Naohide Yamashita

Regulatory Dendritic Cells Protect Mice from Murine Acute Graft-versus-Host Disease and Leukemia Relapse

We have established a novel immunotherapeutic approach involving dendritic cells (DCs) with potent immunoregulatory property (designated as regulatory DCs [rDCs]) for acute graft-versus-host disease (GVHD) and leukemia relapse in allogeneic bone marrow (BM) transplantation (BMT) in mice bearing leukemia. rDCs displayed high levels of MHC molecules and extremely low levels of costimulatory molecules. A single injection of rDCs following allogeneic BMT controlled the ability of the transplanted T cells to induce acute GVHD and graft-versus-leukemia (GVL) effect in the recipients bearing leukemia, and that resulted in protection from the lethality caused by acute GVHD and tumor burden. Thus, the use of rDCs may be therapeutically useful for the treatment of acute GVHD and leukemia relapse in allogeneic BMT.

Dnm3os, a non‐coding RNA, is required for normal growth and skeletal development in mice

Dnm3os, a gene that is transcribed into a non‐coding RNA (ncRNA), contains three micro RNAs (miRNAs), miR‐199a, miR‐199a*, and miR‐214, whose functions remain unknown in mammals. In this study, we introduced the lacZ gene into the Dnm3os locus to recapitulate its expression pattern and disrupt its function. Dnm3os+/lacZ heterozygous embryos showed β‐galactosidase activity, which reflected the authentic expression pattern of Dnm3os RNA. Most of the Dnm3oslacZ/lacZ homozygous pups died within one month of birth. After birth, Dnm3oslacZ/lacZ mice exhibited several skeletal abnormalities, including craniofacial hypoplasia, defects in dorsal neural arches and spinous processes of the vertebrae, and osteopenia. Importantly, the expression of miR‐199a, miR‐199a*, and miR‐214 was significantly down‐regulated in Dnm3oslacZ/lacZ embryos, supporting the assumption that Dnm3os serves as a precursor of these three miRNAs. Thus, Dnm3os has emerged as an miRNA‐encoding gene that is indispensable for normal skeletal development and body growth in mammals. Developmental Dynamics 237:3738–3748, 2008.

Angiotensin II and vasopressin stimulate calcium-activated chloride conductance in rat mesangial cells.

In an attempt to clarify the mechanisms by which angiotensin II (AII) and arginine vasopressin (AVP) regulate mesangial cell function, we examined the membrane potential change of mesangial cells and found that cells contracted and membrane potential depolarized in response to AII and AVP. The depolarization was associated with decreased input resistance. Ca ionophore A23187 caused similar mesangial cell contraction and depolarization. The reversal potential (Vr) of the depolarization response to AII and AVP was -29 +/- 3 and -25 +/- 7 mV (mean +/- SD), respectively. Not only the Vr of the AII-induced depolarization but also Vr of the Ca ionophore-induced response was dependent upon the extracellular Cl- concentration. Further, AII and AVP caused cell contraction and membrane depolarization in Ca++-free medium containing 0.5 mM EGTA. These data suggest the presence of Ca++ -activated Cl- channels in the mesangial cells and that AII and AVP increase Cl- permeability via an elevation of [Ca++]i released from the intracellular organellae.

Periodic hyperpolarizing responses in hamster and mouse eggs fertilized with mouse sperm

1. The zona‐free hamster egg allows multiple entries of heterologous as well as homologous sperm. The hamster egg inseminated with mouse sperm (M × H egg) showed recurring, transient hyperpolarizing responses (h.r.s) with the peak of ‐70 to ‐80 mV. They were superimposed on a hyperpolarizing shift of the resting potential (h.s.) which gradually reached ‐60 mV in 50 min after insemination.

Human peripheral blood monocyte-derived interleukin-10-induced semi-mature dendritic cells induce anergic CD4(+) and CD8(+) T cells via presentation of the internalized soluble antigen and cross-presentation of the phagocytosed necrotic cellular fragments.

We examined the ability of human monocyte-derived interleukin (IL)-10-induced semi-mature dendritic cells (semi-mDCs) that had been pulsed with soluble protein and necrotic cellular fragments to induce an antigen (Ag)-specific anergy in CD4(+) and CD8(+) T cells. IL-10 converted normal immature DCs (iDCs) into semi-mDCs during the maturation. In contrast to normal iDCs and mature DCs, IL-10-induced semi-mDCs as well as IL-10-treated iDCs not only had reduced their allogeneic T cell-stimulatory capacity, but also induced an allogeneic Ag-specific anergy in T cells. Normal mDCs that had been pulsed with tetanus toxin (TT) or allogeneic necrotic cellular fragments caused further activation of TT-specific CD4(+) T cells or allogeneic fibroblast-specific CD8(+) T cells, Ag-pulsed IL-10-induced semi-mDCs induced an anergic state in both cell types. Thus, our results suggest that IL-10-induced semi-mDCs induce an Ag-specific anergy in CD4(+) and CD8(+) T cells via presentation of the internalized protein and cross-presentation of the phagocytosed cellular fragments.

Results of a phase I clinical study using autologous tumour lysate-pulsed monocyte-derived mature dendritic cell vaccinations for stage IV malignant melanoma patients combined with low dose interleukin-2

We conducted a pilot study to assess the feasibility and efficacy of immunotherapy for stage IV malignant melanoma patients resistant to conventional therapies involving vaccination with mature dendritic cells (mDCs) combined with administration of low dose interleukin-2. Autologous monocytes were harvested from a single apheresis and cultured for 7 days with granulocyte–macrophage colony-stimulating factor and interleukin-4, yielding immature dendritic cells (iDCs), which were then cryopreserved until use. For 4 days prior to vaccination, iDCs were exposed to autologous tumour lysate combined with tumour necrosis factor-α to induce terminal differentiation into mDCs. Patients were then vaccinated weekly with 107 mDCs for 10 weeks and given 350–700 kIU of interleukin-2 three times per week. Of the 10 patients in the study, one showed stable disease, seven showed progressive disease, and two showed mixed responses, including partial tumour regression, and were therefore given 20 additional injections. Only minimal adverse events were noted, including localized skin reactions and mild fever (NIH-CTC grade 0–1). Median survival from the first vaccination was 240 days (range 31–735 days). In vitro, melanoma patient-derived dendritic cells (DCs) showed reduced cell surface expression of CD1a antigen on iDCs and reduced CD86 and HLA-DR expression on mDCs. In addition, antigen uptake, chemotaxis and antigen presentation were all attenuated in DCs from the patients. In summary, although improvement of clinical efficacy will require further research, autologous tumour lysate-pulsed monocyte-derived mDCs could be safely harvested, cryopreserved and administrated to patients without obvious complications.

Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim.

We performed stem cell rescue and allogeneic skin transplantation on a lethally neutron-irradiated nuclear accident victim. HLA-DRB1 mismatched unrelated umbilical cord blood cells (2.08 × 107/kg recipient body weight) were transplanted to an 8–10 Gy equivalent neutron-irradiated patient because of a lack of a suitable bone marrow or peripheral blood donor. Pre-transplant conditioning consisted of anti-thymocyte γ-globulin alone, and GVHD prophylaxis was a combination of cyclosporine (CYA) and methylprednisolone (mPSL). Granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and thrombopoietin (TPO) were concurrently administered after transplantation. The absolute neutrophil count reached 0.5 × 109/l on day 15, the reticulocyte count rose above 1% on day 23, and the platelet count was over 50 × 109/l on day 27, respectively. Cytogenetic studies of blood and marrow showed donor/recipient mixed chimerism. Rapid autologous hematopoietic recovery was recognized after withdrawal of CYA and mPSL. Repeated pathological examinations of the skin revealed no evidence of acute GVHD. Eighty-two days after the irradiation, skin transplantation was performed to treat radiation burns. Almost 90% of the transplanted skin engrafted. Immunological examination after autologous hematopoietic recovery revealed an almost normal T cell count. However, immune functions were severely impaired. The patient died from infectious complication 210 days after the accident.Bone Marrow Transplantation (2002) 29, 197–204. doi:10.1038/sj.bmt.1703356

Cytokine production by primary human macrophages infected with highly pathogenic H5N1 or pandemic H1N1 2009 influenza viruses

Highly pathogenic H5N1 avian influenza viruses have caused infection in humans, with a high mortality rate, since 1997. While the pathogenesis of this infection is not completely understood, hypercytokinaemia and alveolar macrophages are thought to play a role. To gain further insight into the cytokine-mediated pathogenesis of this infection in humans, we measured various cytokines produced by primary human macrophages infected with H5N1, pandemic H1N1 or seasonal influenza viruses. We found that many cytokines were produced at higher levels on infection with the H5N1 strains tested compared with seasonal influenza viruses. Interestingly, the extent of cytokine induction varied among the H5N1 strains and did not correlate with replicative ability in macrophages. Further, a pandemic H1N1 virus induced higher levels of several cytokines compared with seasonal viruses and some H5N1 strains. Our results demonstrate that high cytokine induction is not a universal feature of all H5N1 viruses.

Estrogen receptor β mediates the inhibitory effect of estradiol on vascular smooth muscle cell proliferation

Objectives: It has been demonstrated that 17β-estradiol (E2) has an inhibitory effect on the proliferation of vascular smooth muscle cells (VSMCs) through an estrogen receptor (ER)-dependent pathway. Both ER subtypes, classical ER (ERα) and the newly identified ER subtype (ERβ), are expressed in VSMCs. However, it remains unknown which receptor plays the critical role in the inhibitory effect on VSMC proliferation. Methods and results: We constructed replication-deficient adenoviruses bearing the coding region of human ERα, ERβ, and the dominant-negative form of ERβ (designated AxCAERα, AxCAERβ, and AxCADNERβ, respectively). Prior to infection with the adenoviruses, 100 nmol/l E2 attenuated DNA synthesis by up to 14% and transactivated the estrogen-induced expression of the desired mRNA in rat VSMCs. This was accompanied by increased transcriptional activity of estrogen responsive element in response to E2, and the increase was comparable between AxCAERα and AxCAERβ. When VSMCs were infected with AxCAERβ at a multiplicity of infection of 5 or higher, DNA synthesis as well as cell number decreased by 50% in response to E2, and the effect was abolished by co-infection with AxCADNERβ. In contrast, when VSMCs were infected with AxCAERα, the reduction in DNA synthesis was minimal. Conclusions: Our results indicate that ERβ is more potent than ERα in the inhibitory effect on VSMC proliferation.

Proadrenomedullin NH2-terminal 20 peptide inhibits the voltage-gated Ca2+ channel current through a pertussis toxin-sensitive G protein in rat pheochromocytoma-derived PC 12 cells.

The effect of proadrenomedullin NH2-terminal 20 peptide (PAMP) on the voltage-gated Ca2+ channel current was investigated using the perforated whole-cell clamp technique on NGF-treated PC12 cells. PAMP inhibited the Ba2+ current through N-type Ca2+ channels in a concentration dependent manner. Injection of GDPbetaS into the cell abolished the inhibition while injection of GTPgammaS into the cell made the inhibition irreversible, indicating that the PAMP-induced inhibition of the voltage-gated Ca2+ channel was mediated by a G protein. The inhibition was abolished by pretreating the cells with pertussis toxin, indicating that a pertussis toxin-sensitive G protein was involved in the signal transduction mechanism of PAMP. The present study revealed that the inhibition of catecholamine secretion from sympathetic nerve endings by PAMP could be explained by the inhibition of N-type Ca2+ channels, which was mediated by pertussis toxin-sensitive G protein.