Induced pluripotent stem cell lines derived from human gingival fibroblasts and periodontal ligament fibroblasts.

BACKGROUND AND OBJECTIVE: Human induced pluripotent stem (iPS) cells, which have similar properties to human embryonic stem (hES) cells, have been generated from neonatal and adult human dermal fibroblasts by reprogramming. iPS cells have high pluripotency and differentiation potential, and may be a potential autologous stem cell source for future regenerative therapy. MATERIAL AND METHODS: iPS cell lines from human gingival fibroblasts and, for the first time, from periodontal ligament fibroblasts, were generated by reprogramming using a retroviral transduction cocktail of OCT3/4, SOX2, KLF4 and c-MYC. iPS induction was investigated through expression of the embryonic stem cell markers SSEA4, OCT4, NANOG, GCTM-2, TG30 and TRA-1-60. Following in vitro differentiation, the expression of genes for differentiation markers for ectoderm (SOX1, PAX6), mesoderm [RUNX1, T(Brachyury)] and endoderm (GATA4, AFP) was assessed by real-time RT-PCR. The ability to form teratomas following implantation into mouse testes was assessed by histology. RESULTS: Human gingival fibroblast- and periodontal ligament fibroblast-derived iPS cells showed similar characteristics to hES cells. Both sets of iPS cells displayed colony morphology comparable to that of hES cells and expressed the hES cell-associated cell-surface antigens, SSEA3, SSEA4, GCTM-2, TG30 (CD9) and Tra-1-60, and the hES cell marker genes, OCT4, NANOG and GDF3. These iPS cells showed differentiation potential to form embryoid bodies in vitro and expressed genes for endoderm, ectoderm and mesoderm. Teratoma formation following implantation into mouse testes was observed. CONCLUSION: These results demonstrate that iPS cells can be successfully generated from adult human gingival and periodontal ligament fibroblasts.

Putative stem cells in regenerating human periodontium.

BACKGROUND AND OBJECTIVE: Human postnatal stem cells have been identified in periodontal ligament, with the potential to regenerate the periodontium in vivo. However, it is unclear if periodontal ligament stem cells are present in regenerating periodontal tissues. The aim of this study was to identify and localize putative stem cells in block biopsies and explant cultures of regenerating human periodontal tissues. MATERIAL AND METHODS: Guided tissue regeneration was carried out on the molars of three human volunteers. After 6 wk, the teeth with the surrounding regenerating tissues and bone were surgically removed and processed for immunohistochemistry. The mesenchymal stem cell-associated markers STRO-1, CD146 and CD44 were used to identify putative stem cells. Cell cultures established from regenerating tissue explants were analysed by flow cytometry to assess the expression of these markers. Mineralization, calcium concentration and adipogenic potential of regenerating tissue cells were assessed and compared with periodontal ligament stem cells, bone marrow stromal stem cells and gingival fibroblasts. RESULTS: STRO-1(+), CD44(+) and CD146(+) cells were identified in the regenerating tissues. They were found mainly in the paravascular and extravascular regions. Flow cytometry revealed that cultured regenerating tissue cells expressed all three mesenchymal stem cell associated markers. The regenerating tissue cells were able to form mineral deposits and lipid-containing adipocytes. However, the level of mineralization in these cells was lower than that of periodontal ligament stem cells and bone marrow stromal stem cells. CONCLUSION: Cells with characteristics of putative mesenchymal stem cells were found in regenerating periodontal tissues, implying their involvement in periodontal regeneration.

Stem cells and periodontal regeneration.

Periodontitis is an inflammatory disease which manifests clinically as loss of supporting periodontal tissues including periodontal ligament and alveolar bone. For decades periodontists have sought ways to repair the damage which occurs during periodontitis. This has included the use of a range of surgical procedures, the use of a variety of grafting materials and growth factors, and the use of barrier membranes. To date periodontal regeneration is considered to be biologically possible but clinically unpredictable. Recently, reports have begun to emerge demonstrating that populations of adult stem cells reside in the periodontal ligament of humans and other animals. This opens the way for new cell-based therapies for periodontal regeneration. For this to become a reality a thorough understanding of adult human stem cells is needed. This review provides an overview of adult human stem cells and their potential use in periodontal regeneration.

Direct assessment of international consistency of standards for ground-level ozone: strategy and implementation toward metrological traceability network in Asia.

An international exercise to directly assess consistency of standards for ground-level ozone in East Asia was conducted as part of the East Asian Regional Experiment 2005 (EAREX 2005) in the framework of the Atmospheric Brown Clouds (ABC) project. Ten organizations collaboratively participated in the intercomparison. Four groups representing Japan, Korea, Hong Kong, and Taiwan made comparisons at the Gosan super observatory, Jeju Island, Korea, in March 2005, with ozone instruments calibrated to their national standards, and four Japanese groups made off-site comparisons with laboratory-level standards. All comparisons generally indicated good agreement with the standard reference photometer (SRP) 35, built by the National Institute of Standards and Technology (USA) and maintained by the National Institute for Environmental Studies (Japan). The assessment was expanded to measurement networks contributing to the World Meteorological Organization's Global Atmospheric Watch (WMO/GAW) program as part of off-site comparisons, and excellent agreement was achieved. These efforts contribute to propagating traceability of the national metrology standards among the atmospheric science community, to ensuring comparability of the existing ozone measurements, and to establishing an integrated network of air quality monitoring in Asia.

Synthesis and structure-activity relationships of 5-substituted pyridine analogues of 3.

In an effort to probe the steric influence of C5 substitution of the pyridine ring on CNS binding affinity, analogues of 1 substituted with a bulky moiety--such as phenyl, substituted phenyl, or heteroaryl-were synthesized and tested in vitro for neuronal nicotinic acetylcholine receptor binding affinity. The substituted analogues exhibited Ki values ranging from 0.055 to 0.69 nM compared to a Ki value of 0.15 nM for compound 1. Assessment of functional activity at subtypes of neuronal nicotinic acetylcholine receptors led to identify several agonists and antagonists.

Structure-activity studies on a novel series of cholinergic channel activators based on a heteroaryl ether framework.

Analogs of compound 1 with a variety of azacycles and heteroaryl groups were synthesized. These analogs exhibited Ki values ranging from 0.15 to > 10,000 nM when tested in vitro for cholinergic channel receptor binding activity (displacement of [3H](-) cytisine from whole rat brain synaptic membranes).

Antinociceptive effects of the novel neuronal nicotinic acetylcholine receptor agonist, ABT-594, in mice.

ABT-594 [5-((2R)-azetidinylmethoxy)-2-chloropyridine], a novel neuronal nicotinic acetylcholine receptor agonist, produced significant antinociceptive effects in mice against both acute noxious thermal stimulation--the hot-plate and cold-plate tests--and persistent visceral irritation--the abdominal constriction (writhing) assay (maximally-effective dose in each test 0.62 micromol/kg, i.p.). This effect was not stereoselective since the S-enantiomer, A-98593 [5-((2S)-azetidinylmethoxy)-2-chloropyridine], produced similar antinociceptive effects in this dose range. The effect in the hot-plate test peaked at 30 min after i.p. administration and was still present 60 min, but not 120 min, after injection. ABT-594 was orally active, but 10-fold less potent by this route than after i.p. administration. The antinociceptive effect of ABT-594 was prevented, but not reversed, by the noncompetitive neuronal nicotinic acetylcholine receptor antagonist mecamylamine (5 micromol/kg, i.p.). In contrast, the antinociceptive effect of ABT-594 was not prevented by hexamethonium (10 micromol/kg, i.p.), a neuronal nicotinic acetylcholine receptor antagonist that does not readily enter the central nervous system, nor by naltrexone (0.8 micromol/kg), an opioid receptor antagonist. Thus, initiation of antinociception by ABT-594 involves activation of central nicotinic acetylcholine receptors, but does not require activation of naltrexone-sensitive opioid receptors. The antinociceptive effects of morphine and ABT-594 in the mouse hot-plate test appeared to be additive, but ABT-594 did not potentiate the respiratory depression produced by morphine when the two compounds were coadministered. ABT-594 reduced body temperature and spontaneous exploration in the antinociceptive dose range, but did not reliably impair motor coordination in the rotarod test. Thus, it is unlikely that the antinociceptive effects result simply from impaired motor function. The compound also produced an anxiolytic-like effect in the elevated plus maze (at 0.019 and 0.062 micromol/kg, i.p.). Preliminary safety testing revealed an ED50 for overt seizure production of 1.9 micromol/kg, i.p. and an LD50 of 19.1 micromol/kg i.p. in mice, values 10 and 100 times the minimum effective antinociceptive dose of the compound. ABT-594 increased the duration of ethanol-induced hypnotic effects, tended to increase pentobarbital-induced hypnotic effects (P = 0.0502), and had no effect on pentobarbital-induced lethality. These data indicate that ABT-594 is a centrally acting neuronal nicotinic acetylcholine receptor agonist with potent antinociceptive and anxiolytic-like effects in mice.

ABT-594 [(R)-5-(2-azetidinylmethoxy)-2-chloropyridine]: a novel, orally effective antinociceptive agent acting via neuronal nicotinic acetylcholine receptors: II. In vivo characterization.

The antinociceptive effects of ABT-594, a novel nicotinic acetylcholine receptor (nAChR) ligand, were examined in rats in models of acute thermal (hot box) and persistent chemical (formalin test) pain. Also, the effects of ABT-594 treatment on motor function and electroencephalogram (EEG) were determined. In the hot box and formalin test (i.e., phase 1 and 2), acute treatment with ABT-594 (0.03, 0.1 and 0.3 mumol/kg i.p.) produced significant dose-dependent antinociceptive effects. In the hot box, the efficacy of ABT-594 was maintained after a repeated dosing paradigm (5 days b.i.d.i.p.). ABT-594 was fully efficacious in the formalin test when administered before formalin, and also retained significant efficacy (0.3 mumol/kg i.p.) when administered after formalin injection. The antinociceptive effects of ABT-594 in the hot box and formalin tests were attenuated by pretreatment with the nAChR antagonist, mecamylamine, and in animals treated with the nAChR antagonist chlorisondamine, given centrally (10 micrograms/rat i.c.v. 5 days before), but not in animals pretreated with the opioid receptor antagonist, naltrexone. Acute treatment with ABT-594 produced an initial decrease in open-field locomotor activity, which was absent in animals dosed repeatedly (5 days b.i.d.) with ABT-594. Also, acute treatment with ABT-594 decreased body temperature and decreased the amount of time the animals could maintain balance in an edge-balance test. These effects were no longer present in animals dosed repeatedly with ABT-594. At antinociceptive doses, ABT-594 produced activation of free running EEG in contrast to the sedative-like effects of morphine. Full antinociceptive efficacy was maintained in both the hot box and formalin tests after oral administration, whereas the effects on motoric performance were attenuated. In conclusion, these data demonstrate that ABT-594 is a potent antinociceptive agent with full efficacy in models of acute and persistent pain and that these effects are mediated predominately by an action at central neuronal nAChRs. In addition, antinociceptive effects were maintained after repeated dosing, whereas effects of ABT-594 on motor and temperature measures were attenuated in animals treated repeatedly with ABT-594. Thus, compounds acting at nAChRs may represent a novel approach for the treatment of a variety of pain states.

Identification and initial structure-activity relationships of (R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594), a potent, orally active, non-opiate analgesic agent acting via neuronal nicotinic acetylcholine receptors.

New members of a previously reported series of 3-pyridyl ether compounds are disclosed as novel, potent analgesic agents acting through neuronal nicotinic acetylcholine receptors. Both (R)-2-chloro-5-(2-azetidinylmethoxy)pyridine (ABT-594, 5) and its S-enantiomer (4) show potent analgesic activity in the mouse hot-plate assay following either intraperitoneal (i.p.) or oral (p.o.) administration, as well as activity in the mouse abdominal constriction (writhing) assay, a model of persistent pain. Compared to the S-enantiomer and to the prototypical potent nicotinic analgesic agent (+/-)-epibatidine, 5 shows diminished activity in models of peripheral side effects. Structure-activity studies of analogues related to 4 and 5 suggest that the N-unsubstituted azetidine moiety and the 2-chloro substituent on the pyridine ring are important contributors to potent analgesic activity.

Intermittent intussusception caused by colonic lipoma.

Colonic lipomas are rare, usually small, and occur most often in the right colon, particularly in the cecum. They are most common in elderly women. Intermittent episodes of intussusception are not uncommon in patients with colonic lipoma, but they are usually caused by larger pedunculated lipomas. We report a 43-year-old woman with a large colonic submucosal lipoma that induced intermittent colocolic intussusception. The patient presented with symptoms of peptic ulcer, including intractable upper abdominal pain, which did not resolve with treatment. Abdominal sonography showed typical findings of intussusception caused by a lipoma, but the manifestations on barium enema and computed tomography mimicked a malignant colonic tumor. The patient's abdominal pain disappeared after right hemicolectomy and the tumor was demonstrated to be a lipoma. The postoperative course was uneventful; there was no evidence of recurrence at follow-up 6 months later. Physicians should be aware that surrounding organs should also be evaluated in cases of chronic peptic ulcer with intractable upper abdominal pain.

Synthesis and structure-activity relationships of pyridine-modified analogs of 3-[2-((S)-pyrrolidinyl)methoxy]pyridine, A-84543, a potent nicotinic acetylcholine receptor agonist.

Analogs of 3-[2-((S)-pyrrolidinyl)methoxy]pyridine, (A-84543, 1) with 2-, 4-, 5-, and 6-substituents on the pyridine ring were synthesized. These analogs exhibited Ki values ranging from 0.15 to > 9,000 nM when tested in vitro for neuronal nicotinic acetylcholine receptor binding activity. Assessment of functional activity at subtypes of neuronal nicotinic acetylcholine receptors indicates that pyridine substitution can have a profound effect on efficacy at these subtypes, and several subtype-selective agonists and antagonists have been identified.

Structure-activity studies related to ABT-594, a potent nonopioid analgesic agent: effect of pyridine and azetidine ring substitutions on nicotinic acetylcholine receptor binding affinity and analgesic activity in mice.

Analogs of A-98593 (1) and its enantiomer ABT-594 (2) with diverse substituents on the pyridine ring were prepared and tested for affinity to nicotinic acetylcholine receptor binding sites in rat brain and for analgesic activity in the mouse hot plate assay. Numerous types of modifications were consistent with high affinity for [3H]cytisine binding sites. By contrast, only selected modifications resulted in retention of analgesic potency in the same range as 1 and 2. Analogs of 2 with one or two methyl substituents at the 3-position of the azetidine ring also were prepared and found to be substantially less active in both assays.

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine]: I. A potent and selective cholinergic channel modulator with neuroprotective properties.

Accumulating preclinical and clinical evidence data suggests that compounds that selectively activate neuronal nicotinic acetylcholine receptor (nAChR) subtypes may have therapeutic utility for the treatment of several neurological disorders. In the present study, the in vitro pharmacological properties of the novel cholinergic channel modulator ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine], are described. In radioligand binding studies, ABT-089 was shown to display selectivity toward the high-affinity (-)-cytisine binding site present on the alpha4beta2 nAChR subtype (Ki = 16 nM) relative to the [125I]alpha-bungarotoxin binding site present on the alpha7 (Ki > or = 10,000 nM) and alpha1beta1deltagamma (Ki > 1000 nM) nAChR subtypes. In cation flux and channel current studies, ABT-089 displayed a more complex profile than (-)-nicotine having agonist, partial agonist and inhibitory activities depending on the nAChR subtype with which it interacts. ABT-089 differentially stimulated neurotransmitter release. The compound displayed a similar potency and efficacy to (-)-nicotine to facilitate ACh release (ABT-089, EC50 = 3 microM; (-)-nicotine, EC50 = 1 microM), but was markedly less potent and less efficacious than (-)-nicotine to stimulate dopamine release (ABT-089, EC50 = 1.1 microM; (-)-nicotine, EC50 = 0.04 microM). Additionally, ABT-089 was neuroprotective against the excitotoxic insults elicited by exposure to glutamate in both rat cortical cell cultures (EC50 = 10 +/- 3 microM) and differentiated human IMR32 cells (EC50 = 3 +/- 2 microM). The differential full agonist/partial agonist profile of ABT-089, as compared with (-)-nicotine and ABT-418, illustrates the complexity of nAChR activation and the potential to target responses at subclasses of the neuronal and peripheral receptors.

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride]: II. A novel cholinergic channel modulator with effects on cognitive performance in rats and monkeys.

ABT-089 [2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine dihydrochloride], a novel ligand at neuronal nicotinic acetylcholine receptors with reduced adverse effects and improved oral bioavailability relative to (-)-nicotine, was tested in a variety of cognitive tests in rats and monkeys. Administered acutely, ABT-089 only marginally improved the spatial discrimination water maze performance of septal-lesioned rats. However, more robust improvement (45% error reduction on the last training day) was observed when ABT-089 was administered continuously via subcutaneous osmotic pumps (minimum effective dose: 1.3 micromol/kg/day). Continuous infusion of (-)-nicotine produced comparable improvement in the spatial discrimination water maze performance of septal-lesioned rats, but a 40-fold higher dose of (-)-nicotine was required (62 micromol/kg/day). Continuous infusion of ABT-089 to aged rats enhanced spatial learning in a standard Morris water maze, as indexed by spatial bias exhibited during a probe trial conducted after 4 days of training, but not when they were subsequently trained in a two-platform spatial discrimination water maze. The compound induced a small impairment in young rats on the standard water maze, but not on the two-platform task. A probe trial conducted after additional training in the standard water maze revealed no age or drug effects. ABT-089 did not affect performance of either the aged or young rats during inhibitory (passive) avoidance training. Also, continuous infusion of ABT-089 did not affect responses to acoustic startle or prepulse inhibition of acoustic startle in young, aged or septal-lesioned rats and did not affect locomotor activity in either sham-lesioned or septal-lesioned rats. In monkeys, acute administration of ABT-089 modestly improved the delayed matching-to-sample performance of mature, adult monkeys and more robustly improved performance in aged monkeys. Improved performance in the aged monkeys was restricted to the longest delay intervals and was not accompanied by changes in response latencies.

Structure-activity studies on 2-methyl-3-(2(S)-pyrrolidinylmethoxy) pyridine (ABT-089): an orally bioavailable 3-pyridyl ether nicotinic acetylcholine receptor ligand with cognition-enhancing properties.

2-Methyl-3-(2(S)-pyrrolidinylmethoxy)pyridine, ABT-089 (S-4), a member of the 3-pyridyl ether class of nicotinic acetylcholine receptor (nAChR) ligands, shows positive effects in rodent and primate models of cognitive enhancement and a rodent model of anxiolytic activity and possesses a reduced propensity to activate peripheral ganglionic type receptors. The profiles of S-4, its N-methyl analogue, and the corresponding enantiomers across several measures of cholinergic channel function in vitro and in vivo are presented, together with in vitro metabolism and in vivo bioavailability data. On the basis of its biological activities and favorable oral bioavailability, S-4 is an attractive candidate for further evaluation as a treatment for cognitive disorders.

Quantitative structure-activity relationships of nicotine analogues as neuronal nicotinic acetylcholine receptor ligands.

Quantitative structure-activity relationships of 34 pyrrolidine-modified nicotine agonists are investigated for their binding affinity toward neuronal nicotinic acetylcholine receptor. The results indicate that a large substituent at the R1, R2, and R3 position is detrimental to the binding affinity. Likewise, a large substituent at the R2 alpha or R3 alpha position as well as a hydrogen bond accepting substituent at the R2 beta position are not beneficial to the binding.

Lycaconitine revisited: partial synthesis and neuronal nicotinic acetylcholine receptor affinities.

The norditerpenoid alkaloid lycaconitine (2) was synthesized from lycoctonine (3) and its affinity determined for two neuronal nicotinic acetylcholine receptor subtypes. The structure of 2 was confirmed by a combination of spectroscopic methods.

Novel 3-Pyridyl ethers with subnanomolar affinity for central neuronal nicotinic acetylcholine receptors.

Recent evidence indicating the therapeutic potential of cholinergic channel modulators for the treatment of central nervous system (CNS) disorders as well as the diversity of brain neuronal nicotine acetylcholine receptors (nAChRs) have suggested an opportunity to develop subtype-selective nAChR ligands for the treatment of specific CNS disorders with reduced side effect liabilities. We report a novel series of 3-pyridyl ether compounds which possess subnanomolar affinity for brain nAChRs and differentially activate subtypes of neuronal nAChRs. The synthesis and structure-activity relationships for the leading members of the series are described, including A-85380 (4a), which possesses ca.50 pM affinity for rat brain [(3)H]-(-)-cytisine binding sites and 163% efficacy compared to nicotine to stimulate ion flux at human alpha4beta2 nAChR subtype, and A-84543 (2a), which exhibits 84-fold selectivity to stimulate ion flux at human alpha4beta2 nAchR subtype compared to human ganglionic type nAChRs. Computational studies indicate that a reasonable superposition of a low energy conformer of 4A with (S)-nicotine and (-)-epibatidine can be achieved.

A small modified hammerhead ribozyme and its conformational characteristics determined by mutagenesis and lattice calculation.

A prototypic hammerhead ribozyme has three helices that surround an asymmetrical central core loop. We have mutagenized a hammerhead type ribozyme. In agreement with previous studies, progressive removal of stem-loop II from a three stemmed ribozyme showed that this region is not absolutely critical for catalysis. However, complete elimination of stem II and its loop did reduce, but did not eliminate, function. In a stem-loop II-deleted ribozyme, activity was best preserved when a purine, preferably a G, was present at position 10.1. This G contributed to catalysis irregardless of its role as either one part of a canonical pair with a C residue at 11.1 or a lone nucleotide with C (11.1) deleted. Computational methods using lattices generated 87 million three-dimensional chain forms for a stem-loop II-deleted RNA complex that preserved one potential G.C base pair at positions 10.1 and 11.1. This exhaustive set of chain forms included one major class of structures with G(10.1) being spatially proximal to the GUCX cleavage site of the substrate strand. Strong correlations were observed between colinear arrangement of stems I and III, constraints of base-pairing in the central core loop, and one particular placement of G(10.1) relative to the cleavage site. Our calculations of a stem-loop II-deleted ribozyme indicate that without needing to invoke any other constraints, the inherent asymmetry in the lengths of the two loop strands (3 nt in one and 7 nt in the other) that compose the core and flank G10.1-C11.1 stipulated strongly this particular G placement. This suggests that the hammerhead ribozyme maintains an asymmetry in its internal loop for a necessary structure/function reason.

Ligands for brain cholinergic channel receptors: synthesis and in vitro characterization of novel isoxazoles and isothiazoles as bioisosteric replacements for the pyridine ring in nicotine.

Ligands which activate neuronal nicotinic acetylcholine receptors (nAChRs) represent a potential approach for the palliative treatment for the symptoms of memory loss associated with Alzheimer's disease (AD). Based upon this approach, a series of novel 3,5-disubstituted isoxazoles and isothiazoles were prepared and evaluated in vitro as cholinergic channel activators (ChCAs) of neuronal nAChRs. Many of the 3-substituted 5-(2-pyrrolidinyl)isoxazoles were found to have nanomolar binding affinities comparable to (S)-nicotine (2a) in a preparation of whole rat brain. However, in a paradigm measuring the evoked release of [3H]dopamine from a preparation of rat striatum, there were differences in the agonist potencies and efficacies of these analogues relative to 2a. The differences in agonist potency observed between compounds of comparable binding potency may be due to differences in ligand interactions with various subtypes of neuronal nAChRs.