Serum MMP3 Correlated With IL1ß Messenger RNA Expressions of Peripheral Blood Mononuclear Cells in Patients With Relapsing Polychondritis With Respiratory Involvement.

OBJECTIVE: Respiratory involvement was intimately associated with poorer prognosis in patients with relapsing polychondritis (RP). We previously reported that high serum matrix metalloproteinase-3 (MMP3) was frequently observed in patients with RP with respiratory involvement. Elevated MMP3 secreted through local inflammation may be associated with the development of airway lesions. METHODS: We collected peripheral blood mononuclear cells (PBMCs) and sera from 30 patients with RP and 14 healthy individuals. Interleukin (IL) 1ß, IL6, and tumor necrosis factor (TNF) α messenger RNA (mRNA) expressions were analyzed in freshly isolated and cultured PBMCs with phytohemagglutinin and phorbol myristate acetate stimulation by real-time reverse transcription polymerase chain reaction and serum MMP3 by enzyme-linked immunosorbent assay (ELISA). RESULTS: We confirmed our previous finding that patients with respiratory involvements showed higher serum MMP3 compared with patients lacking respiratory involvement. IL1ß mRNA expression was significantly higher in patients with RP than in healthy individuals after mitogenic stimulation. TNFα mRNA expression after stimulation was significantly lower in patients with RP compared with in healthy individuals. We performed correlation analyses between MMP3 and cytokine mRNA expressions in patients with RP. In patients with respiratory involvement, MMP3 correlated with IL1ß and IL6 after stimulation. In patients without respiratory involvement, no positive correlations between MMP3 and cytokine mRNA expressions were observed regardless of culture condition. We did not find any positive correlations between MMP3 and TNFα mRNA expression in patients with RP. CONCLUSION: It is possible that IL1ß mRNA expression associates by some means with airway inflammation via the secretion of MMP3 in patients with RP. Involvement of proinflammatory cytokines, including IL1ß, was suggested for the pathophysiology of airway lesions in patients with RP.

Roles of Reelin/Disabled1 pathway on functional recovery of hemiplegic mice after neural cell transplantation; Reelin promotes migration toward motor cortex and maturation to motoneurons of neural grafts.

Reelin is a large glycoprotein which regulates central nervous system (CNS) development. Dysfunctions of Reelin were reported on certain neuropsychiatric diseases. We examined involvement of Reelin pathway in functional recovery of hemiplegic mice after neural transplantation. Reelin was expressed 1 day after cryogenic injury of right motor cortex. We transplanted neural stem/progenitor cells (NSPCs) from wild-type mice into ipsilateral striatum of hemiplegic mice. The grafts migrated from the striatum and reached the injured cortex 14 days after transplantation. The transplantation significantly improved their motor functions (P < .05). The NSPCs migrating toward the cortex expressed Reelin receptors, Apoer and Vldlr, and phosphorylated Disabled1 (Dab1), a downstream signaling molecule of Reelin. The grafts expressed Ncadherin and active form of Integrin ß1, both of which were known to become active with Reelin stimulation. At day 28, the grafts expressed Ctip2, Crim1, Foxp2, and Fezf2, all of which were forebrain motoneuron associated markers, and Nfm and Synapsin1 on the damaged cortex. We then transplanted NSPCs of yotari mice (yot/yot genotype) having nonfunctional Dab1 by a mutation of its gene. Majority of the grafts from yotari mice (>80%) did not migrate and thus remained at the striatum. The grafts did not express the forebrain motoneuron associated markers nor the cell adhesion molecules including Ncadherin and active Integrin ß1. Reelin pathway was involved in graft migration by regulating certain adhesion molecules and in their differentiation to functional motoneurons accompanying synapse formation. We suggested involvement of Reelin pathway for neural regeneration and functional recovery of hemiplegic mice in adulthood after neural transplantation.

Neuronal Cell Sheets of Cortical Motor Neuron Phenotype Derived from Human iPSCs.

Transplantation of stem cells that differentiate into more mature neural cells brings about functional improvement in preclinical studies of stroke. Previous transplant approaches in the diseased brain utilized injection of the cells in a cell suspension. In addition, neural stem cells were preferentially used for grafting. However, these cells had no specific relationship to the damaged tissue of stroke and brain injury patients. The injection of cells in a suspension destroyed the cell-cell interactions that are suggested to be important for promoting functional integrity of cortical motor neurons. In order to obtain suitable cell types for grafting in patients with stroke and brain damage, a protocol was modified for differentiating human induced pluripotent stem cells from cells phenotypically related to cortical motor neurons. Moreover, cell sheet technology was applied to neural cell transplantation, as maintaining the cell-cell communications is regarded important for the repair of host brain architecture. Accordingly, neuronal cell sheets that were positive Forebrain Embryonic Zinc Finger (Fez) family zinc finger 2 (FEZF2), COUP-TF-interacting protein 2, insulin-like growth factor-binding protein 4 (IGFBP4), cysteine-rich motor neuron 1 protein precursor (CRIM1), and forkhead box p2 (FOXP2) were developed. These markers are associated with cortical motoneurons that are appropriate for the transplant location in the lesions. The sheets allowed preservation of cell-cell interactions shown by synapsin1 staining after transplantation to damaged mouse brains. The sheet transplantation brought about partial structural restoration and the improvement of motor functions in hemiplegic mice. Collectively, the novel neuronal cell sheets were transplanted into damaged motor cortices; the cell sheets maintained cell-cell interactions and improved the motor functions in the hemiplegic model mice. The motoneuron cell sheets are possibly applicable for stroke patients and patients with brain damage by using patient-specific induced pluripotent stem cells.

Possible association of proinflammatory cytokines including IL1ß and TNFα with enhanced Th17 cell differentiation in patients with Behcet's disease.

We have reported that helper T type 17 (Th17) cells increased in patients with Behcet's disease (BD). It remains obscure how Th17 cells increase in the patients. We here analyzed whether T cells preferentially differentiate into Th17 cells in response to various inflammatory cytokines in patients with BD. Exogenous interleukin (IL)23 sustained the higher Th17 cell frequencies of CD4+CD45RO+ T cells after a 2-day culture in vitro in patients with BD, whereas the T cell subpopulation of normal individuals did not respond to IL23 to sustain/increase Th17 cell frequencies. IL23 receptor positive cell frequencies in freshly isolated BD CD4+CD45RO+ T cells correlated with Th17 cell frequencies assessed by intracellular cytokine staining. After a 2-day culture with IL23, BD CD4+ T cells retained the correlation between IL23 receptor expression level and extent of IL17 secretion (as indicated by Th17 cell frequencies), whereas such correlation was not noted in normal individuals. IL23 signals with its receptor were thus suggested to induce IL17 secretion (Th17 cell frequencies) in a short-time culture in patients with BD. We cultured CD4+CD45RO- T cells for 11 days with various inflammatory cytokines to study which cytokine associated with the enhanced Th17 frequencies in the patients. IL17 production by CD4+CD45RO- T cells of BD patients increased significantly by the supplementation of IL1ß and tumor necrosis factor (TNF)α, in addition to IL23, compared with that of normal individuals. These results suggest that proinflammatory cytokines, such as IL1ß, TNFα, and IL23, may associate with the expansion of Th17 cells in patients with BD. This study was registered with the University Hospital Medical Information Network-Clinical Trials Registry (UMIN000003806).

Cellular and molecular mechanisms of the restoration of human APP transgenic mouse cognitive dysfunction after transplant of human iPS cell-derived neural cells.

Cholinergic neuronal loss is a common finding in patients with Alzheimer's disease (AD) and AD model mice. We previously transplanted neurons derived from human induced pluripotent stem (iPS) cells into the hippocampus of human amyloid precursor protein transgenic AD model mice. In the present study, we examined the cellular and molecular mechanisms involved in the alleviation of cognitive dysfunction in transplanted mice. After transplant, mice showed improvement in cognitive function, confirming our previous findings. Human choline acetyltransferase (ChAT)-positive cholinergic neurons were distributed throughout the cortex of the grafted mice. Human and mouse ChAT-positive neurons and alpha7 nicotinic acetylcholine receptor (α7nAChR)-positive neurons were significantly increased in the cortex and hippocampus of the grafted mice compared with the vehicle-injected mice. In addition, human and mouse vesicular GABA transporter (VGAT)-positive neurons were located mainly in the hippocampus and, though the number was small, human VGAT-positive neurons were observed in the cortex. In the grafted mouse cortex, the number of GABA receptor (GABAR)-positive neurons of both human origin and mouse origin were significantly increased compared with those in the vehicle-injected mouse cortex. The α7nAChR-positive and GABAR-positive neurons expressed phosphorylated Akt and c-fos in the cortex, suggesting that these receptor-expressing neurons were possibly activated by the neurotransmitters secreted from the grafted neurons. Collectively, the grafted and host neurons may form positive feedback loops via neurotransmitter secretion in both the cerebral cortex and hippocampus, leading to alleviation of cognitive dysfunction in dementia model mice.

Restoration of spatial memory dysfunction of human APP transgenic mice by transplantation of neuronal precursors derived from human iPS cells.

PDGF promoter driven amyloid precursor protein (PDAPP) transgenic mice were accompanied by age dependent amyloid ß deposition and progressive spatial memory dysfunction which emerges within a few months of age. We conducted transplantation of neuronal precursors of cholinergic neuron phenotype which were derived from human iPS (hiPS) cells into bilateral hippocampus of PDAPP mice. We first generated neuronal precursors with cholinergic neuron phenotype from hiPS cells by culturing them with retinoic acid (RA), sonic hedgehog (SHH) and noggin-Fc (NOG). Spatial memory function of PDAPP mice was significantly impaired compared to that of nontransgenic littermates at age 8 weeks. After neuronal precursor transplantation, subsequent memory dysfunction of PDAPP mice was significantly improved, compared to that of vehicle injected PDAPP mice. We observed choline acetyltransferase (ChAT) positive cholinergic human neurons and vesicle GABA transporter (VGAT) positive GABAergic human neurons in PDAPP mouse hippocampus 45 days after the transplantation. Neuronal precursors with cholinergic neuron phenotype derived from hiPS cells survived in PDAPP mouse hippocampus and their spatial memory loss was improved. hiPS cells may become applicable for the treatment of patients with dementia.

Aberrant Fas ligand expression in lymphocytes in patients with Behçet's disease.

BACKGROUND: Defects in immune responses have been reported in patients with Behçet's disease (BD). To further characterize the immune dysfunction and its contribution to the pathogenesis, we have studied Fas ligand (FasL) expression in peripheral blood lymphocytes (PBL) and mononuclear cells in the skin lesions in patients with BD. METHODS: FasL expression in PBL was studied with RT-PCR and immunoblotting with rabbit anti-human FasL antibody. We studied the expression of FasL in cryostat sections of biopsy specimens of erythema nodosum lesions from 4 patients with BD and of a genital ulcer lesion in another patient using immunohistochemical staining. Apoptotic cell death was detected with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: We found that FasL mRNA and FasL protein expression was detected marginally in the unstimulated PBL, and was induced upon activation in normal individuals. PBL from patients with BD exhibited an enhanced expression of FasL mRNA and FasL protein without in vitro stimulation. Moreover, mitogen stimulation failed to augment FasL expression of their lymphocytes, suggesting a dysregulation of FasL expression of PBL in patients with BD. The skin biopsy specimens revealed that cells infiltrating into skin lesions expressed FasL and there were several TUNEL staining-positive cells in the lesions, suggesting that Fas/FasL-mediated apoptosis is involved in the development of the skin lesion and thus may be associated with the pathogenesis. CONCLUSIONS: We found an excessive expression of FasL in circulating as well as skin-infiltrating lymphocytes and the presence of apoptotic cells in the skin lesions, suggesting that lymphocytes expressing FasL aberrantly may play a role in the development and pathogenesis of BD.