Discovery of

The new isotope ^{39}Na, the most neutron-rich sodium nucleus observed so far, was discovered at the RIKEN Nishina Center Radioactive Isotope Beam Factory using the projectile fragmentation of an intense ^{48}Ca beam at 345 MeV/nucleon on a beryllium target. Projectile fragments were separated and identified in flight with the large-acceptance two-stage separator BigRIPS. Nine ^{39}Na events have been unambiguously observed in this work and clearly establish the particle stability of ^{39}Na. Furthermore, the lack of observation of ^{35,36}Ne isotopes in this experiment significantly improves the overall confidence that ^{34}Ne is the neutron dripline nucleus of neon. These results provide new key information to understand nuclear binding and nuclear structure under extremely neutron-rich conditions. The newly established stability of ^{39}Na has a significant impact on nuclear models and theories predicting the neutron dripline and also provides a key to understanding the nuclear shell property of ^{39}Na at the neutron number N=28, which is normally a magic number.

Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosa associated tissues.

Antigen-specific B cell responses to mucosally delivered proteins are dependent upon CD4-positive T helper (Th) cells, and the frequency of Th1 and Th2 cell responses after oral immunization may determine the level and isotype of mucosal antibody responses. We have used a protein-based vaccine, tetanus toxoid (TT), together with the mucosal adjuvant cholera toxin (CT), for oral immunization of mice to study the nature of antigen-specific Th cell subsets induced in Peyer's patches (PP) of the gastrointestinal (GI) tract and in the spleen (SP) during peak antibody responses. Mice orally immunized with TT and CT responded with antigen-specific secretory immunoglobulin A (S-IgA) antibodies in the GI tract, and with both IgG and IgA antibody responses in serum. PP and SP CD4+ T cells from mice orally immunized with TT plus CT were cultured with antigen-coated latex microspheres for induction of proliferative responses and for enumeration of cytokine producing CD4+ T cells. Interestingly, both PP and SP CD4+ T cell cultures showed increased numbers of IL-4- and IL-5 (Th2-type)-producing, spot-forming cells (SFCs) after 21 d of immunization, while essentially no interferon-gamma (IFN-gamma) or IL-2 (Th1-type) SFCs were noted. Cytokine-specific Northern blots and RT-PCR also revealed that significant IL-4 and IL-5 mRNA levels, but not IFN-gamma or IL-2 mRNA, were present in CD4+ T cells isolated from antigen-stimulated cultures. However, systemic immunization with TT and CT induced antigen-specific IgG and IgM but not IgA antibodies in serum. Further, both IL-2 and IFN-gamma-producing Th1-type cells as well as IL-4- and IL-5-secreting Th2-type cells were generated in SP. Our results show that oral immunization with TT and the mucosal adjuvant CT selectively induced antigen-specific Th2-type responses which may represent the major helper cell phenotype involved in mucosal IgA responses in the GI tract.

[Knack and pitfall in valve replacement].

We choose prosthetic or bioprosthetic valves according to AHA/ACC guidelines in valve replacement. It is important to remove only the calcification and avoid over-resection to preserve the valve annulus during aortic valve replacement. We leave posterior leaflet as well as basal chordae in mitral valve replacement in case of large mitral annulus. Sutures should be tied-down after those on both adjacent sides are pulled up and the sawing cuff and annulus are firmly attached. Intra-operative transesophageal echocardiography is useful for detecting a stack valve, perivalvular leakage and remnant air in the cardiac chambers. We performed 53 cases of valve replacement in 2009. One patient (1.9%) died because of ventricular arrhythmia during hospital stay. Re-operation was required in 2 cases (3.8%) of infective endocarditis due to prosthetic valve endocarditis. No other major complication was observed.

Gene therapy for attenuating cardiac allograft arteriopathy using ex vivo E2F decoy transfection by HVJ-AVE-liposome method in mice and nonhuman primates.

Cardiac allograft arteriopathy, which limits the long-term survival of recipients, is characterized by diffuse intimal thickening composed of proliferative smooth muscle cells. The transcription factor E2F plays a pivotal role in the coordinated transcription of cell-cycle regulatory genes. To test the hypothesis that double-stranded DNA with specific affinity for E2F (E2F decoy) is effective in preventing intimal hyperplasia, we performed ex vivo single intraluminal delivery of E2F decoy into cardiac allografts of mice and Japanese monkeys using the hemagglutinating virus of Japan (HVJ) artificial viral envelope-liposome method. In murine models, antisense cyclin-dependent kinase 2 (cdk2) kinase oligodeoxynucleotide (ODN) and no transfers were performed to compare the effects. Severe intimal thickening was observed, and multiple cell-cycle regulatory genes were enhanced in untreated allografts. E2F decoy prevented neointimal formation and suppressed these genes for up to 8 weeks, whereas antisense cdk2 kinase ODN had limited effects. In primate models, E2F decoy dramatically prevented neointimal thickening and suppressed multiple cell-cycle regulatory genes, whereas intimal thickening developed in the nontransfected or mismatch decoy-transfected allografts. Gel mobility shift assay proved the specific effects of E2F decoy, and reverse transcriptase-polymerase chain reaction documented that neither complication nor dissemination of HVJ into other organs was observed. We demonstrate that ex vivo gene delivery to allografts is a potent strategy to modify allograft gene expression, resulting in prevention of graft arteriopathy without systemic adverse effects.

Clinicopathological significance of core 2 beta1,6-N-acetylglucosaminyltransferase messenger RNA expressed in the pulmonary adenocarcinoma determined by in situ hybridization.

Cell surface carbohydrates of epithelial cells play important roles in tumor progression. Previously, we have shown that expression of core 2 branched O-glycans in colorectal cancer is closely correlated with the vessel invasion and depth of invasion (K. Shimodaira et al., Cancer Res., 57: 5201-5206, 1997). To test whether this is also the case in human lung cancer, we have examined the expression pattern of core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT) mRNA responsible for the biosynthesis of core 2 branched O-glycans in 41 cases of lung cancer. Using in situ hybridization, C2GnT mRNA was detected in 73.2% of the lung cancer cells, irrespective of the histopathological type; whereas in normal lung tissues, its expression was restricted to the basal cells of bronchial mucosa. These results indicate that the expression level of C2GnT mRNA was significantly enhanced in association with malignant transformation. Statistical analysis between the C2GnT mRNA expressed in pulmonary adenocarcinoma and clinicopathological variables revealed that the expression of C2GnT was correlated with vessel invasion and lymph node metastasis with significant difference (P < 0.05), but expression of sialyl Le(x), which is frequently expressed in the adenocarcinoma, was not significantly correlated with lymph node metastasis. These results indicate that C2GnT mRNA detected by in situ hybridization reflects the malignant potentials of pulmonary adenocarcinoma, because lymph node metastasis is the most affecting factor to the patients' prognosis.

[Tracheobronchial injury].

We studied 6 cases of tracheobronchial injury due to the blunt chest truma in our department. All patients were male of 19 to 60 years of age. Injured sites were main bronchus in 2, tracheobronchial portion in 2, cervical trachea and main bronchus in 1, cervical trachea in 1. In a case of cervical tracheal injury and 2 cases of tracheobronchial injury, emergent operation was performed on the day of accident. Other cases with the main bronchial injury underwent conservative treatment at first, but subsequent bronchoplasty was necessary for them due to the bronchial stenosis. After the surgery for 2 cases of tracheobronchial injury, mechanical ventilation with double lumen tube was continued to reduce the airway pressure for the anastomotic sites. In conclusion, early surgical treatment is recommended for the airway injury and the respiratory management using double lumen tube after surgery may be helpful in preventing trouble at the anastomosis.

Creatine kinase release not associated with myocardial necrosis after short periods of coronary artery occlusion in conscious baboons.

The effects of 15 minute periods of coronary artery occlusion on plasma creatine kinase (CK) and CK-MB isoenzyme activity, regional myocardial function and subsequent myocardial necrosis were studied in six conscious baboons 2 to 3 weeks after recovery from instrumentation. Mid left anterior descending coronary artery occlusion induced complete loss of systolic wall thickening (ultrasound transit time technique) and decreases in epicardial (-93%) and endocardial (-96%) blood flows (microsphere technique). Reperfusion after 15 minutes resulted in complete recovery of regional function 24 hours later. Serial plasma enzyme activity revealed a significant increase in total CK from 71 +/- 11 to 976 +/- 158 U/liter and in CK-MB from levels that were too low to measure to 21.4 +/- 2.9 U/liter. At autopsy, neither gross pathologic evidence (triphenyltetrazolium chloride staining technique) nor histologic evidence of myocardial necrosis was observed. Thus, in the conscious baboon short episodes of myocardial ischemia are associated with a significant appearance of CK and CK-MB in the blood in the absence of cellular necrosis.

Tranilast inhibits cardiac allograft vasculopathy in association with p21(Waf1/Cip1) expression on neointimal cells in murine cardiac transplantation model.

Cardiac allograft vasculopathy is a major complication after cardiac transplantation, often limiting long-term recipient survival. N-(3,4-Dimethoxycinnamoyl)anthranilic acid (tranilast) inhibits cyclin-dependent kinase activity through p21(Waf1/Cip1) induction and arrests vascular smooth muscle cell proliferation in vitro. We tested a hypothesis that tranilast inhibits the vasculopathy characterized by diffuse intimal thickening in a murine heart transplantation model. Hearts from DBA/2 mice were heterotopically transplanted into B10.D2 mice as allografts. Oral administration of tranilast started 3 days before transplantation at doses of 550 or 1040 mg/kg per day until the animals were killed. Cardiac allograft vasculopathy was defined as luminal stenosis caused by neointimal formation. The percentage of luminal stenosis and cardiac rejection were analyzed 14 and 28 days after transplantation. Tranilast administration was associated with a marked reduction in luminal occlusion but with no significant effect on cardiac rejection. Immunohistochemical study of the tranilast-treated graft coronary arteries revealed enhancement of p21(Waf1/Cip1) and decreased expression of proliferating cell nuclear antigen in the neointima. The significant reduction in allograft vasculopathy concomitant with the enhancement of p21(Waf1/Cip1) indicates that tranilast has an antiproliferative effect that could be applicable to clinical treatment of cardiac allograft vasculopathy.

Antisense Bcl-x oligonucleotide induces apoptosis and prevents arterial neointimal formation in murine cardiac allografts.

OBJECTIVE: Cardiac allograft arteriosclerosis, which limits long-term survival of recipients, cannot be prevented by conservative therapies. The arteriopathy is characterized by diffuse intimal thickening comprised of proliferative smooth muscle cells (SMCs). Cell death is a prominent feature of atherosclerosis; Bcl-x is one of the anti-apoptotic mediators. METHODS: To test the hypothesis that antisense bcl-x oligodeoxynucleotide (ODN) is effective in preventing intimal hyperplasia through enhancing apoptosis after cardiac transplantation, we performed single intraluminal delivery of antisense bcl-x ODN into murine cardiac allografts (n = 9). DBA/2 (H-2d) hearts were transplanted into B10.D2 (H-2d) mice. Sense bcl-x ODN (n = 8) and no treatment (n = 8) studies were also performed. RESULTS: Allografts were harvested at 4 weeks after transplantation; all allografts kept beating throughout the period. Coronary intimal thickening had developed in nontreated and sense ODN transfected allografts at 4 weeks after transplantation with enhanced expression of Bcl-x and cell adhesion molecules, and suppressed apoptosis. However, antisense bcl-x ODN prevented neointimal formation through enhanced apoptosis. CONCLUSION: These results indicate that apoptosis of vascular SMCs induced by Bcl-x is associated with initial hyperplasia after heart transplantation. Antisense bcl-x ODN inhibits SMC proliferation by inducing apoptosis in graft coronary arteries.

New perspectives in mucosal immunity with emphasis on vaccine development.

In this review, we have purposely focused on five areas that are currently receiving extensive research attention and will be of major importance for development of mucosal and systemic immunity to oral vaccines. These five areas include the following: (1) helper T-cell (Th) subsets and cytokines for mucosal IgA responses; (2) Th1- and Th2-type subsets in regulation of mucosal IgA responses; (3) antigen uptake and presentation in the mucosal immune system; (4) the importance of memory in mucosal immunity to vaccines; and (5) the determination of whether oral immunization alone induces immunity in all mucosal effector tissues. It is now established that the mucosal immune system can be divided into discrete mucosal inductive sites where vaccines/antigens are encountered and taken up, processed, and presented to B and T cells, and separate areas where immune cells actually function (mucosal effector tissues). Further, through the help provided by Th cells and cytokines, the B cells respond to antigen and undergo expansion including memory cell formation. Following the migration of memory B cells to mucosal effector tissues, the cells rapidly develop into IgA plasma cells, and the prevalence of the latter cell type represents a major characteristic of mucosal effector tissues. It also appears that antigen-specific Th cells and perhaps even CD8+ cytotoxic T lymphocytes can make this circular journey (along with memory/activated B cells) from inductive to mucosal effector sites, and this is termed the common mucosal immune system (CMIS). The major implications of the CMIS for development of vaccines would include each of the five components that are discussed.

Intestinal epithelial cells express and secrete the CD43 glycoform that contains core 2 O-glycans.

Several glycoforms of CD43 are known to regulate cellular interactions in the immune system. One such glycoform, the CD43 that bears core 2 O-glycans, is also known to be expressed on T lymphocytes and natural killer cells, but only after their activation. Previous studies have also shown that when Caco-2 cells, which are derived from human colon carcinoma, differentiate into enterocytes, they also express core 2 O-glycans, though proteins bearing this glycan are unknown. To examine whether CD43 glycosylation is altered during enterocytic differentiation of Caco-2 cells, we conducted immunocytochemical studies with a monoclonal antibody, 1D4, that recognizes a glycoform of CD43 bearing core 2 O-glycans. We found that 1D4 could bind to intracellular granules but not the cell surface of differentiated Caco-2 cells, whereas hematopoietic cells expressed 1D4 epitope on the cell surface as previously shown. The reactivity with this antibody increased as the degree of cell differentiation progressed as shown by the activity of the apical enzyme marker, dipeptidyl peptidase IV. 1D4-reactive CD43 was also found in the culture medium of differentiated Caco-2 cells, suggesting this molecule may be stored and secreted. The production and secretion of this CD43 glycoform by enterocyte-like Caco-2 cells was enhanced, and most 1D4 epitope converted to a soluble form when bacterial lipopolysaccharide was present. These observations strongly support the possibility that core 2 O-glycans on mucins such as CD43 are important to primary defense on the intestinal epithelium against infection.

Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors.

A fundamental obstacle in gene therapy for cancer is the specific delivery of an anticancer gene product to a solid tumor, and yet no systemic delivery system that specifically targets solid tumors currently exists. A strain of domestic bacteria, Bifidobacterium longum, which is nonpathogenic and anaerobic, selectively localized and proliferated in several types of mouse solid tumors after systemic application. In this report, we further describe a novel approach to cancer gene therapy in which genetically engineered Bifidobacterium is used as a tumor-specific vector. Similarly to wild-type B. longum, genetically engineered B. longum could be detected in tumor tissue only and was not found in a large survey of normal mouse tissues after intravenous injection. This finding strongly suggests that obligate anaerobic bacteria such as Bifidobacterium can be used as highly specific gene delivery vectors for cancer gene therapy.

Prevention of cardiac allograft arteriosclerosis using antisense proliferating-cell nuclear antigen oligonucleotide.

Cardiac allograft arteriosclerosis limits long-term survival of recipients and is characterized by intimal thickening comprised of proliferative smooth muscle cells. Proliferating-cell nuclear antigen (PCNA) plays a pivotal role in the cell cycle regulatory genes involved in smooth muscle cell proliferation. To test the hypothesis that antisense PCNA oligodeoxynucleotide (ODN) can prevent allograft arterial intimal hyperplasia, we performed single intraluminal delivery of the antisense or sense PCNA ODN or no transfer into murine cardiac allografts. DBA/2 murine hearts were transfected and transplanted into B10.D2 mice; the allografts were harvested 4 weeks later. Severe intimal thickening with enhanced expression of PCNA was observed in untransfected and sense PCNA ODN-treated allografts, whereas antisense PCNA ODN prevented neointimal formation.

Cytokinetic study of aortocoronary bypass vein grafts in place for less than six months.

To evaluate the initial mechanism involving atherosclerotic changes of the saphenous vein graft implanted for coronary artery revascularization in the early stage, immunocytochemical analysis was performed to determine the cell components and kinetics of saphenous vein grafts. Specimens of saphenous vein grafts were obtained from 7 necropsy patients who died at 4 days to 6 months after surgery. Monoclonal antibodies specific for smooth muscle cells (HHF35) and macrophages (HAM56) were used for analysis of the cell components. Migration of macrophages into the intima and the media was observed on the fourth postoperative day. Intimal thickening was characterized by the proliferation of smooth muscle cells, and scattered macrophages were present in the subendothelial layer 1 month after surgery. At 2 months, intimal thickening became prominent and macrophages were recognized circumferentially throughout the layer. At 5 to 6 months, some of the saphenous vein grafts were almost occluded by severe intimal thickening due to proliferation of the smooth muscle cells. Macrophages were also observed both inside and outside of the internal elastic lamina; these are rarely found in the artery. These results suggest that compared with the arterial graft, cytokinesia of the saphenous vein graft contributes to the development of early graft failure because of its rapidity in progression and severity.

Macroscopic extranodal invasion is a risk factor for tumor recurrence in papillary thyroid cancer.

Papillary thyroid cancer patients, upon whom curative operation was performed, were investigated to clarify whether or not macroscopic extranodal invasion is a risk factor for recurrence. They were divided into three groups: group A, patients whose primary tumor showed extrathyroidal invasion (n=31); group B, those whose metastatic lymph nodes showed extranodal invasion (n=6); group C, those who showed both extrathyroidal and extranodal invasion (n=9). Recurrence was significantly higher in groups B and C than in group A (P<0.05). It was concluded that macroscopic extranodal invasion to the adjacent structures was a risk factor for recurrence in patients with papillary thyroid cancer.

The relationship between multiple intrathyroidal involvement in papillary thyroid carcinoma and chronic non-specific thyroiditis.

The relationship between multiple intrathyroidal involvement (MII) and chronic non-specific thyroiditis (CT) was investigated in 69 papillary thyroid carcinoma patients who received a subtotal or total thyroidectomy. The overall incidence of MII in patients with CT and the incidence of MII in the affected lobe of the patients with CT were significantly higher than that without CT (P = 0.0012 and 0.0425, respectively). Because Hashimoto's thyroiditis is believed not to carry the increased risk of associated thyroid malignancy, the high incidence of MII in the affected lobe in the case with CT is postulated to be caused by intraglandular metastases.

Novel V184E MEN1 germline mutation in a Japanese kindred with familial hyperparathyroidism.

We studied the MEN1 gene in a kindred where three patients (the proposita and two of her sons) were affected with hyperparathyroidism. By polymerase chain reaction (PCR)-based direct sequencing of 10 exons of MEN1, a novel germline mutation was identified in the proposita. This mutation, a T-to-A transition at codon 184 in exon 3, predicts an amino acid change from valine to glutamine (V184E). PCR-single-strand conformational polymorphism (PCR-SSCP) analysis of exon 3 followed by sequencing showed the same mutation in the two sons, and in two clinically normal granddaughters of an affected son. Since the T-to-A substitution segregated with the disorder in the kindred except for the granddaughters and it was not detected in 100 alleles from 50 normal individuals, the change observed in MEN1 is not a polymorphism, but causes familial hyperparathyroidism. Thus the two grandchildren with the mutation were diagnosed as presymptomatic carriers.

Mutational analysis of the p73 gene localized at chromosome 1p36.3 in colorectal carcinomas.

Genetic alteration of p53, which monitors DNA damage and operates cellular checkpoints, is a major factor in the development of human colorectal carcinoma (CRC). Recently, p73, a novel family member of p53, has been identified and found, like p53, to activate p21Waf1/Cip1 and to induce apoptosis. The p73 gene was mapped at chromosome 1p36.3 which is a region frequently deleted in CRCs and other cancers including neuroblastoma. To assess whether or not p73 is a tumor suppressor gene of CRC, we performed mutational analysis of p73 in 82 colorectal tumor tissues paired with constitutional DNA. Using a microsatellite marker for p73, the loss of heterozygosity (LOH) study was performed and allelic loss of p73 was found in 17% of the CRCs. RT-PCR single strand conformation polymorphism analysis showed no mutation except three polymorphisms in the p73 coding region. In addition, p73 was expressed at higher levels in the CRC tissues than in the normal mucosa or neuroblastoma tissues, though the transcripts were detectable only by the RT-PCR method. Our results suggest that, in CRCs, p73 may not play a role as a tumor suppressor, at least not in a classic Knudson manner.

Coronary artery involvement in Takayasu's arteritis. Collective review and guideline for surgical treatment.

Coronary artery disease is a rare involvement of Takayasu's arteritis. From 1961 to 1989, 63 patients, including our five, have been reported to undergo operations for coronary artery disease resulting from Takayasu's arteritis. Most of the patients were Japanese (86%) and female (86%). The initial clinical manifestation was angina pectoris in 71%. Among 92 lesions, coronary ostia were most frequently involved (73%) followed by nonostial proximal lesions (18.5%). Forty-two of 62 (67.7%) ostial lesions of the left main coronary artery had more than 90%, or complete, stenosis. Aortic regurgitation was associated in 28 patients (44.4%). Myocardial revascularization was performed in 49, and transaortic endarterectomy in 12. Concomitant aortic valve replacement was done in 16 patients. Operative mortality was five (7.9%), and late deaths were reported in three patients. Postoperative steroid therapy was performed in 22. Operation was repeated in two patients because of graft failure. Thus coronary artery disease resulting from Takayasu's arteritis should be suspected in young Asian women with angina pectoris. The timing preferred for surgical intervention is during an inactive phase. Two procedures are commonly chosen for surgical intervention, either transaortic endarterectomy or coronary revascularization with vein grafts. Postoperative steroid therapy is strongly recommended to those patients who are operated on in the clinically or histologically active stage.

Effect of calcium antagonist diltiazem on renal function in open heart surgery.

STUDY OBJECTIVE: Acute renal failure, which is a serious complication following open heart surgery, has a high mortality rate. Previous reports have shown that the calcium antagonist diltiazem is beneficial either as an adjunct to cardioplegic solution or perioperative treatment for preservation of postoperative cardiovascular function. We studied the effects of diltiazem on renal function, plasma atrial natriuretic peptide levels, and the renin-angiotensin-aldosterone system in patients who had undergone coronary artery bypass grafting. PATIENTS AND MEASUREMENTS: Diltiazem was administered, 0.1 mg/kg, in a bolus injection followed by continuous infusion at a rate of 2 micrograms/kg/min during surgery, and 30 mg through a nasogastric tube at every 8 h. Hemodynamics, renal function, and plasma hormone levels were measured in the diltiazem-treated group (n = 13) and the nontreated group (n = 10). RESULTS: Heart rate, mean arterial pressure, and systemic vascular resistance index in the diltiazem-treated group were significantly lower than those in the nontreated group following cardiopulmonary bypass. Urine volume, creatinine clearance, and free water clearance were well preserved in the diltiazem-treated group. However, plasma renin activity and aldosterone levels were significantly higher in the diltiazem-treated group with the same changes in plasma atrial natriuretic peptide levels. CONCLUSION: Perioperative treatment with diltiazem has a beneficial effect on postoperative renal function, and reflex sympathetic activation induced by peripheral vasodilation activated the renin-angiotensin-aldosterone system.