TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells.

We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b(+) myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells 'iPS-ML', and the iPS-ML-derived APC 'ML-DC'. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8(+) T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8(+) T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

Successful treatment of nonunion with teriparatide after failed ankle arthrodesis for Charcot arthropathy.

We describe a case of successful treatment to nonunion after multiple arthrodesis operations for Charcot arthropathy with teriparatide. We describe the case of a 25-year-old woman with severe Type I diabetes mellitus that resulted in nonunion after multiple arthrodesis operations for Charcot arthropathy. The woman sustained a femoral shaft fracture for which she underwent surgery with intramedullary nail fixation. Immediately after surgery, an empiric course of teriparatide was initiated. Femoral shaft fracture healing was observed after 2 weeks, and the woman was able to walk 12 weeks after the surgery, at which point plain film and computed tomography images revealed complete union of the ankle.

Cognitive and affective impairments of a novel SCA/MND crossroad mutation Asidan.

BACKGROUND: A variety of hereditary spinocerebellar ataxia (SCA) develops a broad spectrum of both ataxia and non-ataxia symptoms. Cognitive and affective changes are one such non-ataxia symptoms, but have been described only in hereditary SCAs with exonic CAG gene expansion. METHODS: We newly found intronic hexanucleotide GGCCTG gene expansion in NOP56 gene as the causative mutation (=SCA36) in nine unrelated Japanese familial SCA originating from Asida river area in the western part of Japan, thus nicknamed Asidan for this mutation. These patients show unique clinical balance of cerebellar ataxia and motor neuron disease (MND), locating on the crossroad of these two diseases. In the nine families, 14 patients were clinically examined and genetically confirmed to Asidan. In the present study, we examined cognitive and affective analyses on 12 patients (seven men and five women) who agreed to join the examination with average age at onset of 53.1 ± 3.2 years, average duration of 12.1 ± 5.2 years, and current average age at 65.1 ± 6.2 years. RESULTS: The 12 Asidan patients demonstrated a significant decrease in their frontal executive functions measured by frontal assessment battery (FAB) and Montreal cognitive assessment (MoCA) compared with age- and gender-matched controls, whilst mini-mental state examination (MMSE) and Hasegawa dementia score-revised (HDS-R) were within normal range. The decline of frontal executive function was related to their disease duration and scale for the assessment and rating of ataxias (SARA). They also demonstrated mild depression and apathy. Single-photon emission tomography (SPECT) analysis showed that these Asidan patients showed decline of regional cerebral blood flow (rCBF) in a particular areas of cerebral cortices such as Brodmann areas 24 and 44-46. CONCLUSION: These data suggest that the patients with Asidan mutation show unique cognitive and affective characteristics different from other hereditary SCAs with exonal CAG expansion or MND.

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.

This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid ß (Aß), the causal protein of Alzheimer's disease. The scFv-transfectant iPS-MP exhibited efficient Aß-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

Lectin panning method: the prospective isolation of mouse neural progenitor cells by the attachment of cell surface N-glycans to Phaseolus vulgaris erythroagglutinating lectin-coated dishes.

Retrospective isolation of neural progenitor cells (NPCs) may cause deterioration of the phenotype during the long-term cultivation. Therefore, prospective isolation is essential for understanding the exact characteristics of intact NPCs in the brain. However, few suitable specific cell surface antigens on NPCs that could be used for their prospective isolation are available. The present study demonstrated that within 60 min after initial plating, embryonic day 12 (E12) brain cells firmly attach to several types of lectin-coated culture wells, including Phaseolus vulgaris erythroagglutinating lectin (E-PHA), concanavalin A (Con A) and wheat germ agglutinin (WGA). Approximately 80% of the cells isolated from E-PHA-coated wells expressed the nestin antigen, which is a specific intracellular marker for NPCs and the ratio of 5-bromo-2'-deoxyuridine (BrdU)-positive/nestin-positive cells to the cells attached on the E-PHA-coated wells was significantly higher than that of the cells attached on the wells coated with other adhesive substrates. The cells that were isolated from the E-PHA-coated wells continued to attach to the well for 1 week, while those isolated from Con A- and WGA-coated wells lost their attachment after 6 days and 1 day, respectively. Furthermore, the cells isolated from the E-PHA-coated wells grew quite satisfactorily and formed numerous attached neurospheres. Their growth rate was almost equal to that observed in suspension cultures. These results indicate that the lectin panning method enables the prospective, quick and easy isolation of mouse NPCs without requiring a fluorescence-activated cell sorter (FACS) system.

Hippocalcin protects hippocampal neurons against excitotoxin damage by enhancing calcium extrusion.

Hippocalcin, which is a member of the neuronal calcium-sensor protein family, is highly expressed in hippocampal pyramidal cells. Recently, it was demonstrated that hippocalcin deficit caused an increase in neuronal cell death in the field CA3 of Ammon's horn (CA3) region of the hippocampus following the systemic injection of kainic acid. Treatment with kainic acid results in seizure-induced cell death in CA3. In the present study, we injected quinolinic acid, which is an N-methyl-d-aspartate receptor agonist, into the hippocampal field CA1 of Ammon's horn (CA1) region in hippocalcin-knockout (-/-) mice, a procedure which mimics transient ischemia. Although significant pyknotic changes were observed at the injected site in wild-type (+/+) mice 24 h after injection, the area of pyknotic cells extended throughout the hippocampus in -/- mice. The quantification of cell numbers in Nissl-stained sections indicated that the cell damage in -/- mice was more severe than that in +/+ mice. The density of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling-positive cells roughly paralleled that of Nissl-stained pyknotic cells. Primary cultures of hippocampal neurons showed that the number of surviving neurons from -/- mice after 7 days in culture was smaller than the number from +/+ mice. The measurement of intracellular calcium concentrations in single cells revealed that the calcium extrusion from -/- neurons was slower than that from +/+ neurons. The involvement of hippocalcin in the upkeep of calcium extrusion was confirmed using hippocalcin-expressing COS7 cells. These results suggest that hippocalcin plays an important role in calcium extrusion from neurons and, in turn, helps to protect them against calcium-dependent excitotoxin damage in the hippocampus.

FOXO3 is essential for CD44 expression in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most fatal types of cancer and the 5-year survival rate is only 5%. Several studies have suggested that cancer stem cells (CSCs) are thought to be involved in recurrence and metastasis and so it is essential to establish an approach targeting CSCs. Here we have demonstrated that cyclic guanosine monophosphate (cGMP) suppressed CD44 expression and the properties of CSCs in PDAC. Microarray analysis suggested that cGMP inhibited Forkhead box O3 (FOXO3), which is known as a tumor suppressor. Surprisingly, our data demonstrated that FOXO3 is essential for CD44 expression and the properties of CSCs. Our data also indicated that patients with high FOXO3 activation signatures had poor prognoses. This evidence suggested that cGMP induction and FOXO3 inhibition could be ideal candidates for pancreatic CSC.

Serum lipoprotein(a) dynamics before/after menopause and long-term effects of hormone replacement therapy on lipoprotein(a) levels in middle-aged and older Japanese women.

AIM: To assess lipoprotein(a) Lp(a) dynamics before and after menopause and to examine long-term changes during hormone replacement therapy (HRT) in middle-aged and older Japanese women. METHODS: (1) Serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and Lp(a) concentrations of 526 patients were compared. The patients were divided into 3 groups on the basis of menopausal status (premenopause, perimenopause, postmenopause). (2) Serum markers of lipid metabolism were measured at baseline and at 6-month intervals in 161 postmenopausal women who continuously received HRT with conjugated equine estrogen (CEE) and medroxyprogesterone acetate (MPA) for 4 years. (3) Changes in serum concentrations of markers were compared among 120 women with hypercholesterolemia who were randomly assigned to receive HRT (CEE plus MPA, or transdermal estradiol plus MPA) or pravastatin. RESULTS: (1) Lp(a) concentrations were significantly higher in the postmenopausal women than in the premenopausal or perimenopausal women. (2) The mean Lp(a) concentration after 6 months of HRT decreased by about 19%, and similar levels were maintained for 4 years (3). The mean Lp(a) concentration after 6 months of HRT decreased by 19.9% in the CEE plus MPA group, but did not change significantly in the transdermal estradiol plus MPA group or the pravastatin group. CONCLUSION: Our results suggest that HRT with CEE plus MPA is useful for the management of elevated serum Lp(a) concentrations in middle-aged and older women. However, follow-up studies are needed to determine whether this finding is related to the future prevention of coronary heart disease events.

Glycosphingolipids of various human ovarian tumors: a significantly high expression of I3SO3GalCer and Lewis antigen in mucinous cystadenocarcinoma.

Among several human ovarian tumors, which include mucinous cystadenocarcinoma, serous cystadenocarcinoma, and clear cell adenocarcinoma, the mucinous cystadenocarcinoma showed a unique glycosphingolipid composition. In particular, more than 90% of the acidic glycosphingolipids in the mucinous cystadenocarcinoma is comprised of sulfolipids, which are hardly detected in normal ovary and are contained in concentrations of less than 40% in the other type of ovarian tumors. By means of negative ion fast atom bombardment mass spectrometry and gas liquid chromatography, the major sulfolipid in mucinous cystadenocarcinoma is confirmed to be I3SO3-GalCer with N-cerebronoyl phytosphingosine, that which contrasts with I3SO3-GalCer with N-nonhydroxy fatty acyl sphingosine as the major molecular species in the other ovarian cancers. In mucinous cystadenocarcinoma, galactosylceramide is found in the relatively high concentration and is also composed of N-cerebronoyl phytosphingosine. In addition, the concentrations of glycolipids with Le(a) and Le(b) antigenicities are significantly higher in mucinous cystadenocarcinoma than those in normal ovary and the other ovarian tumors.

Characterization of tumor-associated neural cell adhesion molecule in human serum.

In human serum, at least two molecular species of the neural cell adhesion molecule (NCAM) with molecular weights of 110,000-130,000 and 150,000-180,000, respectively, can be identified by Western blotting. Both are characterized by the absence of epitopes for monoclonal antibodies KD11 and MG5, which specifically recognize intracellular domains of the human NCAM transmembrane isoforms, NCAM-140 and NCAM-180. In contrast to the M(r) 110,000-130,000 molecule also detectable in serum samples from healthy blood donors, the M(r) 150,000-180,000 molecule appears to be tumor associated. The only difference between these two species is shown to be the presence of long chains of alpha-(2,8)-linked N-acetylneuraminic acids, which are characteristic for the so-called embryonic NCAM form. After treatment with endoneuraminidase N, the M(r) 150,000-180,000 molecule can no longer be discriminated from the M(r) 110,000-130,000 molecule in Western blotting as well as gel and anion exchange chromatography experiments. The experimental data clearly show that only the embryonic NCAM molecule carrying the poly-alpha-(2,8)-linked N-acetylneuraminic acid moiety can be regarded as a specific serum marker for small cell lung cancer.

Human uterine endometrial adenocarcinoma: characteristic acquirement of synthetic potentials for II3SO3-LacCer and ganglio series sulfoglycosphingolipids after transfer of the cancer cells to culture.

The acidic glycosphingolipid composition of human uterine endometrial adenocarcinoma was compared with those of normal uterine endometrium at the proliferative and the secretory phases. Upon chemical composition analysis, no significant transformation-associated change of these glycolipids was observed. However, when cancer cells from the patients with human uterine endometrial adenocarcinoma were transferred to culture, the composition of glycosphingolipids, particularly sulfoglycosphingolipids, was significantly altered after the 70th doubling time. I3SO3-GalCer, which was contained in the original tissues of uterine endometrial adenocarcinomas, disappeared completely from the cultured cells at the 70th doubling time, whereas II3SO3-LacCer and ganglio series sulfoglycosphingolipids, which were originally contained in a trace amount or not present at all in the cancer tissues, became the major components in the total acidic glycosphingolipids in the cultured cells. Also, among cell lines established from several gynecological cancers, which include uterine cervical squamous carcinoma, uterine endometrial adenocarcinoma, ovarian clear cell carcinoma, choriocarcinoma, uterine sarcoma, ovarian sarcoma, and vulvar melanoma, only those cells derived from uterine endometrial adenocarcinoma expressed II3SO3-LacCer and ganglio series sulfoglycosphingolipids and the synthetic activities of these sulfoglycolipids, indicating that uterine endometrial adenocarcinoma cells characteristically lose the sulfotransferase to GalCer and acquire the sulfotransferase to LacCer after being transferred to culture in vitro. Thus, the unique sulfoglycosphingolipids and sulfotransferase are useful markers for the characterization of uterine endometrial adenocarcinoma among human gynecological cancers.

Light and glutamate-induced degradation of the circadian oscillating protein BMAL1 during the mammalian clock resetting.

Recently discovered mammalian clock genes are believed to compose the core oscillator, which generates the circadian rhythm. BMAL1/CLOCK heterodimer is the essential positive element that drives clock-related transcription and self-sustaining oscillation by a negative feedback mechanism. We examined BMAL1 protein expression in the rat suprachiasmatic nuclei (SCN) by immunoblot analysis. Anti-BMAL1 antiserum raised against rBMAL1 recognized 70 kDa mBMAL1b and detected a similar immunoreactivity (IR) as a major band in rat brains. Robust circadian BMAL1-IR oscillations with nocturnal peaks were detected in the SCN during a light/dark cycle and under constant darkness. A short duration light exposure at night acutely reduced BMAL1-IR in the SCN during photoentrainment. This might be attributable to the degradation of BMAL1 protein. Application of glutamate and NMDA to the SCN slices at projected night, a procedure mimicking photic phase delay shift, also acutely reduced BMAL1-IR in a similar manner. A rapid decrease of BMAL1 protein suggests that BMAL1 protein might be implicated in the light-transducing pathway within the SCN.

Luteal phase-characteristic induction of I3SO3-GalCer in human cervical epithelia and uterine endometria, and follicular phase-characteristic formation of a ganglioside-derived negative charge gradient in different regions of fallopian tubes.

In a series of experiments on the hormone-dependent molecular alteration in the human genital tract during the menstrual cycle, we focused our attention on a change in the negative charge due to the sulfuric acid- and sialic acid-containing glycosphingolipids. Although a ganglioside-derived negative charge was maintained in the cervical epithelia and uterine endometria at a relatively constant concentration throughout the luteal and follicular phases, I3SO3GalCer in both tissues characteristically increased in the luteal phase, indicating that the synthesis of I3SO3-GalCer in both tissues is associated with the menstrual cycle. However, I3SO3-GalCer in mucosae of the fallopian tubes in both phases was present in a concentration similar to that in the uterine endometrium in the luteal phase, and the change in the concentration did not associated with the menstrual cycle. On the other hand, although the concentrations of I3SO3-GalCer and II3NeuAc-LacCer, a major ganglioside, were similar in different regions, that is, the isthmus, ampulla and fimbriae of the fallopian tubes in the luteal phase, II3NeuAc-LacCer was present in a gradually increasing concentration from the isthmus to the fimbriae in the follicular phase, giving a gradually decreasing ratio of I3SO3GalCer to ganglioside from the uterus to the fimbriae. These findings indicate that the metabolism of sulfo- and sialoglycosphingolipids in the human genital tract is strictly controlled by estrogen and progesterone.

Site-directed mutagenesis of the response regulator DmsR for the dmsCBA operon expression in Rhodobacter sphaeroides f. sp. Denitrificans: An essential residue of proline-130 in the linker.

DmsR protein is a member of the OmpR response regulator subfamily that activates the transcription of the dmsCBA operon in Rhodobacter sphaeroides f. sp. denitrificans. By site-directed mutagenesis some functional amino acid residues were investigated in DmsR, which consists of the N-terminal regulatory and the C-terminal DNA-binding domains and the linker connecting the two domains. The substitution of P130S in the linker caused decreases of both DNA-binding and transcriptional activator activities. Introducing additional substitutions of R129P or D131P to the DmsR-P130S derivative recovered both activities, demonstrating necessity of proline residue at one of the positions 129-131 in the linker. Substitutions of D12A, D55A, and K104M, at residues conserved in the phosphorylation region, caused no production of DMSO reductase, but retained DNA-binding ability, suggesting that unphosphorylated DmsR also has high affinity to its target nucleotide sequence of DNA. Substitutions in the C-terminal domain suggested the presence of a winged helix-turn-helix structure observed in the DNA-binding domain of the Escherichia coli OmpR.

Molecular cloning of a novel calcium-binding protein structurally related to hippocalcin from human brain and chromosomal mapping of its gene.

A cDNA clone (hHLP2) encoding a novel calcium-binding protein structurally related to hippocalcin has been isolated from the human hippocampus cDNA library. The primary structure consists of 193 amino acids, and contains three EF-hand structures and a possible NH2-terminal myristoylation site. A single transcript at a position corresponding to 1.7 kilobases was detected only in the brain. The hHLP2 gene was mapped to human chromosome 2.

Structural characteristics of the ceramides of neutral glycosphingolipids in the human female genital tract--their menstrual cycle-associated change in the cervical epithelium and uterine endometrium, and their dissociation in the mucosa of the fallopian tube with the menstrual cycle.

In human cervical epithelium, uterine endometrium, and mucosa of the fallopian tubes, neutral glycosphingolipids were exclusively represented by the globo-series glycosphingolipids, such as CMH, LacCer, Gb3Cer and Gb4Cer, but the molecular species of their ceramide moieties were characteristically altered in the cervical epithelium and uterine endometrium during the menstrual cycle. Individual neutral glycosphingolipids in the cervical epithelium and the uterine endometrium at the follicular phase gave two bands on TLC, whereas those at the luteal phase displayed three bands, the third being the lower migrating one. Neutral glycosphingolipids migrating to the same positions as these lower-migrating bands were constantly detected in the mucosa of the fallopian tubes, independent of the menstrual cycle. The lower-migrating bands for the cervical epithelium and the uterine endometrium at the luteal phase were due to molecules mainly constructed of phytosphingosine with alpha-hydroxy fatty acids having chain lengths of 18-24 and 4-sphingenine with alpha-hydroxy fatty acids having chain lengths of 16-22, whereas those in the mucosa of the fallopian tubes were exclusively N-alpha-hydroxypalmitoyl 4-sphingenine.

Hippocalcin-deficient mice display a defect in cAMP response element-binding protein activation associated with impaired spatial and associative memory.

Hippocalcin is a member of the neuronal calcium sensor (NCS) protein family that is highly expressed in hippocampal pyramidal cells and moderately expressed in the neurons of cerebral cortex, cerebellum and striatum. Here we examined the physiological roles of hippocalcin using targeted gene disruption. Hippocalcin-deficient (-/-) mice displayed no obvious structural abnormalities in the brain including hippocampal formation at the light microscopic level. Deletion of hippocalcin did not result in up-regulation of the hippocalcin-related proteins; neural visinin-like Ca(2+)-binding proteins (NVP) 1, 2, and 3. The synaptic excitability of hippocampal CA1 neurons appeared to be normal, as estimated by the shape of field excitatory postsynaptic potentials elicited by single- and paired-pulse stimuli, and by tetanic stimulation. However, N-methyl-d-aspartate stimulation- and depolarization-induced phosphorylation of cAMP-response element-binding protein (CREB) was significantly attenuated in -/- hippocampal neurons, suggesting an impairment in an activity-dependent gene expression cascade. In the Morris water maze test, the performance of -/- mice was comparable to that of wild-type littermates except in the probe test, where -/- mice crossed the previous location of the platform significantly less often than +/+ mice. Hippocalcin-deficient mice were also impaired on a discrimination learning task in which they needed to respond to a lamp illuminated on the left or right side to obtain food reinforcement. No abnormalities were observed in motor activity, anxiety behavior, or fear learning. These results suggest that hippocalcin plays a crucial role in the Ca(2+)-signaling pathway that underlies long-lasting neural plasticity and that leads to spatial and associative memory.

A randomized comparison of locking and non-locking palmar plating for unstable Colles' fractures in the elderly.

This study compared the effectiveness of locking and non-locking palmar plating for unstable Colles' fractures in the elderly. The patients treated with locking plates included 4 men and 18 women with a mean age of 68 years (Group A) and those treated with non-locking plates included 3 men and 28 women with a mean age of 74 years (Group B). Radiographic parameters, including palmar tilt, radial inclination and radial length were measured before surgery, after surgery and at final followup. There were no significant differences in respect of any of the radiographic parameters between the two groups pre-operatively. After surgery, all of the radiographic parameters were improved in both groups and there were no significant differences between the two groups at final followup.

Isolation and characterization of the gene encoding mouse tax-responsive element-binding protein (TREB) 5.

TREB5/hXBP-1/HTF is a basic region leucine zipper protein which binds to a cyclic AMP responsive element (CRE)-like element in both human T-cell leukemia virus type 1 and human major histocompatibility complex (MHC) class II genes. To analyze the structure and transcription regulation of the TREB5 gene, we isolated the mouse TREB5 gene and cDNA. The mouse TREB5 gene contains five exons and four introns and spans approximately 5 kb. The deduced amino acid sequence of mouse TREB5 exhibited 77% and 94% homology to human and rat TREB5, respectively. The b-zip structure is completely conserved in mouse, rat and human. Southern blot analysis of the mouse genomic DNA demonstrated that positive bands exactly coincide with those expected from sequences of the cloned genes, indicating that the mouse TREB5 gene is present as a single copy. The transcription start site of the mouse TREB5 gene was mapped to -15 bp upstream from the ATG initiation codon. Promoter analysis of serial deletion mutants revealed that the -142 bp upstream region is the minimum sequence to promote mouse TREB5 gene expression and that the -1.0 kb upstream region is required for full promoter activity.