Oral administration of an HSP90 inhibitor, 17-DMAG, intervenes tumor-cell infiltration into multiple organs and improves survival period for ATL model mice.

In the peripheral blood leukocytes (PBLs) from the carriers of the human T-lymphotropic virus type-1 (HTLV-1) or the patients with adult T-cell leukemia (ATL), nuclear factor kappaB (NF-κB)-mediated antiapoptotic signals are constitutively activated primarily by the HTLV-1-encoded oncoprotein Tax. Tax interacts with the I κB kinase regulatory subunit NEMO (NF-κB essential modulator) to activate NF-κB, and this interaction is maintained in part by a molecular chaperone, heat-shock protein 90 (HSP90), and its co-chaperone cell division cycle 37 (CDC37). The antibiotic geldanamycin (GA) inhibits HSP90's ATP binding for its proper interaction with client proteins. Administration of a novel water-soluble and less toxic GA derivative, 17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride (17-DMAG), to Tax-expressing ATL-transformed cell lines, C8166 and MT4, induced significant degradation of Tax. 17-DMAG also facilitated growth arrest and cellular apoptosis to C8166 and MT4 and other ATL cell lines, although this treatment has no apparent effects on normal PBLs. 17-DMAG also downregulated Tax-mediated intracellular signals including the activation of NF-κB, activator protein 1 or HTLV-1 long terminal repeat in Tax-transfected HEK293 cells. Oral administration of 17-DMAG to ATL model mice xenografted with lymphomatous transgenic Lck-Tax (Lck proximal promoter-driven Tax transgene) cells or HTLV-1-producing tumor cells dramatically attenuated aggressive infiltration into multiple organs, inhibited de novo viral production and improved survival period. These observations identified 17-DMAG as a promising candidate for the prevention of ATL progression.

Hypoxia-enhanced derivation of iPSCs from human dental pulp cells.

Hypoxia enhances the reprogramming efficiency of human dermal fibroblasts to become induced pluripotent stem cells (iPSCs). Because we showed previously that hypoxia facilitates the isolation and maintenance of human dental pulp cells (DPCs), we examined here whether it promotes the reprogramming of DPCs to become iPSCs. Unlike dermal fibroblasts, early and transient hypoxia (3% O2) induced the transition of DPCs to iPSCs by 3.3- to 5.1-fold compared with normoxia (21% O2). The resulting iPSCs closely resembled embryonic stem cells as well as iPSCs generated in normoxia, as judged by morphology and expression of stem cell markers. However, sustained hypoxia strongly inhibited the appearance of iPSC colonies and altered their morphology, and anti-oxidants failed to suppress this effect. Transient hypoxia increased the expression levels of NANOG and CDH1 and modulated the expression of numerous genes, including those encoding chemokines and their receptors. Therefore, we conclude that hypoxia, when optimized for cell type, is a simple and useful tool to enhance the reprogramming of somatic cells to become iPSCs.

Dental pulp cells for induced pluripotent stem cell banking.

Defined sets of transcriptional factors can reprogram human somatic cells to induced pluripotent stem (iPS) cells. However, many types of human cells are not easily accessible to minimally invasive procedures. Here we evaluated dental pulp cells (DPCs) as an optimal source of iPS cells, since they are easily obtained from extracted teeth and can be expanded under simple culture conditions. From all 6 DPC lines tested with the conventional 3 or 4 reprogramming factors, iPS cells were effectively established from 5 DPC lines. Furthermore, determination of the HLA types of 107 DPC lines revealed 2 lines homozygous for all 3 HLA loci and showed that if an iPS bank is established from these initial pools, the bank will cover approximately 20% of the Japanese population with a perfect match. Analysis of these data demonstrates the promising potential of DPC collections as a source of iPS cell banks for use in regenerative medicine.

Characterization of dental pulp stem cells of human tooth germs.

In previous studies, human dental pulp stem cells (hDPSCs) were mainly isolated from adults. In this present study, we characterized hDPSCs isolated from an earlier developmental stage to evaluate the potential usage of these cells for tissue-regenerative therapy. hDPSCs isolated at the crown-completed stage showed a higher proliferation rate than those isolated at a later stage. When the cells from either group were cultured in medium promoting differentiation toward cells of the osteo/odontoblastic lineage, both became alkaline-phosphatase-positive, produced calcified matrix, and were also capable of forming dentin-like matrix on scaffolds in vivo. However, during long-term passage, these cells underwent a change in morphology and lost their differentiation ability. The results of a DNA array experiment showed that the expression of several genes, such as WNT16, was markedly changed with an increasing number of passages, which might have caused the loss of their characteristics as hDPSCs.

Structural diversity and evolution of the Rf-1 locus in the genus Oryza.

The Rf-1 locus in rice is agriculturally important as it restores fertility in plants with BT-type cytoplasmic male sterility (CMS). The Rf-1 locus contains several duplicated copies of the gene responsible for restoration of fertility. We analyzed the genomic structure of the Rf-1 locus in the genus Oryza to clarify the structural diversity and evolution of the locus. We identified six genes (Rf-1A to Rf-1F) with homology to Rf-1 at this locus in Oryza species with an AA genome. The Rf-1 locus structures in the rice accessions examined were very complex and fell into at least six classification types. The nucleotide sequences of the duplicated genes and their flanking regions were highly conserved suggesting that the complex Rf-1 locus structures were produced by homologous recombination between the duplicated genes. The fact that complex Rf-1 locus structures were common to Oryza species that have evolved independently indicates that a duplication of the ancestral Rf-1 gene occurred early in rice evolution and that homologous recombination resulted in the diversification of Rf-1 locus structures. Additionally, the amino acid sequences of each duplicated gene were conserved between species. This suggests that the duplicated genes in the Rf-1 locus may have divergent functions and may act by controlling mitochondrial gene expression in rice as occurs in the restoration of CMS.

The role of inducible co-stimulator (ICOS)/B7-related protein-1 (B7RP-1) interaction in the functional development of Peyer's patches.

T-cell co-stimulatory molecule, inducible co-stimulator (ICOS)/B7-related protein-1 (B7RP-1) interactions play an essential role of T-cell-dependent B-cell activation in peripheral lymphoid organs such as spleen and lymph nodes. Here, we investigate the role of ICOS/B7RP-1 interactions in the development of Peyer's patches (PPs). In ICOS(-/-) mice, the number of PPs was not decreased, although PPs in ICOS(-/-) mice were significantly reduced in size. Phenotypic analysis showed no obvious differences between ICOS(-/-) and ICOS(+/-) mice in the distribution of T-cells, B-cells, macrophages and dendritic cells. However, PNA(+) cells characteristic of intestinal germinal centers were totally absent in ICOS(-/-) mice. Moreover, production of IgA and IgG, but not IgM was significantly reduced in PPs in ICOS(-/-) mice. These data suggest that ICOS/B7RP-1 interactions may not affect the organogenesis, but involve in the functional development of PPs.

Cold-induced ascites in broiler chickens and its improvement by temperature-controlled rearing.

Pulmonary hypertension syndrome (PHS), also known as ascites, in broiler chickens prevailed in the local area of Ibaraki prefecture, Japan, and was investigated epidemiologically, serologically, and pathologically. PHS developed in chickens older than 35 days of age when rapid increase of body weight started. Approximately 90% of affected birds were males, in which weight increase was greater than in females. Serologic test revealed that PHS broilers had an increase of hematocrit value. Pathologic studies indicated that the heart of affected birds had an obese-induced pressure and cold exposure triggered congestion in the right ventricle/cava and an increase in peritoneal fluid. These changes were consistent with the previous reports of PHS, so we designed the experiment of effects on cold-induced PHS birds in a temperature-controlled house. After the 10 PHS birds at 55 days were reared for 14 days in a temperature-controlled house at 20 +/- 5 C, ascites disappeared in eight birds and hematocrit values decreased to normal range in nine birds. Our finding indicated that temperature-controlled environment may be one solution to reduce mortality in PHS birds.

Myxoid leiomyosarcoma of the gizzard in a broiler chicken.

A leiomyosarcoma was found in the gizzard of a 57-day-old female broiler chicken weighing 1.8 kg. Grossly, the tumor mass, 13.0 x 8.5 x 10.0 cm, enveloped the gizzard and had a gelatinous appearance due to the rich production of mucin. Miliary metastatic tumors were noted in the liver. Histopathologically, there was marked production of mucus throughout the tumor tissue, and densely or loosely arranged long spindle-shaped leiomyosarcoma cells proliferated. The tumor cells had a low rate of mitosis, showed slight cellular atypia, and, immunohistochemically, were positive for actin, alpha-smooth muscle actin, and desmin. Electron microscopically, various amounts of microfibrils with focal densities, dense patches, and basal plates were observed.

Opposing effects of anti-activation-inducible lymphocyte-immunomodulatory molecule/inducible costimulator antibody on the development of acute versus chronic graft-versus-host disease.

The functional role of inducible costimulator (ICOS)-mediated costimulation was examined in an in vivo model of alloantigen-driven Th1 or Th2 cytokine responses, the parent-into-F(1) model of acute or chronic graft-vs-host disease (GVHD), respectively. When the Ab specific for mouse ICOS was injected into chronic GVHD-induced mice, activation of B cells, production of autoantibody, and development of glomerulonephritis were strongly suppressed. In contrast, the same treatment enhanced donor T cell chimerism and host B cell depletion in acute GVHD induced host mice. Blocking of B7-CD28 interaction by injection of anti-B7-1 and anti-B7-2 Abs inhibited both acute and chronic GVHD. These observations clearly indicate that the costimulatory signal mediated by CD28 caused the initial allorecognition resulting in the clonal expansion of alloreactive T cells, whereas the costimulatory signal mediated by ICOS played a critical role in the functional differentiation and manifestation of alloreactive T cells. Furthermore, treatment with anti-ICOS Ab selectively suppresses Th2-dominant autoimmune disease.

Surgical outcome of pulmonary resection in chronic necrotizing pulmonary aspergillosis.

BACKGROUND: Surgical treatment of chronic necrotizing pulmonary aspergillosis is hazardous and controversial. METHODS: Ten patients (8 men, 2 women; mean age, 50 years) with chronic necrotizing pulmonary aspergillosis underwent pulmonary resection between 1989 and 2000. Single segmentectomy or lobectomy, pneumonectomy, or bilobectomy and multisegmentectomy were performed. Clinicopathologic features of these patients were reviewed to clarify the role of surgical intervention for chronic necrotizing pulmonary aspergillosis. RESULTS: The mean time from the onset of clinical symptoms to operation was 5.3 years. Surgical intervention was undertaken because of prolonged illness in 4 patients and hemoptysis in 6 patients. All patients survived. Three major complications (1 late empyema, 2 bronchopleural fistulas) occurred in the large dead space in the right pleural cavity. All survivors were free of aspergillosis at a mean follow-up time of 4.8 years, and only 1 patient required antifungal drugs for relapse during the follow-up period. CONCLUSIONS: Aggressive pulmonary resection in chronic necrotizing pulmonary aspergillosis should be considered when patients have prolonged illness or frequent hemoptysis. Empyema and bronchopleural fistula are the main complications. Concomitant thoracoplasty or intrathoracic transposition of the chest wall musculature is recommended in cases involving a large residual pleural cavity on the right side.

ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement.

We investigated the relationship between ICOS, CD28, CTLA-4, and IL-2 to gain a better understanding of this family of costimulatory receptors in the immune response. Using magnetic beads coated with anti-CD3 and varying amounts of anti-ICOS and anti-CTLA-4 Abs, we show that CTLA-4 ligation blocks ICOS costimulation. In addition to inhibiting cellular proliferation, CTLA-4 engagement prevented ICOS-costimulated T cells from producing IL-4, IL-10, and IL-13. Both an indirect and direct mechanism of CTLA-4's actions were examined. First, CTLA-4 engagement on resting cells was found to indirectly block ICOS costimulation by interferring with the signals needed to induce ICOS cell surface expression. Second, on preactivated cells that had high levels of ICOS expression, CTLA-4 ligation blocked the ICOS-mediated induction of IL-4, IL-10, and IL-13, suggesting an interference with downstream signaling pathways. The addition of IL-2 not only overcame both mechanisms, but also greatly augmented the level of cellular activation suggesting synergy between ICOS and IL-2 signaling. This cooperation between ICOS and IL-2 signaling was explored further by showing that the minimum level of IL-2 produced by ICOS costimulation was required for T cell proliferation. Finally, exogenous IL-2 was required for sustained growth of ICOS-costimulated T cells. These results indicate that stringent control of ICOS costimulation is maintained initially by CTLA-4 engagement and later by a requirement for exogenous IL-2.

AILIM/ICOS: its expression and functional analysis with monoclonal antibodies.

Activation-inducible lymphocyte immuno-mediatory molecule (AILIM/ICOS) is the third member of the co-stimulatory molecule CD28/CTLA-4 (CD152) family, and an inducible cell surface glycoprotein expressed on lymphocytes following activation. To determine the expression profile of the molecule, we generated monoclonal antibodies (MAbs) against human, rat, and mouse AILIM/ICOS. None of the MAbs bound to AILIM/ICOS of other species. The numbers of AILIM/ICOS-positive cells among human peripheral blood mononuclear cells (PBMC), and rat and mouse splenocytes were very low (0.5, 0.4, and 1.2%, respectively), and the cells included many CD4-positive T cells except in the case of rat. Rat AILIM/ICOS-positive cells among splenocytes included many CD45RA-positive B cells, although the expression on lymph node cells was similar to that on human PBMC and mouse splenocytes. Among rat thymocytes, the AILIM/ICOS expression was mainly localized on CD4- and CD8-double positive T cells. The binding of AILIM/ICOS to B7h-Ig, which is the ligand-Fc chimeric protein, was inhibited by all AILIM/ICOS-specific MAbs except for SG430. The potency of the co-stimulatory activity of CD3 and AILIM/ICOS as to T-cell proliferation was found to be substantial in human. Interestingly, the levels of stimulation with the two types of MAbs were equal to that with CD3 and CD28 despite the different functions of the two MAbs in the AILIM/ICOS-B7h interaction. On the other hand, the potencies in rat and mouse, although two independent MAbs were tested, were relatively lower than that of CD28-mediated co-stimulation.

Evaluation of autonomic nervous function during upper gastrointestinal endoscopy using heart rate variability.

To investigate autonomic nervous function during upper gastrointestinal endoscopy, we analyzed R-R interval variability from electrocardiograms obtained during endoscopy. Holter electrocardiogram recordings were made before and after premedication, and during endoscopy. Time- and frequency-domain analyses of heart rate variability were performed in 54 subjects premedicated with scopolamine butylbromide (SB group) and in 66 subjects premedicated with glucagon (G group). To determine the effect of autonomic imbalance on arrhythmia generation during endoscopy, subjects with arrhythmias (A group) were compared with subjects without arrhythmias (N group). In the SB group, high frequency spectral power (HF power; 0.15 to 0.40 Hz), which reflects parasympathetic activity, decreased significantly after premedication, and decreased further during endoscopy (P < 0.01). Moreover, HF power before premedication or during endoscopy in the A group was significantly lower than that in the N group (P < 0.01). This study suggests that the measurement of HF power prior to endoscopy can identify subjects with reduced HF power. This should allow the prevention of cardiovascular complications related to premedication and endoscope insertion.

Identification and characterization of rat AILIM/ICOS, a novel T-cell costimulatory molecule, related to the CD28/CTLA4 family.

Activation-inducible lymphocyte immuno-mediatory molecule (AILIM) is an inducible cell surface glycoprotein expressed on thymocytes and activated lymphocytes. Specific monoclonal antibody to rat AILIM induced the cell aggregation of a rat thymoma cell line and ConA-activated splenocytes. In the present study, we identified the primary structure of two species of rat AILIM by expression cloning. We also cloned mouse and human AILIM homologues and the predicted amino acid sequences were identical to those of the inducible costimulator ICOS/CRP-1, which belongs to the CD28/CTLA4 family. Although the human and mouse AILIM/ICOS molecule is localized on T-cells, the major population of AILIM/ICOS-positive cells in rat splenocyte was CD45RA-positive B-cells. The expression level of AILIM/ICOS on T-cells was relatively low; however, its expression was drastically induced by the treatment with PMA plus Ca-ionophore or the engagement of CD3 and these costimulatory molecules. Almost all T-cells exhibited potency as to its expression. Functional analysis of AILIM/ICOS demonstrated that AILIM-mediated costimulation was relatively weak compared to that of human.

Free 4-hydroxyproline content in serum of bedridden aged people is elevated due to fracture.

Hydroxyproline (Hyp) content in biological fluids is used as a parameter of collagen catabolism, especially bone resorption. In this paper, we examined the relationship between aging and serum free 4-Hyp and proline (Pro) content using a newly introduced analytical technique which is chemiluminescence determination with electrogenerated tris (2,2'-bipyridine) ruthenium(III). Serum was collected from 50 bedridden aged people and 131 normal subjects living in Yamato-son, Amamioshima, Kagoshima. In the free 4-Hyp content of normal serum, the change in females is more gentle than that males with aging. The free 4-Hyp content of bedridden aged serum is significantly (p<0.05) elevated compared to normal aged serum. Bedridden aged females and males also show levels significantly (p<0.05) elevated in comparison to normal. These changes are due to bone resorption based on bedridden patients' atrophy. In bedridden aged people, free 4-Hyp of those with a fracture was significantly (p<0.05) elevated than in people without a fracture. No relationship between low bone density and high free 4-Hyp in serum was observed. It seems that bone resorption or bedridden atrophy may be enhanced in bedridden aged people by a fracture. In free Pro content in serum, no significant change was observed. These results show that the free 4-Hyp content in serum is useful in the clinical or biological inspection of bone resorption, especially in aged people.

Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture.

Recently, we cloned a messenger RNA (mRNA) predominantly expressed in chondrocytes from a human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, by differential display PCR and found that its gene, named hcs24, was identical with that of connective tissue growth factor (CTGF). Here we investigated CTGF/Hcs24 function in the chondrocytic cell line HCS-2/8 and rabbit growth cartilage (RGC) cells. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA (mRNA) proliferated more rapidly than HCS-2/8 cells transfected with control adenoviruses. HCS-2/8 cells transfected with recombinant adenoviruses that generate CTGF/Hcs24 sense RNA expressed more mRNA of aggrecan and type X collagen than the control cells. To elucidate the direct action of CTGF/Hcs24 on the cells, we transfected HeLa cells with CTGF/Hcs24 expression vectors, obtained stable transfectants, and purified recombinant CTGF/Hcs24 protein from conditioned medium of the transfectants. The recombinant CTGF/Hcs24 effectively promoted the proliferation of HCS-2/8 cells and RGC cells in a dose-dependent manner and also dose dependently increased proteoglycan synthesis in these cells. In addition, these stimulatory effects of CTGF/Hcs24 were neutralized by the addition of anti-CTGF antibodies. Furthermore, the recombinant CTGF/Hcs24 effectively increased alkaline phosphatase activity in RGC cells in culture. Moreover, RT-PCR analysis revealed that the recombinant CTGF/Hcs24 stimulated gene expression of aggrecan and collagen types II and X in RGC cells in culture. These results indicate that CTGF/Hcs24 directly promotes the proliferation and differentiation of chondrocytes.

AILIM/ICOS: a novel lymphocyte adhesion molecule.

The adhesive interaction between T cells and antigen-presenting cells is required for the formation of the immunological synapse. Inducible co-stimulator (ICOS) is a third member of the CD28 family of co-stimulatory molecules. Here we describe a novel lymphocyte adhesion molecule, of relative molecular mass 47,000, designated AILIM, that is a rat homolog of ICOS. Rat AILIM was constitutively expressed on thymocytes and was induced on naive T cells after activation. Human thymoma cells bound to purified AILIM. Furthermore, cells transfected with the AILIM gene aggregated in an AILIM-dependent manner. These results suggest a novel function for AILIM/ICOS as an adhesion molecule, which plays an important role in T cell activation.

Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo.

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.

Toward creation of a universal NMR database for the stereochemical assignment of acyclic compounds: the case of two contiguous propionate units.

[formula: see text] Using triol 1 as a representative example of natural products containing two contiguous propionate units, 13C and 1H NMR databases for the stereochemical assignment of acyclic compounds have been created. Chemical shift increments due to the presence of additional functional groups as well as solvent effects are discussed.