Masayuki Oki

Gene expression profiles of endothelial progenitor cells by oligonucleotide microarray analysis.

Among the many tissue stem or progenitor cells recently being unveiled, endothelial progenitor cells (EPCs) have attracted particular attention, not only because of their cardinal role in vascular biology and embryology but also because of their potential use in the therapeutic development of a variety of postnatal diseases, including cardiovascular and peripheral vascular disorders and cancer. The aim of this study is to provide some basic and comprehensive information on gene expression of EPCs to characterize the cells in molecular terms. Here, we focus on EPCs derived from CD34-positive mononuclear cells of human umbilical cord blood. The EPCs were purified and expanded in culture and analyzed by a high-density oligonucleotide microarray and real-time RT-PCR analysis. We identified 169 up-regulated and 107 down-regulated genes in the EPCs compared with three differentiated endothelial cells of human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (LMEC) and human aortic endothelial cells (AoEC). It is expected that the obtained list include key genes which are critical for EPC function and survival and thus potential targets of EPC recognition in vivo and therapeutic modulation of vasculogenesis in cancer as well as other diseases, in which de novo vasculogenesis plays a crucial role. For instance, the list includes Syk and galectin-3, which encode protein tyrosine kinase and β-galactoside-binding protein, respectively, and are expressed higher in EPCs than the three control endothelial cells. In situ hybridization showed that the genes were expressed in isolated cells in the fetal liver at E11.5 and E14.5 of mouse development.

Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells.

OBJECTIVE To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs. MATERIALS AND METHODS We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens. RESULTS CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors. CONCLUSION Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.

Extensive and long-term ex vivo production of dendritic cells from CD34 positive umbilical cord blood or bone marrow cells by novel culture system using mouse stroma

We previously developed a system using murine strome (HESS-5), which could expand umbilical cord blood (UCB) stem and progenitor cells, especially CD34+/38- cells, in the presence of human recombinant cytokines. In this study, the ability of expanded UCB- or bone marrow (BM)-CD34+ cells to differentiate into dendritic cells (DCs) was examined. DCs could be induced either from short or long term cultured CD34+ cells after switching the cytokines from Flk-2/Flt-3 ligand, stem cell factor (SCF), thrombopoietin (TPO) to granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) (immature type) plus tumor necrosis factor (TNF)-alpha with stimulation by CD40L transfectant (mature type). Each immature or mature UCB-DCs showed a dextran uptake or a potent allo-T lymphocytes proliferative ability, respectively. Furthermore, those DCs from BM significantly stimulated auto-T lymphocytes in an antigen (varicella zoster virus) specific manner. In conclusion, a novel culture system using HESS-5 is useful to support a rapid and sustained generation of primitive myeloid cells which can develop into functional DCs.

Reconstitution of functional human B lymphocytes in NOD/SCID mice engrafted with ex vivo expanded CD34+ cord blood cells

OBJECTIVE Functional capacity of B cells developed from ex vivo expanded hematopoietic stem cells has not been fully evaluated. Therefore, we investigated the antigen-specific antibody production in human B cells maturated from ex vivo expanded cord blood (CB) CD34(+) cells in NOD/Shi-scid (NOD/SCID) mice. MATERIALS AND METHODS CB CD34(+) cells were cultured for 5 days in the presence of human cytokines and the murine stromal cell line HESS-5, and transplanted into irradiated NOD/SCID mice. These mice, reconstituted with human hematopoietic cells, were challenged with T-cell-independent (TI) or T-cell-dependent (TD) antigens after CD19(+) cells appeared at 6 weeks. RESULTS Three months later, anti-dinitrophenol (DNP)-specific antibody was detected in both mice immunized with DNP-Ficoll (TI) and those immunized with DNP-keyhole limpet hemocyanin or DNP-ovalbumin (TD). The anti-DNP antibody was mainly immunoglobulin M, but a small amount of immunoglobulin G also was detected. In the spleen, the majority of CD19(+) cells expressed mature B-cell markers such as CD40, immunoglobulin M, immunoglobulin D, cytoplasmic Cmu, and light chains kappa, and lambda. CONCLUSIONS These results indicate that human B cells develop from CD34(+) cells in NOD/SCID mice to produce antigen-specific antibody with in vivo primary stimulation. This system provides a powerful and versatile tool for studying the entire process of human B-lymphocyte development and producing specific human monoclonal antibodies.

A case of scrub typhus with acalculous cholecystitis, aseptic meningitis and mononeuritis multiplex.

We present an unusual case of a patient with scrub typhus who developed acalculous cholecystitis, aseptic meningitis and mononeuritis multiplex. The patient was successfully treated with oral minocycline. To our knowledge, this is the first report of mononeuritis multiplex caused by scrub typhus.

Epstein-Barr Virus Gastritis

A 20-year-old Japanese woman with a 1-year history of ulcerative colitis presented with a fever, sore throat, diarrhea, and abdominal pain that persisted for 1 week. For 3 months prior to admission, she had received treatment with infliximab and undergone granulocytopheresis. One month prior to admission, the prednisone dose was tapered and was eventually discontinued. However, her abdominal symptoms worsened despite azathioprine maintenance therapy, which she continued to take until admission. Colonoscopy yielded unremarkable findings, but gastroscopy showed multiple ulcerative nodular lesions (Picture 1). Hematoxylin-Eosin staining of a biopsy specimen of the gastric mucosa showed lymphocyte infiltration (Picture 2). In situ hybridization for Epstein-Barr virus (EBV)-encoded RNA (Picture 3) revealed signals consistent with those observed for CD20-positive lymphocytes (Picture 4). The peripheral blood EBV load was 4,378 copies/μg DNA on admission; the EBV antibody titers were as follows: EBV capsid antigen (VCA) immunoglobulin G (IgG), 160; EBV-VCA IgM, <10; and EBV nuclear antigen (EBNA) IgG, <10. Therefore, we diagnosed

A Case of Helicobacter pylori-negative Advanced Gastric Cancer with Massive Eosinophilia

The incidence of Helicobacter pylori-negative gastric cancer is very low. A 60-year-old man was referred to Tokai University Hospital from a local clinic because of eosinophilia. The laboratory data revealed prominent eosinophilia, with a white blood cell count of 7,900 /μL and increased eosinophil granulocyte level of 1,659 /μL. After an examination for secondary eosinophilia, esophagogastroduodenoscopy showed an enlarged gastric fold in the corpus, suggesting type 4 gastric cancer. Repeated esophagogastroduodenoscopy (EGD) and a re-biopsy demonstrated poorly differentiated adenocarcinoma and signet ring cell carcinoma. The patient was negative for Helicobacter pylori infection according to the serum anti-Helicobacter pylori antibody, culture and histopathological findings.

Irreversible metronidazole encephalopathy in an elderly woman with primary biliary cholangitis

An 82‐year‐old woman with primary biliary cholangitis was diagnosed with an irreversible neurological disorder, caused by metronidazole (MNZ)‐induced encephalopathy. Although the disorder is a reversible pathological condition, in rare cases, it can cause serious sequelae or could even be fatal. Therefore, medications should be administered carefully, particularly in patients who require long‐term administration of large doses or those with liver dysfunction.

A case of subclinical primary aldosteronism and subclinical Cushing's syndrome without risk factors of cardiovascular disease

• The critical issue is why excessive aldosterone secretion did not affect the cardiovascular status of this patient. In this regard, Ito et al. recently reviewed several possible mechanisms of normotensive PA (termed subclinical PA) [ Ito Y, Takeda R, Takeda Y. Subclinical primary aldosteronism. Best Pract Res Clin Endocrinol Metab. 2012; 26: 485495.]. They demonstrated that five possibilities could explain the causes of this normotensive form of PA: (a) an early state of the disease, (b) a mild form of the disease, (c) a low spontaneous baseline blood pressure level, (d) low sodium intake and/or high consumption of green tea, or (e) a low body mass index (BMI).