Effects of intravenous administration of umbilical cord blood CD34(+) cells in a mouse model of neonatal stroke.

Neonatal stroke occurs in approximately 1/4000 live births and results in life-long neurological impairments: e.g., cerebral palsy. Currently, there is no evidence-based specific treatment for neonates with stroke. Several studies have reported the benefits of umbilical cord blood (UCB) cell treatment in rodent models of neonatal brain injury. However, all of the studies examined the effects of administering either the UCB mononuclear cell fraction or UCB-derived mesenchymal stem cells in neonatal rat models. The objective of this study was to examine the effects of human UCB CD34(+) cells (hematopoietic stem cell/endothelial progenitor cells) in a mouse model of neonatal stroke, which we recently developed. On postnatal day 12, immunocompromized (SCID) mice underwent permanent occlusion of the left middle cerebral artery (MCAO). Forty-eight hours after MCAO, human UCB CD34(+) cells (1×10(5)cells) were injected intravenously into the mice. The area in which cerebral blood flow (CBF) was maintained was temporarily larger in the cell-treated group than in the phosphate-buffered saline (PBS)-treated group at 24h after treatment. With cell treatment, the percent loss of ipsilateral hemispheric volume was significantly ameliorated (21.5±1.9%) compared with the PBS group (25.6±5.1%) when assessed at 7weeks after MCAO. The cell-treated group did not exhibit significant differences from the PBS group in either rotarod (238±46s in the sham-surgery group, 175±49s in the PBS group, 203±54s in the cell-treated group) or open-field tests. The intravenous administration of human UCB CD34(+) cells modestly reduced histological ischemic brain damage after neonatal stroke in mice, with a transient augmentation of CBF in the peri-infarct area.

Characterization of 5C11-positive activated interferon-producing cells in patients with systemic lupus erythematosus.

OBJECTIVE: 5C11 antibody is a novel monoclonal antibody against human BST2 and can be used to detect activation of interferon-producing cells (IPCs). Activated IPCs, which produce large amounts of interferon-α (IFNα), are considered to play an important role in the pathogenesis of systemic lupus erythematosus (SLE). We investigated the characterization of 5C11-positive cells in patients with SLE. METHODS: The proportions of 5C11-positive cells among blood dendritic cell antigen 2 (BDCA-2)-, CD3-, CD19- and CD14-positive cells in peripheral blood from SLE patients (SLE-PBMCs) and healthy controls (control-PBMCs) were analyzed by flow cytometry. The effect of 5C11 antibody on IFNα production from SLE-PBMCs under stimulation with cytosine-phosphate-guanosine (CpG2216, bacterial oligonucleotide motif) was also examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: The proportions of 5C11-positive cells among BDCA-2-, CD3- and CD19-, but not CD14-positive cells in SLE-PBMCs were significantly increased compared to those in control-PBMCs (p < 0.0001, all). Especially, the number of 5C11-positive cells among BDCA-2-positive cells was significantly increased in SLE-PBMCs by about six-fold compared to that in control-PBMCs (p < 0.0001). 5C11 antibody inhibited IFNα production by SLE-PBMCs induced by CpG and the inhibition rates was 27% (p < 0.001). CONCLUSION: SLE patients had a significantly higher proportion of 5C11-positive cells among CD3 and CD19 cells, and especially BDCA-2 positive cells. The ability of 5C11 antibody to inhibit IFNα production from SLE-PBMCs warrants further investigation for its possible clinical application for the treatment of SLE.

Synthesis, release ability and bioactivity evaluation of chitin beads incorporated with curcumin for drug delivery applications.

Spherical curcumin-incorporated chitin porous beads, with porosity exceeding 98%, were successfully fabricated by combining a coagulation method with freeze-drying. Tween 20 was used to enhance the solubility of curcumin in the chitin beads during the incorporation step. The internal pores and the outer non-porous shell structure of the chitin beads determined the curcumin-releasing behaviour: the internal porous structure could dramatically increase the uptake of the releasing medium, while the non-porous outer skin layer dominated the slow releasing behaviour of curcumin. In addition, the amount of Tween 20 also played an important role in the release characteristic of curcumin from the chitin matrix. Bioactivity evaluation of the obtained chitin beads was conducted by immersion in a simulated body fluid (SBF). It could be postulated the entrapment of curcumin into the cores of Tween 20 micelles encapsulated in the chitin shell could be a promising candidate for drug delivery devices.

Insulin-like growth factor-binding protein-2 and -3 in gingival crevicular fluid.

BACKGROUND AND OBJECTIVE: Insulin-like growth factor-binding proteins (IGFBPs) are crucial regulators of insulin-like growth factor (IGF). They enhance or inhibit IGF functions, but also exhibit IGF-independent effects. In a previous study, we detected, qualitatively, IGFBP-2 and -3 in gingival crevicular fluid using a cytokine antibody array. Here we extended these results using an ELISA to determine the concentrations of IGFBP-2 and -3 in gingival crevicular fluid. In addition, we explored whether the expression of IGFBP-2 and IGFBP-3 correlates with periodontal disease severity. MATERIAL AND METHODS: Gingival crevicular fluid samples from 92 sites of 12 patients affected with periodontal disease and from 100 sites of 19 healthy volunteers, were collected, divided into two groups and analyzed by ELISA for IGFBP-2 and -3 expression. The potential correlation among probing depth, gingival index and the concentrations of IGFBP-2 and -3 was analyzed. RESULTS: Positive correlations were observed between the concentration of IGFBP-2 and probing depth and gingival index, but not for IGFBP-3. The IGFBP-2 concentrations at bleeding on probing-positive sites and at sites with a probing depth of ≥ 4 mm were higher than at bleeding on probing-negative sites and at sites with a probing depth of ≤ 3 mm. CONCLUSION: These results indicate that IGFBP-2 is a potential novel marker for periodontal disease progression. As IGFBP-2 modulates bone metabolism and cell migration, IGFBP-2 in the gingival crevicular fluid may reflect periodontal disease activity.

TGF-ß signaling in gingival fibroblast-epithelial interaction.

The underlying mechanism and the therapeutic regimen for the transition of reversible gingivitis to irreversible periodontitis are unclear. Since transforming growth factor (TGF)-ß has been implicated in differentially regulated gene expression in gingival fibroblasts, we hypothesized that TGF-ß signaling is activated in periodontitis-affected gingiva, along with enhanced collagen degradation, that is reversed by TGF-ß inhibition. A novel three-dimensional (3D) gel-culture system consisting of primary human gingival fibroblasts (GF) and gingival epithelial (GE) cells in collagen gels was applied. GF populations from patients with severe periodontitis degraded collagen gels, which was reduced by TGF-ß-receptor kinase inhibition. Up-regulation of TGF-ß-responsive genes was evident in GF/GE co-cultures. Furthermore, the TGF-ß downstream transducer Smad3C was highly phosphorylated in periodontitis-affected gingiva and 3D cultures. These results imply that TGF-ß signaling is involved in fibroblast-epithelial cell interaction in periodontitis, and suggest that the 3D culture system is a useful in vitro model for therapeutic drug screening for periodontitis.

Reproducibility of 24-hour dietary recall for vitamin intakes by middle-aged Japanese men and women.

OBJECTIVE: To examine the reproducibility of 24-hour dietary recall for estimating dietary vitamin intakes by middle-aged Japanese men and women. METHODS AND RESULTS: The subjects were 132 men and 130 women aged 40-69 years, selected from participants in cardiovascular risk surveys conducted in 4 communities. The reproducibility of the 24-hour dietary recall was tested by comparing nutrient and food intake for two recalls conducted on the same season 1 year apart, designated as recalls 1 and 2. Differences in mean values between two recalls were tested using analysis of variance, and Spearman rank correlation coefficients for the two recalls were calculated for nutrient and food intakes. Mean values of energy and vitamins for both sexes were generally similar for the two recalls. The reproducibility of recall by men was high for vitamin B2, folate, pantothenic acid, and vitamin C and by women for vitamin B2, moderate by men for vitamins A, E, K, B1, B6 and niacin, and by women for vitamins A, E, K, B1, B6 and niacin, folate, pantothenic acid and vitamin C, and low by both men and women for vitamins D and B12. The reproducibility during 1985-1999 was generally lower than that of 1973-1984, but that for folate, pantothenic acid and vitamin C remained to be moderate in 1984-1999. CONCLUSIONS: Although the reproducibility of 24-hour dietary recall varies among vitamins, moderate and sustained reproducibility was observed for folate, vitamin C and pantothenic acid.

Overexpression of suppressor of cytokine signalling-5 augments eosinophilic airway inflammation in mice.

BACKGROUND: Enhanced expression of the suppressor of cytokine signalling (SOCS)-5 might be of therapeutic benefit for T-helper type 2 (Th2) dominant diseases, as its expression is reported to result in a reduction of Th2 differentiation in vitro due to the inhibition of IL-4 signalling. OBJECTIVE: To investigate the regulatory role of SOCS-5 in vivo, we explored the phenotype of an experimental asthma model developed in SOCS-5 transgenic (Tg) mice. METHODS: The SOCS-5 Tg mice or wild-type (WT) mice were sensitized and repeatedly challenged with ovalbumin (OVA). We examined bronchoalveolar lavage fluid (BALF), lung specimens, and airway hyperresponsiveness (AHR) to methacholine. RESULTS: The production of IFN-gamma by CD4(+) T cells from unprimed SOCS-5 Tg mice was significantly increased in comparison with unprimed wild-type mice, indicating that SOCS-5 Tg mice have a Th1-polarizing condition under natural conditions. However, in an asthma model, significantly more eosinophils in the airways and higher levels of IL-5 and IL-13 in BALF were observed in the SOCS-5 Tg than the wild-type mice. AHR in the asthma model of SOCS-5 Tg was also more enhanced than that of wild-type mice. OVA-stimulated CD4(+) T cells from the primed SOCS-5 Tg mice produced significantly more IL-5 and IL-13 than CD4(+) T cells from wild-type mice. CONCLUSION: Our results demonstrate that the overexpression of SOCS-5 does not inhibit Th2 response, but rather augments the phenotype of the asthma model in vivo. This finding throws into question the therapeutic utility of using enhancement of SOCS-5 expression for Th2-dominant disease.

Cytokine profile in synovial fluid from patients with internal derangement of the temporomandibular joint: a preliminary study.

OBJECTIVES: Temporomandibular joint disorders (TMD) comprise a group of chronic painful conditions of mastication in the temporomandibular joint (TMJ). Although the association between TMD and internal derangement of the TMJ is well documented, the functional relevance is still unclear. Increased concentrations of inflammatory mediators have been identified in the synovial fluid of affected patients with TMD, suggesting an underlying degenerative or inflammatory process. The aim of this study was to generate a comprehensive cytokine expression profile in TMD. METHODS: 15 samples from patients with internal derangement of TMJ were analysed using a novel cytokine array that enables the analysis of 79 different cytokines simultaneously. RESULTS: Cytokine levels were correlated with the presence of joint effusion (JE) determined by MRI. In the majority of synovial fluid samples, angiogenin (Ang), fibroblast growth factor (FGF)-9, insulin-like growth factor-binding protein (IGFBP)-3, interleukin (IL)-1alpha, IL-1beta, IL-8, inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1beta, osteoprotegerin (OPG), transforming growth factor (TGF)-beta2, tissue inhibitor of metalloproteinase (TIMP)-1, TIMP-2, tumour necrosis factor (TNF)-beta and vascular endothelial growth factor (VEGF) were detectable. Furthermore, the expression levels of Ang, brain-derived neurotrophic factor (BDNF), FGF-4, FGF-9, IGFBP-2, IL-8, MIP-1beta, OPG, pulmonary and activation-regulated protein (PARC), TGF-beta2, TIMP-2 and VEGF were significantly associated with the presence of JE; among these, nine cytokines (Ang, BDNF, FGF-4, FGF-9, IGFBP-2, MIP-1beta, PARC, TGF-beta2 and TIMP-2) were hitherto not described in TMD. CONCLUSIONS: This study confirmed previous reports of elevated cytokine levels in TMD. Additionally, we identified previously undescribed cytokines that were upregulated and correlated significantly with the presence of JE. We were able to identify novel cytokines that have hitherto not been described in TMD. Strategies targeting the identified cytokines may represent a novel therapy option in TMD.

Mitogen-activated protein kinase/extracellular signal-regulated protein kinase activation of cultured human dental pulp cells by low-power gallium-aluminium-arsenic laser irradiation.

AIM: To examine whether low-power laser irradiation (LPLI) promotes cellular proliferation of human dental pulp-derived fibroblast-like cells (dental pulp cells). METHODOLOGY: Dental pulp cells were obtained by primary culture of human dental pulp tissues from extracted third molar teeth. The phosphorylation of the mitogen-activated protein kinase (MAPK) family after LPLI of these cells was investigated by Western blotting. By using a specific MAPK/ERK kinase (MEK) inhibitor (PD098059), the specific effect of LPLI on the MAPK pathway was also investigated by Western blotting as described above. The incorporation of [3H]thymidine into the cells after LPLI was determined, and statistical analysis was performed by Wilcoxon signed-ranks test. RESULTS: Extracellular signal-regulated protein kinase (ERK) 1/2 was phosphorylated between 5 and 30 min after LPLI. Moreover, PD098059 inhibited LPLI-mediated ERK1/2 activation. LPLI did not affect p38 MAPK or c-Jun N-terminal kinase (JNK) phosphorylation. But LPLI did not stimulate [3H]thymidine incorporation into these cells. CONCLUSIONS: These results indicated that LPLI activated MAPK/ERK, a signal for proliferation, differentiation and survival, but did not activate the stress signals p38 MAPK and JNK in human dental pulp cells.

Early esophageal carcinoma: endoscopic ultrasonography using the Sonoprobe.

BACKGROUND: Almost all cases of superficial esophageal carcinoma are curable by endoscopic mucosal resection (EMR), but a precise diagnosis of the depth of tumor invasion is necessary to assess the indication for EMR. Although endoscopy has a high rate of accuracy for diagnosing the depth of tumor invasion, it depends on the experience of the examiner in interpreting surface information of the lesions. Today, endoscopic ultrasonography (EUS) is one of the most powerful techniques for obtaining objective tomographic images of a tumor. The high-frequency ultrasound probe is appropriate for EUS in cases of superficial esophageal carcinoma because of its excellent near-field resolution that provides precise ultrasound images under direct control of the endoscope. METHODS: We performed EUS with the Sonoprobe System in 85 cases of superficial esophageal carcinoma before treatment and evaluated the resected specimens histopathologically. We interpreted the depth of tumor invasion based on our fundamental studies of ultrasonograms taken with a 20-MHz probe. RESULTS: The clinical usefulness of the Sonoprobe with linear and radial scanning modes is due to its capacity to differentiate between mucosal and submucosal carcinoma by means of analyses of the muscularis mucosae. Although a clear assessment of microinvasion and lymphoid hyperplasia surrounding the tumor of interest remains speculative, the diagnostic accuracy rate for 96 lesions of superficial esophageal carcinoma reached 93% in terms of differentiating between mucosal from submucosal carcinoma. CONCLUSION: EUS with the Sonoprobe can play an important role in the pretreatment diagnosis of superficial esophageal carcinomas.

Activity-dependent change in the protein level of brain-derived neurotrophic factor but no change in other neurotrophins in the visual cortex of young and adult ferrets.

Neurotrophins are suggested to play a role in activity-dependent plasticity of visual cortex during the critical period of postnatal development. Thus, the concentration of neurotrophins in the cortex is expected to change with development and/or with alteration in neuronal activities. To test this, we measured protein levels of nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and neurotrophin-4/5 in visual cortex of young (postnatal day 38-46, at the peak of the critical period) and adult ferrets with two-site enzyme-immunoassay systems. Measurements were carried out also in somatosensory cortex, hippocampus and cerebellum as control. With development the level of brain-derived neurotrophic factor did not significantly change, while those of the other neurotrophins changed in the visual cortex. A blockade of visual inputs for 24 h by an injection of tetrodotoxin into both eyes significantly decreased brain-derived neurotrophic factor protein level in the visual cortex, but not in the other regions in both young and adult ferrets. On the other hand, no significant decrease was seen in the protein level of the other neurotrophins in the visual cortex of young and adult ferrets. A monocular injection of tetrodotoxin in young ferrets resulted in the reduction of brain-derived neurotrophic factor by approximately half that by binocular injection. The degree of the decrease in the contralateral cortex to the injected eye was significantly larger than that in the ipsilateral cortex, reflecting that the contralateral eye is dominantly represented in the cortex in ferrets. Blockade of cortical neuronal activities by a GABA(A) receptor agonist led to a remarkable reduction of brain-derived neurotrophic factor protein in the visual cortex. These results suggest that the level of brain-derived neurotrophic factor protein in visual cortex is regulated by activities of cortical neurons.

Inducible cross-tolerance to herbicides in transgenic potato plants with the rat CYP1A1 gene.

A gene of the enzyme involved in xenobiotic metabolism in mammalian liver was introduced into potato to confer inducible herbicide tolerance. A rat cytochrome P450 monooxygenase, CYP1A1 cDNA, was kept under the control of the tobacco PR1a promoter in order to apply the system of chemical inducible expression using the plant activator Benzothiadiazole (BTH). Transgenic plants were obtained based on the kanamycin resistance test and PCR analysis. Northern-blot analysis revealed the accumulation of mRNA corresponding to rat CYP1A1 in the transgenic plants treated with BTH (3.0 micro mol/pot), whereas no accumulation of the corresponding mRNA occurred without BTH treatment. These transgenic plants also produced a protein corresponding to CYP1A1 in the leaves by BTH treatment. The transgenic plants with BTH application showed a much-higher tolerance to the phenylurea herbicides chlortoluron and methabenzthiazuron than non-transgenic plants. These findings indicated that the ability of metabolizing the two herbicides to less-toxic derivatives was displayed in the transgenic plants after BTH treatment. Transgenic plants harboring the CYP1A1 cDNA fused with the yeast P450 reductase (YR) gene under the control of PR1a were also produced. Although the plants showed a lower expression level of the fused gene than transgenic plants with CYP1A1 cDNA alone, they were tolerant to herbicides. These facts suggested that the CYP1A1 enzyme fused with YR showed a higher specific activity than CYP1A1 alone. This study demonstrated that the mammalian cDNA for the de-toxification enzyme of herbicides under the control of the PR1a promoter conferred chemical-inducible herbicide tolerance on potato.

Brain-derived neurotrophic factor induces long-lasting potentiation of synaptic transmission in visual cortex in vivo in young rats, but not in the adult.

Brain-derived neurotrophic factor (BDNF) rapidly enhances excitatory synaptic transmission in cortical slices. To date, however, a question of how long such an action persists remains unanswered as it is hard to record synaptic responses longer than several hours in slice preparations. To address this question and to investigate possible age-dependency of the action, we analysed effects of a brief application of BDNF and nerve growth factor (NGF) on field potentials of visual cortex in rats of postnatal days 13-17 and 19-24 and in the adulthood for 10-24 h. Evoked potentials to stimulation of the lateral geniculate nucleus were recorded simultaneously from two cortical sites into which the neurotrophin and control solution were injected. An application of BDNF induced a slowly developing increase in the field potential amplitude in young rats. The amplitude attained a plateau level 3-4 h after the infusion; 139 +/- 26% (mean +/- SD) and 132 +/- 21% of the baseline in the rats at P13-17 and P19-24, respectively. This potentiation remained stable from 4 to 8 h, then gradually decreased to the baseline 15-16 h after the infusion. NGF applied in the same way did not induce potentiation. An inhibitor of BDNF receptors blocked the potentiation when it was applied immediately after the BDNF application, but was not effective about 2 h later. In the adults, BDNF did not potentiate field potentials. These results indicate that BDNF induces synaptic potentiation lasting for several hours only in the developing cortex through processes downstream of receptor activation.

High- and moderately high-methionine uptake demonstrated by PET in a patient with a subacute cerebral infarction.

In patients with cerebral tumors, high accumulations of L-methyl-11C-methionine (11C-Met) have been reported in some cases of cerebral ischemic disease, but no high accumulations of 11C-Met in areas where only transient arterial occlusions are most likely to occur have been reported. Herein we present a case of a high accumulation of 11C-Met in an area of frontal interhemispheric cerebral infarction and a moderately high accumulation with an unclear margin in a distant frontal convexity area. A craniotomy revealed a subacute stage of cerebral infarction in the interhemispheric lesion, and an ischemic change in the distant convexity area. Sixteen months after onset, CT scans demonstrated an infarction area in the interhemispheric lesion only, and no atrophic changes were observed in the distant convexity area indicating that no serious tissue damage had occurred.

Melatonin therapy for REM sleep behavior disorder.

Rapid eye movement sleep behavior disorder (RBD) is a parasomnia with clinical symptoms that include punching, kicking, yelling and leaping out of bed in sleep. Polysomnographic (PSG) finding showed REM sleep without muscle atonia. Clonazepam is generally used for treating RBD symptoms but melatonin was reported to be effective so we reconfirmed the effect of melatonin on RBD patients in the present study. We used melatonin (3-9 mg/day) which could ameliorate problem sleep behaviors remarkably, as well as %tonic activity in PSG variables. In the present study, melatonin was reconfirmed to be effective in RBD symptoms, especially for patients with low melatonin secretion, while its mechanism was not clearly known in the present study.

Hepatocyte growth factor secreted by periodontal ligament and gingival fibroblasts is a major chemoattractant for gingival epithelial cells.

To ascertain whether periodontal fibroblasts could be involved in the pathogenesis of periodontal pocket formation, the chemotactic activity of periodontal ligament fibroblast-conditioned medium (PLF-CM) and gingival fibroblast-conditioned medium (GF-CM) for gingival epithelial cells was examined using a modified Boyden chamber assay. Both PLF-CM and GF-CM possessed significant chemotactic activity, which was decreased markedly by treatment with anti-human hepatocyte growth factor (HGF) neutralizing antibody. Furthermore, the chemotactic activity of PLF-CM and GF-CM was well correlated with HGF content. These results show that PLF and GF secrete an HGF-like factor, and suggest that such a factor derived from periodontal fibroblasts might play a role in epithelial apical migration in periodontitis.

Characteristics of an ammonia-oxidizing bacterium with a plasmid isolated from alkaline soils and its phylogenetic relationship.

An ammonia-oxidizing bacterium, strain TCH716, was isolated from alkaline soil at Harbin city, China. The cells of strain TCH716 are lobate (0.8-1.5 x 1.0-2.0 microm), gram-negative, obligately aerobic, and nonmotile. Colonies (1-2 mm in diameter) on gellan gum plate culture are reddish, circular, and smooth. The G + C content of DNA is 54.78 mol%. Its percentage of 16S rRNA gene sequence similarity (%) to Nitrosolobus multiformis ATCC 25196T (type strain) is 98.56%. This bacterium has an optimal growth temperature and pH at 30 degrees C and 8.0-8.5, respectively. The concentration of ammonium sulfate in the HEPES medium for optimum growth of this bacterium is 38 mM. Strain TCH716 was found to have a plasmid (approximately 6.5 kbp) that possessed a plasmid-linked gene for sulfonamide resistance. Phosphoglycerate kinase, RubisCO and PEPC were found to possess high specific activities compared to the activities of these enzymes in strain ATCC 25978T. In identification of strain TCH716, both morphological characteristics (compartmentalized cells) and the phylogenetic relationship based on 16S rRNA gene sequence are important. Based on results obtained, strain TCH716 belongs to the genus Nitrosolobus, and designated as Nitrosolobus sp. TCH716.

Hepatocyte growth factor is a predominant chemoattractant for gingival epithelial cells produced by radicular cyst-derived fibroblast-like cells.

To clarify whether fibroblasts could be involved in the pathogenesis of periodontal pocket formation, the chemotactic activity of radicular cyst-derived fibroblast-like cell (RCF)-conditioned medium (RCF-CM) for gingival epithelial cells was examined using a modified Boyden chamber assay. RCF-CM possessed significant chemotactic activity, which was decreased markedly by treatment with anti-human hepatocyte growth factor (HGF) antibody. Furthermore, the chemotactic activity of RCF-CM was well correlated with HGF content. These results show that the RCF secrete an HGF-like factor, and suggest that such a factor derived from periodontal fibroblasts might play a role in epithelial apical migration in periodontitis.