Immortalization of bone marrow-derived human mesenchymal stem cells by removable simian virus 40T antigen gene: analysis of the ability to support expansion of cord blood hematopoietic progenitor cells

Human marrow-derived mesenchymal stem cells (MSC), which have the potential to differentiate into mesenchymal tissues, such as bone, cartilage, adipose and bone marrow stroma, were transduced with a retroviral vector carrying the simian virus 40 large T antigen, hygromycin-resistant gene and herpes simplex virus thymidine kinase gene, that can be excised by Cre/loxP site-specific recombination. This resulted in establishment of an MSC cell line, HMSC-1, which retained original surface characteristics and differentiation potential, and exhibited a higher proliferative capacity than parental cells. HMSC-1 expressed mRNAs of BMP-4, Jagged-1, and SCF that are known to promote hematopoiesis. Human CB CD34+ hematopoietic progenitor cells (HPC) cultured on a layer of HMSC-1 cells showed high expansion of CD34+CD38- immature HPC, capable of reconstituting human hematopoiesis in non-obese diabetic/severe combined immunodeficient disease (NOD/SCID) mice. This cell line may be of value for developing strategies for ex vivo expansion of human HPC.     

Resveratrol suppresses growth of Ishikawa cells through down-regulation of EGF

Resveratrol and other polyphenols have anti-carcinogenic and anti-tumorigenic activities in various carcinomas. However, such studies are limited in endometrial cancer. We hypothesize that resveratrol suppresses cancer growth through modulation of cell cycle and cell growth regulatory genes. To test this hypothesis, we treated endometrial cancer cells (Ishikawa cell line) with resveratrol (1, 10, 50 and 100 micro M) for 1, 3, 5 and 7 days, and analyzed for growth signal genes (EGF and VEGF), cell cycle regulatory genes (p53 and p21), and apoptosis-related genes (bcl-2 and bax). Results of these experiments demonstrate that after resveratrol treatment, the growth of Ishikawa cells was inhibited in a dose dependent manner. The gene and protein expression data suggest that resveratrol treatment significantly decreased EGF, whereas VEGF was up-regulated in Ishiwaka cell lines. Interestingly, protein expressions of p21 and Bax were decreased, even though their mRNA expressions did not show significant changes. The present study suggests that resveratrol can suppress proliferation of Ishikawa cells through down-regulation of EGF.     

CYP1B1 gene polymorphisms have higher risk for endometrial cancer,and positive correlations with estrogen receptor alpha and estrogen receptor beta expressions

The estradiol metabolites by CYP1B1 received particular attention because of their causative role in malignant transformation of endometrium. We hypothesize that polymorphisms of CYP1B1 gene can predict higher incidence of endometrial cancer. To test this hypothesis, the genetic distributions of six different CYP1B1 gene polymorphisms were investigated, by sequence-specific PCR and direct DNA sequencing, in 113 Japanese endometrial cancer patients and 202 healthy controls. We also investigated whether the expression of estrogen receptors (ERalpha and ERbeta), progesterone receptor, and androgen receptor genes are influenced by the CYP1B1 genotypes in endometrial cancer. The results of our study demonstrated that the distributions of CYP1B1 genotypes at codons 119 and 432 were significantly different between endometrial cancer patients and healthy normal controls. The relative risks of 119T/T and 432G/G in endometrial cancer were calculated as 3.32 and 2.49 compared with wild-types. The 119T/T showed significant correlation for positivities of ERalpha and ERbeta. The 432G/G also showed weak correlations for ERalpha positivity. Other loci, intron 1, codon 48, and codon 449 were not different between endometrial cancer patients and healthy normal control. This is the first report that demonstrates that the rare polymorphisms at codons 119 and 432 of CYP1B1 gene have higher risk for endometrial cancer, and positive correlations with ERalpha and ERbeta expressions in endometrial cancer.     

Mediastinal cavernous hemangioma in a child: report of a case

We present herein a rare case of mediastinal cavernous hemangioma in a 5-year-old boy. The patient was referred to our hospital for an evaluation of cough and high fever. On admission, a chest computed tomogram and magnetic resonance imaging revealed a large tumor arising from the left-sided mediastinum which compressed the left main pulmonary artery and left lung. The tumor, measuring 105 × 60 × 60 mm in size and weighing 170 g, was completely resected without any major bleeding, and a pathological examination confirmed the diagnosis of cavernous hemangioma. Received: October 29, 2001 / Accepted: May 7, 2002 Reprint requests to: J. Hanaoka     

Engineering of human hepatocyte lines for cell therapies in humans: prospects and remaining hurdles

Hepatocyte-based biological therapies are increasingly envisioned for temporary support in acute liver failure and provision of specific-liver functions in liver-based metabolic deficiency. One of the hurdles to develop such therapies is severe shortage of human livers for hepatocyte isolation. To address the issue, we have focused on reversible immortalization of human hepatocytes. Such technology can allow rapid preparation of functional and uniform human hepatocytes. Here we present our strategy to construct transplantable human hepatocyte cell lines.     

The differential effects of midazolam and diazepam on intracellular Ca2+ transients and contraction in adult rat ventricular myocytes

We investigated the direct effects of midazolam and diazepam on cardiac excitation-contraction coupling in adult rat ventricular myocytes. Freshly isolated rat ventricular myocytes were loaded with fura-2/AM and field-stimulated at 28 degrees C. Intracellular Ca(2+) transients (340:380 ratio) and myocyte shortening (video edge detection) were simultaneously monitored in individual cells. Midazolam (3-100 micro M) caused a dose-dependent decrease in both peak intracellular Ca(2+) and cell shortening. Diazepam (30 and 100 micro M) increased myocyte shortening and peak Ca(2+) concomitant with a decrease in time to peak Ca(2+). A larger concentration of diazepam (>300 micro M) nearly abolished intracellular Ca(2+) and cell shortening. Midazolam (100 micro M) and diazepam (300 micro M) decreased the amount of Ca(2+) released from intracellular stores in response to caffeine. Diazepam (30 micro M), but not midazolam (10 micro M), caused a downward shift in the dose-response curve to extracellular Ca(2+) for shortening, with no concomitant effect on peak intracellular Ca(2+) transient. These results indicate that midazolam and diazepam have different inotropic effects on cardiac excitation-contraction coupling at the cellular level, which is mediated by altering the availability of intracellular-free Ca(2+). However, the benzodiazepines have no direct influence on excitation-contraction coupling in rat ventricular myocytes, except at very large doses. Inhibition of Ca(2+) release from caffeine-sensitive intracellular Ca(2+) stores may play some part in myocardial depression at the larger concentrations of benzodiazepines. Diazepam, but not midazolam, decreased myofilament responsiveness to Ca(2+). IMPLICATIONS: Midazolam and diazepam differentially alter the cardiac excitation-contraction coupling at the cellular level, which is mediated by altering the availability of intracellular free Ca(2+) in adult rat ventricular myocytes. In addition, diazepam, but not midazolam, decreases myofilament Ca(2+) sensitivity. However, the benzodiazepines have no direct influence on excitation-contraction coupling, except at very large doses.     

Empyema necessitatis into the retroperitoneal space

Empyema necessitatis is a rare complication of tuberculous empyema. We present a very rare case of empyema necessitatis into the retroperitoneal space through the diaphragm. The fistula between the thoracic empyema cavity and the retroperitoneal abscess was clearly identified by magnetic resonance imaging.     

Gene therapy for lung cancer

Surgical resection is so far the most reliable therapy for localized non-small-cell lung cancer (NSCLC). Although the combination of several modalities such as chemotherapy or radiotherapy is used to treat advanced or recurrent disease, the results have not been satisfactory. A new therapeutic strategy is thus required to improve lung cancer treatment. Gene therapy is one such new therapeutic strategy. Several clinical trials of gene therapy protocols have been conducted and patient eligibility and being efficacy are evaluated. Representative studies of gene therapy for lung cancer and an interim report on a clinical trial using adenovirus vector expressing wild-type p53 in non-small cell lung cancer in Japan are introduced in this paper. Problems encountered and future regimens are also discussed.