Inhibition of heart transplant injury and graft coronary artery disease after prolonged organ ischemia by selective protein kinase C regulators

OBJECTIVE: Transplanted hearts subjected to prolonged ischemia develop ischemia-reperfusion injury and graft coronary artery disease. To determine the effect of delta-protein kinase C and -protein kinase C on ischemia-reperfusion injury and the resulting graft coronary artery disease induced by prolonged ischemia, we used a delta-protein kinase C-selective inhibitor peptide and an -protein kinase C-selective activator peptide after 30 or 120 minutes of ischemia. METHODS: Hearts of piebald viral glaxo (PVG) rats were heterotopically transplanted into allogeneic August Copenhagen Irish (ACI) rats. After cardioplegic arrest of the donor heart, -protein kinase C activator was injected antegrade into the coronary arteries. Hearts were procured and bathed in -protein kinase C activator, and before reperfusion, delta-protein kinase C inhibitor was injected into the recipient inferior vena cava. Controls were treated with saline. To analyze ischemia-reperfusion injury, grafts were procured at 4 hours after transplantation and analyzed for superoxide generation; myeloperoxidase activity; tumor necrosis factor alpha, interleukin 1beta, and monocyte/macrophage chemoattractant protein 1 production; and cardiomyocyte apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and caspase 2, 3, 8, and 9 activity. To analyze graft coronary artery disease, another set of animals underwent equal ischemic times and treatment strategies and then after 90 days were analyzed for graft coronary artery disease indexes. RESULTS: All measures of ischemia-reperfusion injury and graft coronary artery disease after 120 minutes of ischemia in the saline-treated group were significantly increased relative to those observed after 30 minutes of ischemia. It is important to note that all ischemia-reperfusion injury parameters and graft coronary artery disease indexes decreased significantly in the protein kinase C regulator-treated group in comparison to saline-treated controls; additionally, these values were equivalent to those in saline-treated controls with 30 minutes of ischemia. CONCLUSIONS: Combined treatment with -protein kinase C activator and delta-protein kinase C inhibitor reduces ischemia-reperfusion injury and decreases the resulting graft coronary artery disease induced by prolonged ischemia.     

Diffuse large B-cell lymphoma with extra Bcl-2 gene signals detected by FISH analysis is associated with a "non-germinal center phenotype"

Amplification and translocation of the Bcl-2 gene has been detected in a certain subset of diffuse large B-cell lymphomas (DLBCL). The correlations among Bcl-2 protein expression, gene translocation or amplification, and the molecular signature determined by cDNA array are poorly understood. This study examined 25 cases with de novo nodal DLBCL. Interphase fluorescence in situ hybridization (FISH) analysis was performed to evaluate the Bcl-2 gene using IGH/BCL2 and CEP18 centromere probes (Vysis). When extra Bcl-2 gene signals were observed in each tumor cell and when these signals were in proportion to the extra CEP18 probe signals, we regarded the findings as indicating the presence of an additional chromosome 18; when extra Bcl-2 signals were observed but additional CEP18 signals were not, we regarded the findings as indicating the presence of gene amplification. A panel of 3 antigens (CD10, Bcl-6, and MUM-1) was applied to categorize each case as either a "germinal center B-cell (GCB) phenotype" or a "non-GCB phenotype." Of the 25 cases examined, 8 cases (32%) were classified as "GCB phenotype" and 17 cases (68%) were classified as "non-GCB phenotype." A FISH analysis revealed that t(14;18) was detected in 2 of the 8 cases (25%) with the "GCB phenotype" but in none of the 17 "non-GCB phenotype" cases. Extra Bcl-2 gene signals were detected in 7 of the 25 (28%) cases examined: n = 5 for an additional chromosome 18, n = 1 for gene amplification, and n = 1 for additional chromosome 18 + gene amplification. Extra Bcl-2 gene signals were exclusively detected in DLBCL with the "non-GCB phenotype"; these cases, with the exception of one, stained strongly positive for Bcl-2. The DLBCLs with Bcl-2 protein overexpression were classified into at least two heterogeneous molecular groups, based on the results of the FISH analysis.     

Inhibitory effects of brotizolam,a new thienodiazepine,on limbic forebrain and neostriatal dopaminergic systems in vivo and in vitro

Studies were performed to elucidate the effects of brotizolam, a newly synthesized thienodiazepine, chemically related to the benzodiazepines. on dopamine turnover in the limbic forebrain and neostriatum. Intraperitoneally administered brotizolam retarded the rate of α-methyl-p-tyrosine-induced depletion of dopamine in the olfactory tubercle (OT), nucleus accumbens (NA) and caudate nucleus (CN). Significant retardation was observed with brotizolam in doses ranging from 0.1–10 mg/kg in the olfactory tubercle, and from 1–10 mg/kg in the nucleus accumbens and caudate nucleus. These inhibitory effects of brotizolam were antagonized by bicuculline, a GABA antagonist, in all of the regions examined. Using slices of the olfactory tubercle, nucleus accumbens and caudate nucleus, the effects of brotizolam on dopaminergic nerve terminals were examined _{in vitro}. Basal release of dopamine was not affected by brotizolam in concentrations up to 10^?6 M; however. K⁺-stimulated release of dopamine was significantly reduced by brotizolam at 10^?7M or above. The reduction of K⁺-stimulated release of dopamine was antagonized by bicuculline, added in the superfusion medium. These data suggest that brotizolam inhibits the release of dopamine in the limbic forebrain and neostriatal systems probably through mechanisms including a facilitation of GABAergic action on dopaminergic nerve terminals.     

A case of idiopathic sclerosing mediastinitis in a 7-year-old Japanese boy

Sclerosing mediastinitis is a very rare benign disorder characterised by the development of dense fibrous tissue within the mediastinum. Affected patients are typically young adults with infant cases being uncommon especially in areas without endemic histoplasmosis. We report a Japanese boy with markedly elevated serum inflammatory markers for more than 1 year in the absence of any clinical manifestations. ⁶⁷Ga-scintigraphy demonstrated an accumulation in the mediastinal region and an open biopsy revealed a hard fibrous mass in the anteriosuperior mediastinum. Thus, a diagnosis of idiopathic sclerosing mediastinitis was made. Conclusion:To the best of our knowledge, this case is the youngest patient reported with this disorder. In patients with mediastinal mass lesions the diagnosis of sclerosing mediastinitis should be considered as well as infectious, autoimmune or neoplastic disease even in children.     

Successful unmanipulated haploidentical bone marrow transplantation from an HLA 2-locus-mismatched mother for Wiskott-Aldrich syndrome after unrelated cord blood stem cell transplantation

The authors describe a boy with Wiskott-Aldrich syndrome (WAS) who was diagnosed immediately after birth using flow cytometric and genetic analysis. At 1 year of age he received unrelated cord blood stem cell transplantation (UCBSCT); however, the sex chromosomes of the peripheral blood mononuclear cells showed that the recipient type was over 70%. This rate gradually increased to over 90% after immunosuppressant therapy was discontinued. Clinical manifestations, including high fever, graft-versus-host disease (GVHD)-like eruptions, and signs of infection recurred. Results of flow cytometric and genetic analysis of mononuclear cells from the boy's mother were normal with no mutation. Three months after UCBSCT, he received an unmanipulated HLA-haploidentical 2-locus-mismatched bone marrow transplant (BMT) from his mother. The prophylaxis against GVHD was tacrolimus and short-term methotrexate. Hematopoietic reconstitution was rapid and fluorescence in situ hybridization analysis revealed sustained engraftment. Grade II acute GVHD developed but improved rapidly with the administration of methylprednisolone. The patient is progressing well and displays complete chimerism 2 years after the BMT. This case suggests that unmanipulated haploidentical BMT from the mother might be feasible not only for malignant disease but also for immunodeficiency disease patients who urgently need stem cell transplants and have no HLA-identical donors.     

Successful treatment of chemoresistant stage 3 neuroblastoma using irinotecan as a single agent

The authors describe a 1-year-old boy who was diagnosed with neuroblastoma by mass screening at age 6 months. The tumor originated from the left retroperitoneum and extended over the midline, involving major vessels and invading the spine with compression of the spinal cord. Although seven courses of chemotherapy consisting of vincristine sulfate, cyclophosphamide, pirarubicin hydrochloride, and cisplatin were administered, there was no reduction in tumor size or decrease in tumor markers. The patient received irinotecan 180 mg/m per day for 3 days. Approximately 3 weeks later the tumor had regressed remarkably, and tumor markers normalized after the second course of irinotecan. This therapy was given a total of four courses every 4 weeks, with the tumor shrinking successively in each session. Four years after treatment there is no sign of recurrence and the patient is doing well. This case may be the first report showing the dramatic efficacy of irinotecan in the treatment of chemoresistant neuroblastoma without the use of other antitumor agents. Irinotecan might be a promising drug in the management of patients with high-risk neuroblastoma.     

Quantitative analysis of binding parameters of [3H]N-methylscopolamine in central nervous system of muscarinic acetylcholine receptor knockout mice

We have studied binding parameters (Kd, Bmax) of [3H]N-methylscopolamine ([3H]NMS) in various brain regions and spinal cord of wild-type (WT) and muscarinic acetylcholine receptor (mAChR) subtype (M1-M5) knockout (KO) mice. In the M1-M4 KO mice, the number of [3H]NMS binding sites (Bmax) was decreased throughout the central nervous system (CNS) with significant regional differences. Our results collectively suggest that M1 receptor was present in a relatively high density in the cerebral cortex and hippocampus, and the densities of M1 and M4 subtypes were highest in the corpus striatum. M2 receptor appeared to be the major subtype in the thalamus, hypothalamus, midbrain, pons-medulla, cerebellum and spinal cord. These findings may contribute significantly not only to the further understanding of the physiological roles of mAChR subtypes in the central cholinergic functions, but also to the development of selective therapeutic agents targeting specific subtype.     

Assessment of chromosome and gene analysis for the diagnosis of the fragile X syndrome in Japan: annual incidence

We assessed the utilization of diagnostic analyses for fragile X syndrome by a mail-in questionnaire on 1) the number of patients analyzed and diagnosed with the syndrome in the past year, 2) types of diagnostic analyses used, 3) clinical features that made physicians to decide analyses, 4) purpose of analyses, and 5) informed consent for analyses. Facilities for the mentally handicapped, as well as hospitals and physicians specialized in genetics, completed our questionnaire. Among 101 responders, total of 543 cases underwent analyses. Nine cases (including 3 cases over 20 years old) were finally diagnosed in a year. The rate of positive findings was 0.6% for chromosomal analyses, and 8% for gene analyses. Physicians dicided to make analyses based on clinical features such as mental retardation, characteristic face, and autistic features, in order to find the cause (s) of the subjects' condition. For gene analyses, more than a half of physicians obtained a form of informed consent. Specialists should have interest in this syndrome because the analyses identified new adult cases. Establishment of a guideline for diagnosis of this syndrome requires gene analyses based on evidence and informed consent.