[Show abstract][Hide abstract] ABSTRACT: Macrophages are involved in low-grade inflammation in diabetes, and play pathogenic roles in proliferative diabetic retinopathy (PDR) by producing proinflammatory cytokines. T cells as well as other cells are also activated by proinflammatory cytokines, and infiltration into the vitreous of patients with PDR has been shown. In this study, we measured helper T (Th) cell-related cytokines in the vitreous of PDR patients to define the characteristics of Th-mediated immune responses associated with PDR. The study group consisted of 25 type 2 diabetic patients (25 eyes) with PDR. The control group consisted of 27 patients with epiretinal membrane (ERM), 26 patients with idiopathic macular hole (MH), and 26 patients with uveitis associated with sarcoidosis. Vitreous fluid was obtained at the beginning of vitrectomy, and centrifuging for cellular removals was not performed. Serum was also collected from PDR patients. IL-1β, IL-4, IL-6, IL-10, IL-17A, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, IFN-γ, soluble sCD40L, and TNFα in the vitreous and serum samples were measured. Both percent detectable and levels of IL-4, IL-6, IL-17A, IL-21, IL-22, and TNFα in the vitreous were significantly higher than those in the serum in PDR patients. Vitreous levels of these cytokines and IL-31 were significantly higher in PDR than in ERM or MH patients. Vitreous levels of IL-4, IL-17A, IL-22, IL-31, and TNFα in PDR patients were also significantly higher than those of sarcoidosis patients. In PDR patients, vitreous IL-17A level correlated significantly with vitreous levels of IL-22 and IL-31, and especially with IL-4 and TNFα. Although it is unclear whether these cytokines play facilitative roles or inhibitory roles for the progression of PDR, the present study indicated that Th2- and Th17-related immune responses are involved in the pathogenesis of PDR.
PLoS ONE 09/2015; 10(9):e0137358. DOI:10.1371/journal.pone.0137358 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recently mesenchymal stem cells have demonstrated a potential for neurotrophy and neurodifferentiation. We have recently isolated mobilized dental pulp stem cells (MDPSCs) using granulocyte-colony stimulating factor (G-CSF) gradient which have high neurotrophic/angiogenic potential. The aim of this study is to investigate the effects of MDPSCs transplantation on peripheral nerve regeneration. Effects of MDPSCs transplantation were examined in a rat sciatic nerve defect model and compared with autografts and control conduits containing collagen scaffold. Effects of conditioned medium of MDPSCs were also evaluated in vitro. Transplantation of MDPSCs in the defect demonstrated regeneration of myelinated fibers, whose axons were significantly higher in density compared with those in autografts and control conduits only. Enhanced revascularization was also observed in the MDPSCs transplants. The MDPSCs did not directly differentiate into Schwann cell phenotype; localization of these cells near Schwann cells induced several neurotrophic factors. Immunofluorescence labelling demonstrated reduced apoptosis, and increased proliferation in resident Schwann cells in the MDPSCs transplant compared with control conduits. These trophic effects of MDPSCs on proliferation, migration and anti-apoptosis in Schwann cells were further elucidated in vitro. The results demonstrate that MDPSCs promote axon regeneration through trophic functions, acting on Schwann cells, and promoting angiogenesis.
[Show abstract][Hide abstract] ABSTRACT: Ascorbic acid is an effective antioxidant and free radical scavenger. Therefore, it is expected that ascorbic acid should act as a radioprotectant. We investigated the effects of post-radiation treatment with ascorbic acid on mouse survival. Mice received whole body irradiation (WBI) followed by intraperitoneal administration of ascorbic acid. Administration of 3 g/kg of ascorbic acid immediately after exposure significantly increased mouse survival after WBI at 7 to 8 Gy. However, administration of less than 3 g/kg of ascorbic acid was ineffective, and 4 or more g/kg was harmful to the mice. Post-exposure treatment with 3 g/kg of ascorbic acid reduced radiation-induced apoptosis in bone marrow cells and restored hematopoietic function. Treatment with ascorbic acid (3 g/kg) up to 24 h (1, 6, 12, or 24 h) after WBI at 7.5 Gy effectively improved mouse survival; however, treatments beyond 36 h were ineffective. Two treatments with ascorbic acid (1.5 g/kg × 2, immediately and 24 h after radiation, 3 g/kg in total) also improved mouse survival after WBI at 7.5 Gy, accompanied with suppression of radiation-induced free radical metabolites. In conclusion, administration of high-dose ascorbic acid might reduce radiation lethality in mice even after exposure.
PLoS ONE 02/2015; 10(2):e0117020. DOI:10.1371/journal.pone.0117020 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the efficacy of spectral domain optical coherence tomography (SD-OCT) in monitoring the development of mouse experimental autoimmune uveoretinitis (EAU) as an animal model of endogenous uveitis, and to develop an OCT-based grading system for EAU severity.
C57BL/6 mice were immunised with human interphotoreceptor retinoid-binding protein (amino acid sequence 1-20) peptide and complete Freund's adjuvant to induce EAU. The development of EAU was monitored by SD-OCT serially throughout the disease course, and the images were graded from 1 to 4 and compared with the clinical and histopathological grades.
SD-OCT images depicted retinal lamella structures including the inner segment/outer segment (IS/OS) line in normal mice. Retinal structural changes were observed on SD-OCT images in mice that developed EAU clinically scored as grade 1 or higher, which precisely corresponded to the pathological findings. The SD-OCT images of EAU were graded as follows: grade 1, a few infiltrating cells in the vitreous and retina; grade 2, increased vitreous cells, retinal vasculitis, and granulomatous lesion; grade 3, cell infiltration into the whole retina, disappearance of IS/OS line, and destruction of the retinal layer structure; and grade 4, disappearance of the outer retina. The SD-OCT grade of EAU based on these criteria correlated significantly with both the clinical grade (R(2)=0.282, p<0.005) and histopathological grade (R(2)=0.846, p<0.0001).
SD-OCT is useful for evaluating the development and severity of mouse EAU. The SD-OCT scoring system we developed accurately reflects clinical and histopathological changes.
The British journal of ophthalmology 02/2014; 98(6). DOI:10.1136/bjophthalmol-2013-304421 · 2.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Non-alcoholic steatohepatitis (NASH) is characterized by the presence of steatosis, inflammation, and fibrosis and is believed to develop via a "two-hit process"; however, its pathophysiology remains unclear. Fibroblast growth factors (FGFs) are heparin-binding polypeptides with diverse biological activities in many developmental and metabolic processes. In particular, FGF5 is associated with high blood pressure. We investigated the function of FGF5 in vivo using spontaneously Fgf5 null mice and explored the role of diet in the development of NASH. Mice fed a high-fat diet gained little weight and had higher serum alanine transaminase, aspartate amino transferase, and non-high-density lipoprotein-cholesterol levels. Liver histology indicated marked inflammation, focal necrosis, fat deposition, and fibrosis, similar to the characteristics of NASH. FGF5 and a high-fat diet play significant roles in the pathophysiology of hepatic fibrosis and Fgf5 null mice may provide a suitable model for liver fibrosis or NASH.
[Show abstract][Hide abstract] ABSTRACT: Treatment of deep caries with pulpitis is a major challenge in dentistry. Stem cell therapy represents a potential strategy to regenerate the dentin-pulp complex, enabling conservation and restoration of teeth. The objective of this study was to assess the efficacy and safety of pulp stem cell transplantation as a prelude for the impending clinical trials. Clinical-grade pulp stem cells were isolated and expanded according to good manufacturing practice conditions. The absence of contamination, abnormalities/aberrations in karyotype, and tumor formation after transplantation in an immunodeficient mouse ensured excellent quality control. After autologous transplantation of pulp stem cells with granulocyte-colony stimulating factor (G-CSF) in a dog pulpectomized tooth, regenerated pulp tissue including vasculature and innervation completely filled in the root canal, and regenerated dentin was formed in the coronal part and prevented microleakage up to day 180. Transplantation of pulp stem cells with G-CSF yielded a significantly larger amount of regenerated dentin-pulp complex compared with transplantation of G-CSF or stem cells alone. Also noteworthy was the reduction in the number of inflammatory cells and apoptotic cells and the significant increase in neurite outgrowth compared with results without G-CSF. The transplanted stem cells expressed angiogenic/neurotrophic factors. It is significant that G-CSF together with conditioned medium of pulp stem cells stimulated cell migration and neurite outgrowth, prevented cell death, and promoted immunosuppression in vitro. Furthermore, there was no evidence of toxicity or adverse events. In conclusion, the combinatorial trophic effects of pulp stem cells and G-CSF are of immediate utility for pulp/dentin regeneration, demonstrating the prerequisites of safety and efficacy critical for clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Sebaceous adenomas are found mainly in elderly individuals and are usually tan, pink, or yellow nodules or papules, usually approximately 5 mm in the largest size.
A 65-year-old man presented with a progressively enlarging exophytic lesion in the right eyelid for 3 months. External examination revealed a yellowish-pink growth measuring 18 × 13 × 14 mm. The lesion surface was covered by palpebral conjunctiva with fine papillary projections, vascularity, crusting, and ulceration. Two weeks later, the growth enlarged to 20 × 14 × 14 mm, and ulceration also expanded. An excisional biopsy with clear resection margins was performed. No malignancy was found in the stump. Histopathologically, the lesion was located principally within the cutaneous compartment and composed of multiple circumscribed sebaceous lobules, separated, and exhibiting no cytologic atypia. Cystic change was not evident, and no infiltrative growth pattern, pagetoid lesions, mitotic figures, and lymphovascular space invasion were observed. The Ki-67 nuclear antigen was detected in 10%-15% of cells located in the basal zone of the nodule. Fluorescence in situ hybridization showed low human epidermal growth factor receptor 2 amplification, suggesting no genetic changes. The clinical findings, lack of infiltrative border, low Ki-67 index, and low proliferative ability support a diagnosis of sebaceous adenoma.
Sebaceous adenoma that shows excessively rapid growth due to hyperplasia may appear to be malignant. Histopathology, fluorescence in situ hybridization, and Ki-67 were useful to the diagnosis of the adenoma. Excisional biopsy with clear resection margins must be performed in rapidly growing tumors.
[Show abstract][Hide abstract] ABSTRACT: Purpose:
To determine whether retinal pigment epithelial (RPE) cells can inhibit mature dendritic cells (mDCs).
Cultured RPE cells were established from C57BL/6 mice. DCs were established from bone marrow cells of normal mice, and mDCc were induced by culture in medium containing granulocyte macrophage-colony-stimulating factor (GM-CSF) and IL-4 in the presence of lipopolysaccharide and TNF-α. Activation of mDCs was assessed by a proliferation assay and ELISA to measure the production of pro-inflammatory cytokines (TNF-α, IL-1β, and IL-12p40). Expression of major histocompatibility complex (MHC) class II, CD11c, and costimulatory molecules such as CD80, CD86, programmed cell death 1 ligand 1 (PD-L1), and PD-L2 on mDCs or RPE-exposed mDCs was evaluated by immune staining and flow cytometry. Production of IL-1 receptor antagonist (IL-1Ra) by RPE cells was evaluated by oligonucleotide microarray or ELISA. Anti-IL-1Ra neutralizing antibodies or RPE cells from IL-1Ra knockout donors were used for the assay.
Cultured RPE cells greatly suppressed the activation of mDCs, especially the production of pro-inflammatory cytokines, and the expression of cell-surface molecules. Moreover, RPE cells significantly suppressed mixed lymphocyte reactions by mDCs. In an examination of immunoregulatory candidate molecules, RPE cells expressed much higher levels of IL-1Ra as compared with control cells, and RPE cells pretreated with recombinant TNF-α and/or IL-1β produced high levels of IL-1Ra. RPE cells in the presence of anti-IL-1Ra antibodies, but not other candidate factors, failed to suppress activation by mDCs. In addition, RPE cells from IL-1Ra null donors failed to suppress mDC activation.
Our results suggest that ocular resident cells can produce pro-inflammatory cytokine antagonist that suppresses antigen-presenting cell activation.
[Show abstract][Hide abstract] ABSTRACT: We compared the phototoxicity of indocyanine green (ICG) and Brilliant Blue G (BBG) in cultured RPE cells under fluorescent lamp illumination imitating ambient light.
Cultured human RPE line cells were stained with ICG or BBG solution at concentrations of clinical use, and cultured in a colorless medium for 24 hours in the dark or under illumination from a fluorescent lamp. After culture, cell morphology and TUNEL-positive apoptotic cells were observed. Cell viability and cell death rate were evaluated. Absorption spectral changes of BBG before and after incubation were measured.
ICG-stained cells cultured under illumination changed to an oval morphology with increased number of apoptotic cells, whereas ICG-stained cells cultured in the dark, and BBG-stained cells cultured under illumination and dark conditions maintained a flat morphology without increase in apoptotic cells. Cell viability decreased and cell death rate increased only in cells stained by ICG followed by culture under illumination. Staining cells with ICG at one-tenth concentration of clinical usage induced no cytotoxicity after culture under illumination. Approximately 30% of total BBG retained in the stained cells was released into the culture supernatant after incubation for 24 hours. The absorption spectrum of BBG did not change after fluorescent light irradiation.
Illumination with a fluorescent lamp caused cell death via apoptosis in ICG-exposed, but not in BBG-exposed cultured RPE cells. BBG may be a safer dye than ICG because of low light-induced cytotoxicity and rapid elution from stained cells.
[Show abstract][Hide abstract] ABSTRACT: Loss of pulp due to caries and pulpitis leads to loss of teeth and reduced quality of life. Thus, there is an unmet need for regeneration of pulp. A promising approach is stem cell therapy. Autologous pulp stem/progenitor (CD105(+)) cells were transplanted into a root canal with stromal cell-derived factor-1 (SDF-1) after pulpectomy in mature teeth with complete apical closure in dogs. The root canal was successfully filled with regenerated pulp including nerves and vasculature by day 14, followed by new dentin formation along the dentinal wall. The newly regenerated tissue was significantly larger in the transplantation of pulp CD105(+) cells with SDF-1 compared with those of adipose CD105(+) cells with SDF-1 or unfractionated total pulp cells with SDF-1. The pulp CD105(+) cells highly expressed angiogenic/neurotrophic factors compared with other cells and localized in the vicinity of newly formed capillaries after transplantation, demonstrating its potent trophic effects on neovascularization. Two-dimensional electrophoretic analyses and real-time reverse transcription-polymerase chain reaction analyses demonstrated that the qualitative and quantitative protein and mRNA expression patterns of the regenerated pulp were similar to those of normal pulp. Thus, this novel stem cell therapy is the first demonstration of complete pulp regeneration, implying novel treatment to preserve and save teeth.
Tissue Engineering Part A 03/2011; 17(15-16):1911-20. DOI:10.1089/ten.TEA.2010.0615 · 4.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To investigate the phototoxicity of persistent indocyanine green (ICG) under continuous visible light illumination and to determine whether blocking peak absorbance wavelengths of ICG is cytoprotective.
Cultured quail Müller cells were exposed to 0 to 5 mg/mL ICG for 30 seconds or 10 minutes and then were cultured in a colorless medium for 24 hours with or without continuous fluorescent lamp illumination. Cells exposed to 5 mg/mL ICG for 10 minutes were cultured under illumination filtered through a dichroic mirror that blocks red to near-infrared, green, or blue wavelengths. After microscopic observation, cell viability and cell death were evaluated.
ICG exposure followed by illuminated culture induced severe morphologic changes in cells, significant reductions in cell viability, and increases in cell death from apoptosis compared with exposure to ICG or illumination alone or with no exposure. Although ICG exposure at higher concentrations caused cell damage in a dose- and time-dependent manner, an increase in cell viability was noted for cells exposed to lower ICG concentrations. Blocking red to near-infrared wavelengths prevented the decrease in cell viability and the increase in cell death in the culture exposed to ICG followed by illuminated culture.
Continuous fluorescent lamp illumination enhanced the cytotoxicity of persistent ICG on Müller cells in a dose- and exposure time-dependent manner. Blocking peak absorbance wavelengths of ICG prevented photodynamic cytotoxicity of persistent ICG under continuous visible light illumination in vitro. This culture system could be used to study the mechanisms of prevention of unfavorable outcomes in ICG-assisted surgery.
[Show abstract][Hide abstract] ABSTRACT: Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. Angiogenesis is essential for reparative dentin formation during pulp wound healing. The mechanism of angiogenesis, however, still remains unclear. We hypothesized that certain MMPs expressed during pulp wound healing may support recovery processes. To address this issue, a rat pulp injury model was established to investigate expression of MMPs during wound healing. Real-time RT-PCR analysis showed that expression MMP-3 and MMP-9 (albeit lower extent) was up-regulated at 24 and 12 hours after pulp injury, respectively, whereas expression of MMP-2 and MMP-14 was not changed. MMP-3 mRNA and protein were localized in endothelial cells and/or endothelial progenitor cells in injured pulp in vivo. In addition, MMP-3 enhanced proliferation, migration, and survival of human umbilical vein endothelial cells in vitro. Furthermore, the topical application of MMP-3 protein on the rat-injured pulp tissue in vivo induced angiogenesis and reparative dentin formation at significantly higher levels compared with controls at 24 and 72 hours after treatment, respectively. Inhibition of endogenous MMP-3 by N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid resulted in untoward wound healing. These results provide suggestive evidence that MMP-3 released from endothelial cells and/or endothelial progenitor cells in injured pulp plays critical roles in angiogenesis and pulp wound healing.
American Journal Of Pathology 11/2009; 175(5):1905-14. DOI:10.2353/ajpath.2009.080705 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The juxta-oral organ is a bilateral organ in the mammalian bucca. It consists of epithelial cords with surrounding mesenchyme. It develops from embryonic oral epithelium, but its macroscopic morphology in mice is less studied and seems to be very different from that of humans. The juxta-oral organ in mice extends more widely from the subcutaneous tissue of the mandible near the lateral fascia of the masseter to the submucosa of the soft palate. In this paper, we report that the mutant mouse allele Bmp7(lacZ) presented intense lacZ expression in the epithelial component of the juxta-oral organ in its homo- and heterozygous states. The main aims of this study were to show that this mutant mouse allele is suitable for observing macroscopic structure of the juxta-oral organ and to describe the development of this organ during embryonic and postnatal stages. Whole-mount beta-gal staining of this strain of mouse showed that the juxta-oral organ in mice appeared at E12.0 from oral epithelium and lost connection with it before E12.5. Then, the juxta-oral organ extended anteriorly to the lateral fascia of the masseter and posteriorly to the submucosal layer of the soft palate via the orbit. The mature juxta-oral organ had no connection to other epithelia such as those of the bucca and parotid duct. It persisted until adulthood and there seemed to be no tendency to regress. Transmission electron microscopy showed that each part of the juxta-oral organ was an epithelial cord surrounded by a basement membrane and mesenchymal tissue.
Journal of Anatomy 07/2009; 215(4):452-61. DOI:10.1111/j.1469-7580.2009.01118.x · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To clarify which factors are involved in postnatal lacrimal gland development, the expressions of several growth factors and their receptors were analyzed in purified lacrimal gland epithelial and mesenchymal cells. Reconstruction of acinarlike structures in three-dimensional culture conditions from this epithelial cell population was attempted.
Lacrimal gland epithelial and mesenchymal cells were isolated from newborn mice and purified using nylon mesh and collagenase. By real-time reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blotting, the expressions of several growth factors and their receptors were analyzed. Responses of epithelial cells to the growth factors were analyzed by the addition of these factors to the culture medium.
Fibroblast growth factor (FGF)10 and hepatocyte growth factor (HGF) were more intensely expressed in mesenchymal cells than in epithelial cells, and their receptors (FGFR2IIIb and c-Met, respectively) were less intensely expressed, whereas the expression of epidermal growth factor (EGF) and its receptor showed no significant difference between cell types. In the monolayer culture, cell viability was activated by the addition of EGF or HGF to epithelial cells, but no response was observed when FGF10 was added. The epithelial cells formed clusters with lumina when cultured on basement membrane matrix. These clusters contained secretion granules and showed positive immunostaining for aquaporin-5.
EGF and HGF were considered to act in an autocrine/paracrine manner in and around the postnatal lacrimal gland, whereas epithelial cells did not respond to FGF10. It was suggested that certain extracellular matrix conditions accommodate these epithelial cells to reconstruct functional acinarlike structures.
[Show abstract][Hide abstract] ABSTRACT: To achieve complete regeneration of dental pulp in vivo by stem/progenitor cells obtained from a fraction of side population (SP) cells from canine pulp.
A subfraction of SP cells, CD31(-)/CD146(-) SP cells, were isolated by flow cytometry from canine dental pulp. The efficiency of this subfraction of SP cells was evaluated in an experimental model of pulp injury in the dog.
The fractionated SP cells formed extensive networks of tube-like structures in vitro. Transplantation of the SP cells into an in vivo model of amputated pulp resulted in complete regeneration of pulp tissue with capillaries and neuronal cells within 14 days. Gene-expression studies demonstrated the expression of pro-angiogenic factors, implying trophic action on endothelial cells.
This investigation demonstrates the potential utility of fractionated SP cells as a source of cells for total pulp regeneration complete with angiogenesis and vasculogenesis.
Regenerative Medicine 05/2009; 4(3):377-85. DOI:10.2217/rme.09.5 · 2.79 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Neonatal exposure to anesthetics that block N-methyl-D-aspartate receptors and/or hyperactivate gamma-aminobutyric acid type A receptor has been shown to cause neuronal degeneration in the developing brain, leading to functional deficits later in adulthood. The authors investigated whether exposure of neonatal mice to inhaled sevoflurane causes deficits in social behavior as well as learning disabilities.
Six-day-old C57BL/6 mice were exposed to 3% sevoflurane for 6 h. Activated cleaved caspase-3 immunohistochemical staining was used for detection of apoptosis. Cognitive functions were tested by pavlovian conditioned fear test. Social behavior was tested by social recognition and interaction tests.
Neonatal exposure to sevoflurane significantly increased the number of apoptotic cells in the brain immediately after anesthesia. It caused persistent learning deficits later in adulthood as evidenced by decreased freezing response in both contextual and cued fear conditioning. The social recognition test demonstrated that mice with neonatal exposure to sevoflurane did not develop social memory. Furthermore, these mice showed decreased interactions with a social target compared with controls in the social interaction test, indicating a social interaction deficit. The authors did not attribute these abnormalities in social behavior to impairments of general interest in novelty or olfactory sensation, because they did not detect significant differences in the test for novel inanimate object interaction or for olfaction.
This study shows that exposure of neonatal mice to inhaled sevoflurane could cause not only learning deficits but also abnormal social behaviors resembling autism spectrum disorder.