Erika Takada

Nicotine inhibits the production of proinflammatory mediators in human monocytes by suppression of I-κB phosphorylation and nuclear factor-κB transcriptional activity through nicotinic acetylcholine receptor α7

Macrophages/monocytes and the proinflammatory mediators, such as tumour necrosis factor (TNF)‐α, prostaglandin E2 (PGE2), macrophage inflammatory protein (MIP)‐1α and MIP‐1α, play a critical role in the progression of immunological disorders including rheumatoid arthritis, Behçet’s disease and Crohn’s disease. In addition, the nicotinic acetylcholine receptor‐α7 (α7nAChR) subunit is an essential regulator of inflammation. In this study, we evaluated the expression of the α7nAChR subunit on human peripheral monocytes and the effect of nicotine on the production of these proinflammatory mediators by activated monocytes. Fluorescein isothiocyanate (FITC)‐labelled α‐bungarotoxin demonstrated the cell surface expression of the α7nAchR subunit. Pretreatment with low‐dose nicotine caused inhibition of TNF‐α, PGE2, MIP‐1α and MIP‐1α production, and mRNA expression of TNF‐α, MIP‐1α and MIP‐1α and COX‐2 in lipopolysaccharide (LPS)‐activated monocytes. These suppressive effects of nicotine were caused at the transcriptional level and were mediated through α7nAChR. Nicotine suppressed the phosphorylation of I‐κB, and then inhibited the transcriptional activity of nuclear factor‐κB. These immunosuppressive effects of nicotine may contribute to the regulation of some immune diseases.

Involvement of Th1 cells and heat shock protein 60 in the pathogenesis of intestinal Behcet's disease.

Involvement of excessive Th1 cell functions and heat shock protein expression in the pathogenesis of Behçets disease (BD) has been reported. In this study we have characterized immune responses in intestinal lesions of BD. Peripheral blood lymphocytes (PBL) of BD and healthy controls (HC) and tissue specimens of intestinal Behçets disease (intestinal BD), Crohns disease (CD) and ulcerative colitis (UC) were analysed for mRNA and protein expression by reverse transcriptase‐polymerase chain reaction (PCR) and immunohistochemistry, respectively. PBL of BD patients expressed the Th1‐related chemokine receptor, CCR5 and CXCR3 preferentially compared with those of healthy controls. Intestinal lesions of BD expressed interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and interleukin (IL)‐12 mRNA, indicating Th1 skewed responses in vivo. mRNA of Txk, a Tec family tyrosine kinase specific to Th1 cells, was expressed in the lesions, suggesting its contribution to the Th1‐dominant responses. In the intestinal samples, CCR5 was detected in all the cases with BD, whereas Th2‐related CCR3 and CCR4 were detected randomly, mainly in the cases with inactive BD and those receiving large amounts of prednisolone, indicating the Th1‐dominant immune responses in the intestinal lesions. As the ligands of CCR5, MIP1α and MIP1β were detected, whereas RANTES was not. Heat shock protein (HSP) 60 was expressed in PBL and intestinal tissues of BD. Th1‐dominant immune responses and HSP60 expression may induce the inflammatory responses and thus be associated with the pathogenesis of intestinal BD.

Excessive expression of Txk, a member of the Tec family of tyrosine kinases, contributes to excessive Th1 cytokine production by T lymphocytes in patients with Behcet's disease

Excessive Th1 cell function is importantly involved in the pathogenesis of Behcets disease (BD). We previously found that Txk, a member of the Tec family of tyrosine kinases, acts as a Th1 cell specific transcription factor. To investigate immune aberration in the pathogenesis of BD, we studied the expression of Txk and Th1 cytokines in peripheral blood lymphocytes (PBL) and skin lesions in patients with BD. Cytokine production by the lymphocytes was assessed using ELISA. PBL produced excessive Th1 associated cytokines including IFN‐γ and IL‐12 spontaneously and in response to exogenous HSP60‐derived peptide stimulation, which was shown to induce proliferation of PBL, in patients with BD. Circulating CD4+ T cells expressed excessive Txk protein. A majority of cells infiltrating into skin lesions expressed IFN‐γ in the BD specimens. IL‐12 and IL‐18 were also expressed in the mononuclear cell aggregates. Lymphocytes accumulating in the skin lesion expressed higher levels of Txk as compared with atopic dermatitis lesions, a typical Th2 disease. IFN‐γ, IL‐18 and Il‐12 were detected in the BD skin lesions, which may induce preferential development of Th1 cells in patients with BD. The mononuclear cell aggregates contained Txk expressing cells in such skin lesions. Collectively, Txk expressing Th1 cells and the Th1 associated cytokines may play a critical role in the development of skin lesions in BD.

Transplantation of myocyte precursors derived from embryonic stem cells transfected with IGFII gene in a mouse model of muscle injury.

Background. Reconstruction of skeletal muscle tissue is hampered by the lack of availability of functional substitution of the tissue. Methods. Embryonic stem (ES) cells were transfected with the insulin-like growth factor (IGF) II gene and were selected with G418. The resultant cell clones were analyzed regarding their myogenic differentiation in vitro and in vivo. Results. The cells expressed early and late myogenic differentiation markers, including myoD, myogenin, and dystrophin in vitro. They had phosphorylated Akt within the cells, suggesting their activation by the secreted IGFII. Transplantation of the cells to injured anterior tibial muscle of mice significantly improved their motor functions compared to injured mice transplanted with undifferentiated ES cells and injured mice given vehicle alone. The transfected cells adapted to the injured muscle, formed myofibers positive for dystrophin and negative for MyoD and myogenin. Trichrome staining and toluidine blue staining support myofiber formation in vivo. The enzymatic activity of acetylcholine esterase suggested the functional activity of the regenerated motor units. The evoked electromyogram of anterior tibial muscle transplanted with the transfected cells showed significantly higher potentials compared to that transplanted with undifferentiated ES cells and that injected with phosphate-buffered saline (control injury). Electron microscopic examination confirmed the myofiber formation in the cells in vivo. Conclusions. Transfection of IGFII gene into ES cells may be applicable for transplantation therapy of muscle damage due to injury and myopathies.

Transfection with pax6 Gene of Mouse Embryonic Stem Cells and Subsequent Cell Cloning Induced Retinal Neuron Progenitors, Including Retinal Ganglion Cell-Like Cells, in vitro

Objective: It is theoretically possible to induce various cell types, including retinal neurons, from embryonic stem cells (ESCs). pax6 regulates early events in eye development, including the generation of retinal ganglion cells (RGCs). We previously reported the successful induction of corneal epithelial cells from ESCs transfected with the pax6 gene. Here, we attempted to establish cloned RGC-like cells from ESCs transfected with the pax6 gene. Methods: Undifferentiated mouse ESCs were transfected with pax6 cDNA by electroporation, followed by selection with G418. We conducted limiting-dilution culture of pax6-transfected cells. We expanded the cloned pax6-transfected cells, which expressed nestin and musashi-1, for further characterization in culture media containing fibronectin. The cells were characterized using RT-PCR, immunostaining, electron microscopy, renal subcapsular transplantation assay and Ca imaging. Results: We obtained clonally expanding pax6-transfected cells, all of which were positive for six3, sonic hedgehog (shh), math5, brn3, thy1 and melanopsin, by using several ESCs. When transplanted into a mouse renal capsule, they differentiated into neurons with elongated axons, expressing βIII tubulin and neurofilament middle chain, and were free from teratoma development. Electron-microscopic examination showed neurotubules and neurofilaments in the axon-like processes of the cloned pax6-transfected cells. High KCl stimulation increased free Ca influx on Ca2+ imaging. Conclusions: ESCs were applicable for the induction of retinal progenitor cells, including RGC-like cells, by transfection with the pax6 gene and subsequent limiting-dilution culture. Cloned cell lines may be useful to analyze the requirements for retinal progenitor cell differentiation, and our study suggests the clinical application of this cell type.

Experimental transplantation of corneal epithelium-like cells induced by Pax6 gene transfection of mouse embryonic stem cells

Purpose: Corneal epithelial stem cells are deficient in cases of limbal disorders, leading to conjunctival epithelial ingrowth, vascularization, and eventually visual disturbance. We introduced the eye development-associated transcription factor pax6 to embryonic stem (ES) cells and tested whether pax6-transfected cells resembling purified corneal epithelial cells were applicable as a cell source for corneal transplantation. Methods: pax6 cDNA with green fluorescence protein was electrotransfected to ES cells and the cells were cultured with G418 for 14 days. They were characterized by reverse transcription-polymerase chain reaction and immunohistochemistry. The cells were transplanted onto experimentally damaged mouse corneas. Histologic reconstitution of the corneal epithelium was assessed. Results: pax6-transfected cells formed a monolayer of epithelium-like cells in vitro. They expressed cytokeratin12, a specific keratin of corneal epithelial cells, E-cadherin, and CD44, which are important adhesion molecules of corneal epithelial cells on the cell membrane. They accumulated to make a colony that gave a staining pattern of reticular configuration for cytokeratin 12, E-cadherin, and CD44. When the cells were transplanted onto damaged cornea, they have been kept alive on the cornea. Conclusions: The purified corneal epithelium-like cells derived from ES cells transfected with pax6 gene adapted to the injured cornea and were kept alive on it. These results suggested application of ES cell-derived corneal epithelial cells for treating corneal injuries.

Bifidobacteria Abundance-Featured Gut Microbiota Compositional Change in Patients with Behcet’s Disease

Gut microbiota compositional alteration may have an association with immune dysfunction in patients with Behcet’s disease (BD). We conducted a fecal metagenomic analysis of BD patients. We analyzed fecal microbiota obtained from 12 patients with BD and 12 normal individuals by sequencing of 16S ribosomal RNA gene. We compared the relative abundance of bacterial taxa. Direct comparison of the relative abundance of bacterial taxa demonstrated that the genera Bifidobacterium and Eggerthella increased significantly and the genera Megamonas and Prevotella decreased significantly in BD patients compared with normal individuals. A linear discriminant analysis of bacterial taxa showed that the phylum Actinobacteria, including Bifidobacterium, and the family Lactobacillaceae exhibited larger positive effect sizes than other bacteria in patients with BD. The phylum Firmicutes and the class Clostridia had large effect sizes in normal individuals. There was no significant difference in annotated species numbers (as numbers of operational taxonomic unit; OTU) and bacterial diversity of each sample (alpha diversity) between BD patients and normal individuals. We next assigned each sample to a position using three axes by principal coordinates analysis of the OTU table. The two groups had a significant distance as beta diversity in the 3-axis space. Fecal sIgA concentrations increased significantly in BD patients but did not correlate with any bacterial taxonomic abundance. These data suggest that the compositional changes of gut microbes may be one type of dysbiosis (unfavorable microbiota alteration) in patients with BD. The dysbiosis may have an association with the pathophysiology of BD.

Role of SDF1/CXCR4 Interaction in Experimental Hemiplegic Models with Neural Cell Transplantation

Much attention has been focused on neural cell transplantation because of its promising clinical applications. We have reported that embryonic stem (ES) cell derived neural stem/progenitor cell transplantation significantly improved motor functions in a hemiplegic mouse model. It is important to understand the molecular mechanisms governing neural regeneration of the damaged motor cortex after the transplantation. Recent investigations disclosed that chemokines participated in the regulation of migration and maturation of neural cell grafts. In this review, we summarize the involvement of inflammatory chemokines including stromal cell derived factor 1 (SDF1) in neural regeneration after ES cell derived neural stem/progenitor cell transplantation in mouse stroke models.

Establishment of retinal progenitor cell clones by transfection with Pax6 gene of mouse induced pluripotent stem (iPS) cells.

We previously reported that transfection of Pax6 gene which regulated early events in eye development into mouse ES cells brought about their differentiation into retinal progenitors. Here, we attempted to establish cloned retinal progenitors which had ability to further differentiate into photoreceptor like cells by transfecting mouse induced pluripotent stem (iPS) cells with Pax6 gene. Undifferentiated iPS cells were transfected with Pax6 cDNA, followed by selection with G418. After limiting dilution culture, we selected cloned Pax6-transfected cells, which simultaneously expressed mRNAs of Nestin, Musashi1, Six3 and Chx10 for further characterization. We obtained totally 8 clonally expanding Pax6-transfected cells. They started to express mRNAs of Brn3b, Cone-rod homeobox (Crx), pkc, CD73, rhodopsin and the γ-subunit of rod cGMP phosphodiesterase (PDEγ). Flow cytometric analysis revealed that almost half of the cells were CD73+, a marker of photoreceptor precursors. Western blotting confirmed cytoplasmic protein expression of rhodopsin. High KCl stimulation increased free Ca influx into the cells on Ca(2+) imaging. iPS cells transfected with Pax6 gene, followed by subsequent limiting dilution culture became retinal progenitors including photoreceptor like cells. The cloned cell lines may be useful for analyzing differentiation requirement of retinal progenitors.

Loss of dopaminoreceptive neuron causes L-dopa resistant parkinsonism in tauopathy

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is a family of inherited dementias caused by tauopathy. A mutation in exon 10 of the tau gene, N279K, causes a particular kindred of FTDP-17, which is predominant for parkinsonism. The disease initially presents as L-dopa resistant parkinsonism which then rapidly progresses. The final pathological features reveal disappearing dopamine (DA) neurons, but the causes remain poorly understood. We previously established a transgenic mouse with human N279K mutant tau as a model for FTDP-17, which showed cognitive dysfunctions caused by the mutant. Here we analyze L-dopa resistant parkinsonism by several behavioral tests, and focus on the distributions and accumulations of the mutant tau in the DA system by immunohistochemistry and Western blot. Interestingly, dopaminoreceptive (DAr) neurons in the striatum showed neurofibrils degeneration and apoptosis through caspase-3 activation by mutant tau accumulation. The DAr neuron loss in the caudoputamen, the target of the nigrostriatal system occurred before DA neuron loss in young symptomatic mice. Residual DA neurons in the mouse functioned in DA transportation, whereas dysregulation of intracellular DA compartmentalization implied an excess level of DA caused by DAr neuron loss. In the final stages, both DAr and DA neurons decreased equally, unlike Parkinsons disease. Therefore, DAr neurons were fundamentally vulnerable to the mutation indicating a critical role for the L-dopa resistant parkinsonism in tauopathy.