.

Generation of two iPSC lines from siblings of a homozygous patient with hearing loss and a heterozygous carrier with normal hearing carrying p.G45E/Y136X mutation in GJB2.

The gap junction beta-2 (GJB2) gene is the most common genetic cause of hereditary deafness worldwide. Among them, the G45E/Y136X mutation in GJB2 is the third most prevalent in Japan. In this study, we generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of siblings with moderate-to-severe hearing loss (patient) or normal hearing (genetic carrier) carrying a homozygous or heterozygous G45E/Y136X mutation in GJB2 gene, respectively. These iPSC lines showed the expression of pluripotency markers and could differentiate into three germ layers. These disease-specific iPSC lines will be a powerful tool for investigating the pathogenesis of GJB2-related deafness.

Generation of two induced pluripotent stem cell lines from PBMCs of siblings carrying c.235delC mutation in the GJB2 gene associated with sensorineural hearing loss.

The gap junction beta-2 (GJB2) gene is the most common genetic cause of hereditary deafness worldwide. Especially, the 235delC mutation in GJB2 is most prevalent in East Asia. In this study, we generated two iPSC lines from PBMCs of siblings carrying homozygous 235delC mutation which exhibits an audiometric phenotype of profound hearing loss. These iPSC lines had normal karyotype, showed expression of pluripotency markers, and could differentiate into three germ layers. These disease specific iPSC lines may be useful for the construction of the disease models and for the elucidation of pathogenesis in GJB2-related deafness.

Generation of the induced pluripotent stem cell (hiPSC) line (JUFMDOi004-A) from a patient with hearing loss carrying GJB2 (p.V37I) mutation.

The gap junction beta-2 (GJB2) gene is the most common genetic cause of hereditary deafness worldwide. Especially, V37I mutation in GJB2 is most prevalent in Southeast Asia including Thailand, Malaysia, and Indonesia. Furthermore, it is the second most prevalent cause in Japan and China, and exhibits an audiometric phenotype of mild-to-moderate hearing loss. In this study, we generated induced pluripotent stem cells (iPSC) from peripheral blood mononuclear cells (PBMCs) of patient with homozygous V37I mutation. This iPSC line will be a powerful tool for investigating the pathogenesis and for developing a treatment for GJB2-related hearing loss.

64Cu-Intraperitoneal Radioimmunotherapy: A Novel Approach for Adjuvant Treatment in a Clinically Relevant Preclinical Model of Pancreatic Cancer.

Pancreatic cancer (PC) has a very poor prognosis. Surgery is the primary treatment for patients with resectable PC; however, local recurrence, hepatic metastasis, and peritoneal dissemination often occur even after extensive surgery. Adjuvant chemotherapy, typically with gemcitabine, has been used clinically but with only a modest survival benefit. To achieve a better outcome, we investigated the efficacy of 64Cu-intraperitoneal radioimmunotherapy (ipRIT) with 64Cu-labeled antiepidermal growth factor receptor antibody cetuximab as an adjuvant treatment after PC surgery using an orthotopic xenografted mouse model. Methods: The efficacy of adjuvant 64Cu-ipRIT was investigated in a human PC mouse model harboring orthotopic xenografts of xPA-1-DC cells. To reproduce the clinical situation, PC xenografts were surgically resected when pancreatic tumors were readily visible but not metastatic tumors. Increasing doses of 64Cu-cetuximab were intraperitoneally injected, and the mice were monitored for toxicity to determine the safe therapeutic dose. For adjuvant 64Cu-ipRIT, the day after tumor resection, the mice were intraperitoneally administered 22.2 MBq of 64Cu-PCTA-cetuximab and the survival was compared with that in surgery-only controls. For comparison, adjuvant chemotherapy with gemcitabine was also examined using the same model. Results: The mouse model not only developed primary tumors in the pancreas but also subsequently reproduced local recurrence, hepatic metastasis, and peritoneal dissemination after surgery, which is similar to the manifestations that occur with human PC. Adjuvant 64Cu-ipRIT with 64Cu-labeled cetuximab after surgery effectively suppressed local recurrence, hepatic metastasis, and peritoneal dissemination in this model. Significant improvement of the survival with minimal toxicity was achieved by adjuvant 64Cu-ipRIT compared with that in control mice that underwent surgery only. Adjuvant chemotherapy with gemcitabine nominally prolonged the survival, but the effect was not statistically significant. Conclusion:64Cu-ipRIT with cetuximab can be an effective adjuvant therapy after PC surgery.

Multiple Administrations of 64Cu-ATSM as a Novel Therapeutic Option for Glioblastoma: a Translational Study Using Mice with Xenografts.

Glioblastoma is the most aggressive malignant brain tumor in humans and is difficult to cure using current treatment options. Hypoxic regions are frequently found in glioblastoma, and increased levels of hypoxia are associated with poor clinical outcomes of glioblastoma patients. Hypoxia plays important roles in the progression and recurrence of glioblastoma because of drug delivery deficiencies and induction of hypoxia-inducible factor-1α in tumor cells, which lead to poor prognosis. We focused on a promising hypoxia-targeted internal radiotherapy agent, 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM), to address the need for additional treatment for glioblastoma. This compound can target the overreduced state under hypoxic conditions within tumors. Clinical positron emission tomography studies using radiolabeled Cu-ATSM have shown that Cu-ATSM accumulates in glioblastoma and its uptake is associated with high hypoxia-inducible factor-1α expression. To evaluate the therapeutic potential of this agent for glioblastoma, we examined the efficacy of 64Cu-ATSM in mice bearing U87MG glioblastoma tumors. Administration of single dosage (18.5, 37, 74, 111, and 148 MBq) and multiple dosages (37 MBq × 4) of 64Cu-ATSM was investigated. Single administration of 64Cu-ATSM in high-dose groups dose-dependently inhibited tumor growth and prolonged survival, with slight and reverse signs of adverse events. Multiple dosages of 64Cu-ATSM remarkably inhibited tumor growth and prolonged survival. By splitting the dose of 64Cu-ATSM, no adverse effects were observed. Our findings indicate that multiple administrations of 64Cu-ATSM have effective antitumor effects in glioblastoma without side effects, indicating its potential for treating this fatal disease.

An autopsy case of the rupture of a giant aneurysm in a saphenous vein graft: 18 years after CABG.

The saphenous vein is a widely used blood vessel for arterial bypass procedures. Failures of saphenous vein aortocoronary bypass grafts are predominantly the result of subsequent vein graft atherosclerotic disease. Rarely saphenous vein grafts undergo aneurysmal degeneration. This report describes a case of a ruptured aneurysm in a saphenous vein graft that occurred in an 82-year-old female who underwent a coronary artery bypass operation 18 years previously. We could not resuscitate her, but describe the autopsy findings in detail.

High ratio of myeloid dendritic cells to plasmacytoid dendritic cells in blood of patients with acute coronary syndrome.

BACKGROUND: Dendritic cells (DCs) stimulate T-cells to participate in the inflammatory processes that promote the destruction of vulnerable plaques. The relationship between circulating levels of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in patients with acute coronary syndrome (ACS) was evaluated. METHODS AND RESULTS: Blood samples were obtained from 39 patients with ACS, 41 patients with stable angina pectoris (SAP) and 43 controls. The proportion of mDCs tended to be lower in the ACS group than in the SAP group and controls. Interleukin-12 levels associated with mDCs were significantly higher in the ACS group than in control group. The proportion of pDCs was significantly lower in the ACS groups than in the other two groups. Interferon-alpha levels secreted by pDCs, however, were not significantly different among the 3 groups. The ratio of mDCs to pDCs >or=4 is an important value for distinguishing ACS from SAP patients and control patients through receiver operating characteristic analysis (sensitivity; 85.0%, specificity; 83.4%). CONCLUSIONS: The ratio of mDCs to pDCs may be a useful marker for detecting ACS and the existence of vulnerable plaques.

Expression levels of Toll-like receptor genes in coronary atherosclerotic lesions of patients with acute coronary syndrome or stable angina pectoris.

BACKGROUND: The differences between acute coronary syndrome (ACS) and stable angina pectoris (SAP) in Toll-like receptor (TLR) expression levels in coronary plaques are not well known. TLR gene expression levels were examined, not only in peripheral blood mononuclear cells (PBMCs), but also in coronary plaques in ACS and SAP patients. METHODS AND RESULTS: TLR gene expression levels were examined in PBMCs using real-time RT-PCR in 27 ACS patients, 45 SAP patients and 28 control subjects. TLR2 and TLR4 expression levels in the PBMCs were significantly higher in the ACS group than in the SAP group. TLR9 expression levels were not significantly different among the 3 groups. TLR gene expression levels were also measured in directional coronary atherectomy (DCA) samples from 9 ACS and 14 SAP patients. The TLR2 expression levels in the DCA samples did not significantly differ between the 2 groups. The TLR4 expression levels were significantly higher in the ACS group than in the SAP group. CONCLUSIONS: The results suggest that TLR4 signaling could be more associated with plaque destabilization than with plaque progression. TLR4 expression control may be a novel target for ACS treatment.

Significant association between score of periodontal disease and coronary artery disease.

Recently, several researchers have demonstrated the association between periodontal disease and coronary artery disease (CAD). Therefore, we herein investigate the association between periodontal diseases and the existence of CAD among the study population who received both coronary angiography and dental examination. A total of 174 consecutive patients with dental examination including radiography and coronary angiography in the same hospitalization were recruited (64.5 +/- 10.3 years, M/F: 94/80). A dentist assessed severity of periodontal status markers (bleeding on probing, probing depth >or=6 mm, teeth lost, alveolar bone resorption >half of root length by radiography and smoking status). We divided these patients into two groups according to whether they had CAD (CAD group, n = 99) or not (non-CAD group, n = 75) according to the results of coronary angiography. The composite periodontal risk scores calculated from periodontal status markers were higher in the CAD group than in the non-CAD group (P = 0.02). The composite periodontal scores were higher in the CAD group of age <60 years old population (P = 0.03) and in the CAD group of patients with normal glucose tolerance (P = 0.04). However, the difference was not significant in the age >or=60 years old population or those with diabetes mellitus or impaired glucose tolerance. In all populations, hypertension, glucose tolerance, statin therapy, and composite of periodontal risk scores were associated with CAD. Multivariate analyses revealed statin therapy, glucose tolerance, and periodontal risk scores were independent and significant risk factors for CAD. Composite periodontal risk scores were independent and significant predictive factors for CAD.

Periodontal status and Prevotella intermedia antibody in acute coronary syndrome.

We performed periodontal examination and measured serum antibody levels against Prevotella intermedia in patients with acute coronary syndrome (ACS). Composite periodontal risk scores were significantly higher in the ACS group than in the coronary artery disease (CAD) group. Serum antibody levels were higher in the ACS group than in the CAD group and those were significantly correlated with the composite periodontal risk scores. These results provided important information about the status of P. intermedia infection in patients with ACS.

Osteopontin expression of circulating T cells and plasma osteopontin levels are increased in relation to severity of heart failure.

BACKGROUND: T cells in peripheral blood reflect the systemic inflammatory response in patients with heart failure (HF). In a rat model of HF, osteopontin is dramatically increased in the left ventricular myocardium, so the association between osteopontin and HF was examined in the present study. METHODS AND RESULTS: Peripheral blood was collected from 93 patients with heart disease and 38 controls. Left ventricular ejection fraction (LVEF) was calculated using a modified Simpson's rule. The 93 patients were classified into 3 classes according to the New York Heart Association (NYHA) functional classification. Osteopontin-expressing CD4+ T cells were quantified by flow cytometry. Plasma osteopontin levels (ng/ml) and the frequencies of osteopontin-expressing CD4+ T cells (%) were higher in patients with HF than in controls (800+/-554, 575+/-229, p=0.016 and 27.3+/-12.2, 16.7+/-10.0, p<0.001). Furthermore, the plasma osteopontin levels and the frequencies of osteopontin-expressing CD4+ T cells increased in proportion to the severity of the NYHA functional class. The frequencies of osteopontin-expressing CD4+ T cells were significantly correlated with LVEF (r=-0.336, p=0.0048) and log plasma brain natriuretic peptide levels (r=0.305, p=0.0025). CONCLUSIONS: Osteopontin expression of circulating CD4+ T cells and plasma osteopontin levels reflect the severity of HF. Osteopontin could be a new target in the assessment of HF.

Relation between CD4+ T-cell activation and severity of chronic heart failure secondary to ischemic or idiopathic dilated cardiomyopathy.

The percentage of CD4(+) T cells in blood is correlated with left ventricular dysfunction and decreased ejection fraction in heart disease. The aim of this study was to determine the relation between activation of CD4(+) T cells and New York Heart Association functional classes in chronic heart failure (HF) and differences in inflammatory activation between ischemic cardiomyopathy (IC) and idiopathic dilated cardiomyopathy (IDC). Blood samples were obtained from 47 patients with HF and 20 controls. Percentages of interferon-gamma-positive CD4(+) T cells (representative type 1 T-helper cells) and interleukin-4-positive CD4(+) T cells (representative type 2 T-helper cells) were analyzed using 3-color flow cytometry. The proportion of interferon-gamma-positive CD4(+) T cells was higher in patients with HF (28.96 +/- 12.90%) than in controls (18.12 +/- 5.28, p = 0.0006), but there was no difference in percentage of interleukin-4-positive CD4(+) T cells between the 2 groups. The proportion of interferon-gamma-positive CD4(+) T cells and plasma B-type natriuretic peptide levels increased with worsening of New York Heart Association functional class in the IC and IDC groups. The proportion of interferon-gamma-positive CD4(+) T cells in the IC group (33.88 +/- 13.33%) was higher than in the IDC group (22.33 +/- 8.88%, p = 0.002); however, plasma B-type natriuretic peptide levels were higher in the IDC group (358.0 pg/ml, 327.5 to 1,325.7) than in the IC group (82.7 pg/ml, 34.7 to 252.9, p = 0.019). In conclusion, we demonstrated pronounced type 1 T-helper cell activation in patients with HF in proportion to severity of HF and that the specificity of T-cell activation differs between patients with IC and those with IDC.

Expression of interferon-gamma and interleukin-4 production in CD4+ T cells in patients with chronic heart failure.

The prevalence of inflammation is high among patients with chronic heart failure (CHF). Reduced ejection fraction was associated with frequency of CD4(+) T cells of leukocytes. Therefore, we investigated inflammatory cytokines of expression markers in CD4(+) T cells in patients with CHF. Blood samples were obtained from 103 patients with CHF, from 83 patients with stable angina (SA), and from 57 controls. Interferon-gamma (IFN-gamma)-positive CD4(+) T cells and interleukin-4 (IL-4)-positive CD4(+) T cells were analyzed using 3-color flow cytometry. The frequency (%) of IFN-gamma-positive CD4(+) T cells increased in patients with CHF compared with those with SA and controls (CHF: 28.3 +/- 13.8, SA: 23.50 +/- 10.38, controls: 19.00 +/- 7.45, P < 0.001). There was no significant difference in the frequency of IL-4-positive CD4(+) T cells among the three groups. The frequencies of CD4(+) T cells that stained for IFN-gamma decreased from 32.37% +/- 16.40% on admission to 26.91% +/- 12.53% after 2 weeks in 26 patients with CHF. B-type natriuretic peptide (pg/ml) and high-sensitivity C-reactive protein (mg/dl) levels decreased from 251.7 +/- 150.4 and 0.64 +/- 0.78 on admission to 208.2 +/- 166.4 and 0.36 +/- 0.34 after 2 weeks in the 26 patients with CHF. We have demonstrated expression of IFN-gamma production of CD4(+) T cells during CHF. Prevention of unwanted T cell activation could represent a new target in the treatment of CHF.