BACKGROUND: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. METHODS: Balb/c (H-2(d)) mice were fed with dendritic cell line, DC2.4 (H-2(b)) every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. RESULTS: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-gamma, IL-5, and TNF-alpha. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. CONCLUSION: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.
Animals ; Cell Proliferation ; Cytokines ; Dendritic Cells ; Ear ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Immunization ; Interleukin-5 ; Lymphocyte Culture Test, Mixed ; Mice ; Plasmids ; RNA, Messenger ; Spleen ; T-Lymphocytes ; Tumor Necrosis Factor-alpha ; Veins

BACKGROUND: A co-inhibitory molecule, B7-H4, is believed to negatively regulate T cell immunity by suppressing T cell proliferation and inhibiting cytokine production. However, the mechanism behind B7-H4-mediated tolerance remains unclear. METHODS: Balb/c (H-2(d)) mice were fed with dendritic cell line, DC2.4 (H-2(b)) every day for 10 days. Meantime, mice were hydrodynamically injected with recombinant plasmid expressing B7-H4 fusion protein (B7-H4.hFc) or hFc via tail vein. One day after last feeding, mice were immunized with allogeneic B6 spleen cells. 14 days following immunization, mice were challenged with B6 spleen cells to ear back and the ear swelling was determined the next day. Subsequently, a mixed lymphocyte reaction (MLR) was also performed and cytokines profiles from the reaction were examined by sandwich ELISA. Frequency of immunosuppressive cell population was assayed with flow cytometry and mRNA for FoxP3 was determined by RT-PCR. RESULTS: Tolerant mice given plasmid expressing B7-H4.hFc showed a significant reduction in ear swelling compared to control mice. In addition, T cells from mice given B7-H4.hFc plasmid revealed a significant hyporesponsiveness of T cells against allogeneic spleen cells and showed a significant decrease in Th1 and Th2 cytokines such as IFN-gamma, IL-5, and TNF-alpha. Interestingly, flow cytometric analysis showed that the frequency of CD4+CD25+FoxP3+ Tregs in spleen was increased in tolerant mice given recombinant B7-H4.hFc plasmid compared to control group. CONCLUSION: Our results demonstrate that B7-H4 synergistically potentiates oral tolerance induced by allogeneic cells by increasing the frequency of FoxP3+ CD4+CD25+ Treg and reducing Th1 and Th2 cytokine production.
Animals ; Cell Proliferation ; Cytokines ; Dendritic Cells ; Ear ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Immunization ; Interleukin-5 ; Lymphocyte Culture Test, Mixed ; Mice ; Plasmids ; RNA, Messenger ; Spleen ; T-Lymphocytes ; Tumor Necrosis Factor-alpha ; Veins

The generation of disease-specific induced pluripotent stem cell (iPSC) lines from patients with incurable diseases is a promising approach for studying disease mechanisms and drug screening. Such innovation enables to obtain autologous cell sources in regenerative medicine. Herein, we report the generation and characterization of iPSCs from fibroblasts of patients with sporadic or familial diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), juvenile-onset, type I diabetes mellitus (JDM), and Duchenne type muscular dystrophy (DMD), as well as from normal human fibroblasts (WT). As an example to modeling disease using disease-specific iPSCs, we also discuss the previously established childhood cerebral adrenoleukodystrophy (CCALD)- and adrenomyeloneuropathy (AMN)-iPSCs by our group. Through DNA fingerprinting analysis, the origins of generated disease-specific iPSC lines were identified. Each iPSC line exhibited an intense alkaline phosphatase activity, expression of pluripotent markers, and the potential to differentiate into all three embryonic germ layers: the ectoderm, endoderm, and mesoderm. Expression of endogenous pluripotent markers and downregulation of retrovirus-delivered transgenes [OCT4 (POU5F1), SOX2, KLF4, and c-MYC] were observed in the generated iPSCs. Collectively, our results demonstrated that disease-specific iPSC lines characteristically resembled hESC lines. Furthermore, we were able to differentiate PD-iPSCs, one of the disease-specific-iPSC lines we generated, into dopaminergic (DA) neurons, the cell type mostly affected by PD. These PD-specific DA neurons along with other examples of cell models derived from disease-specific iPSCs would provide a powerful platform for examining the pathophysiology of relevant diseases at the cellular and molecular levels and for developing new drugs and therapeutic regimens.
Alzheimer Disease/genetics/*pathology ; Cell Differentiation ; Cells, Cultured ; Diabetes Mellitus, Type 1/genetics/*pathology ; Drug Discovery/*methods ; Fibroblasts/cytology/metabolism/pathology ; Gene Expression ; Humans ; Induced Pluripotent Stem Cells/cytology/metabolism/*pathology ; Muscular Dystrophy, Duchenne/genetics/*pathology ; Parkinson Disease/genetics/*pathology

Objective: This study aimed to investigate the blood-brain barrier (BBB) disruption in mild traumatic brain injury (mTBI) patients with post-concussion syndrome (PCS) using dynamic contrast-enhanced (DCE) magnetic resonance (MR) imaging and automatic whole brain segmentation. Materials and Methods: Forty-two consecutive mTBI patients with PCS who had undergone post-traumatic MR imaging, including DCE MR imaging, between October 2016 and April 2018, and 29 controls with DCE MR imaging were included in this retrospective study. After performing three-dimensional T1-based brain segmentation with FreeSurfer software (Laboratory for Computational Neuroimaging), the mean Ktrans and vp from DCE MR imaging (derived using the Patlak model and extended Tofts and Kermode model) were analyzed in the bilateral cerebral/cerebellar cortex, bilateral cerebral/cerebellar white matter (WM), and brainstem. Ktrans values of the mTBI patients and controls were calculated using both models to identify the model that better reflected the increased permeability owing to mTBI (tendency toward higher Ktrans values in mTBI patients than in controls). The Mann-Whitney U test and Spearman rank correlation test were performed to compare the mean Ktrans and vp between the two groups and correlate Ktrans and vp with neuropsychological tests for mTBI patients. Results: Increased permeability owing to mTBI was observed in the Patlak model but not in the extended Tofts and Kermode model. In the Patlak model, the mean Ktrans in the bilateral cerebral cortex was significantly higher in mTBI patients than in controls (p = 0.042). The mean vp values in the bilateral cerebellar WM and brainstem were significantly lower in mTBI patients than in controls (p = 0.009 and p = 0.011, respectively). The mean Ktrans of the bilateral cerebral cortex was significantly higher in patients with atypical performance in the auditory continuous performance test (commission errors) than in average or good performers (p = 0.041). Conclusion BBB disruption, as reflected by the increased Ktrans and decreased vp values from the Patlak model, was observed throughout the bilateral cerebral cortex, bilateral cerebellar WM, and brainstem in mTBI patients with PCS.