Overexpression of mutant HSP27 causes axonal neuropathy in mice.

BACKGROUND: Mutations in heat shock 27 kDa protein 1 (HSP27 or HSPB1) cause distal hereditary motor neuropathy (dHMN) or Charcot-Marie-Tooth disease type 2 F (CMT2F) according to unknown factors. Mutant HSP27 proteins affect axonal transport by reducing acetylated tubulin. RESULTS: We generated a transgenic mouse model overexpressing HSP27-S135F mutant protein driven by Cytomegalovirus (CMV) immediate early promoter. The mouse phenotype was similar to dHMN patients in that they exhibit motor neuropathy. To determine the phenotypic aberration of transgenic mice, behavior test, magnetic resonance imaging (MRI), electrophysiological study, and pathology were performed. Rotarod test showed that founder mice exhibited lowered motor performance. MRI also revealed marked fatty infiltration in the anterior and posterior compartments at calf level. Electrophysiologically, compound muscle action potential (CMAP) but not motor nerve conduction velocity (MNCV) was reduced in the transgenic mice. Toluidine staining with semi-thin section of sciatic nerve showed the ratio of large myelinated axon fiber was reduced, which might cause reduced locomotion in the transgenic mice. Electron microscopy also revealed abundant aberrant myelination. Immunohistochemically, neuronal dysfunctions included elevated level of phosphorylated neurofilament and reduced level of acetylated tubulin in the sural nerve of transgenic mice. There was no additional phenotype besides motor neuronal defects. CONCLUSIONS: Overexpression of HSP27-S135F protein causes peripheral neuropathy. The mouse model can be applied to future development of therapeutic strategies for dHMN or CMT2F.

Blood sphingolipid as a novel biomarker in patients with neuromyelitis optica spectrum disorder.

BACKGROUND: Sphingolipids are signaling molecules and structural components of the axolemma and myelin sheath. Plasma sphingolipid levels may reflect disease status of neuromyelitis optica spectrum disorder (NMOSD). We aimed to examine plasma sphingolipids as disease severity biomarkers for NMOSD and compare their characteristics with those of serum neurofilament light chain (sNfL) and glial fibrillary acidic protein (sGFAP). METHODS: We measured plasma sphingolipids, sNfL, and sGFAP levels in NMOSD cases with anti-aquaporin-4-antibody. An unbiased approach, partial least square discriminant analysis (PLS-DA), was utilized to determine whether sphingolipid profiles differ according to the disease state of NMOSD (presence, moderate-to-severe disability [Expanded Disease Severity Scale, (EDSS) > 3.0], and relapses). RESULTS: We investigated 81 patients and 10 controls. PLS-DA models utilizing sphingolipids successfully differentiated patients with EDSS > 3.0, but failed to identify the presence of disease and relapses. Ceramide-C14-a significant contributor to differentiating EDSS > 3.0-positively correlated with EDSS, while its levels were independent of age and the presence of relapses. This characteristic was unique from those of sNfL and sGFAP, which were affected by age and relapses as well as EDSS. CONCLUSION: Plasma sphingolipids may be useful NMOSD biomarkers for disability with distinct characteristics compared to sNfL and sGFAP.

Tonsil-derived mesenchymal progenitor cells acquire a follicular dendritic cell phenotype under cytokine stimulation.

Tonsils comprise part of the mucosal immune system and contain lymphocytes, macrophages, and follicular dendritic cells (FDCs). FDCs are located in the B cell area of the follicles of secondary lymphoid organs, such as the spleen, tonsils, or lymph nodes, and they trap and retain immune complexes on their surfaces to regulate B cell activation and maturation. Stromal cells from the palatine tonsils are often used for FDC in vitro studies, and it has been reported that human palatine tonsils may be a good source of multipotent mesenchymal cells. Therefore, we assessed whether tonsil-derived mesenchymal stromal cells could differentiate into a FDC-like phenotype. We discovered that stromal cells isolated from human tonsils not only had the potential to differentiate into various cell types of mesenchymal origin, but they also could differentiate into FDC-like cells under cytokine stimulation in vitro.

Tonsil-derived mesenchymal stromal cells: evaluation of biologic, immunologic and genetic factors for successful banking.

BACKGROUND AIMS: Although mesenchymal stromal cells (MSC) from human palatine tonsils (tonsillar MSC, T-MSC) have been isolated, whether T-MSC isolated from multiple donors are feasible for cell banking has not been studied. METHODS: T-MSC before and after a standard protocol of cryopreservation and thawing were assessed regarding several basic characteristics, including colony-forming unit-fibroblast features, MSC-specific surface antigen profiles, and inhibition of alloreactive T-cell proliferation. In vitro mesodermal differentiation potentials to adipocytes, osteocytes and chondrocytes were detected by staining with either cell-specific dyes or antibody after incubation with each appropriate differentiation medium. Expression of mesoderm-specific genes was also quantified by real-time polymerase chain reaction (PCR) assay. Expression profiles of endoderm-specific genes were identified by reverse transcription PCR assay. The feasibility of T-MSC in future engraftment was tested by short tandem repeat (STR) analysis using genomic DNA isolated randomly from three independent subjects. RESULTS: Both fresh and cryopreserved-thawed T-MSC showed a similar high proliferation capacity and expressed primitive cell-surface markers. Hematopoietic cell markers, HLA-DR, co-stimulatory molecules and follicular dendritic cell markers were not detected. In addition to mesodermal differentiation, fresh and cryopreserved-thawed cells also underwent endodermal differentiation, as evidenced by the expression of endoderm-specific genes including forkhead box A2 (FoxA2), SIX homeobox 1 (Six1) and chemokine (C-C motif) ligand 21 (CCL21). Both cells significantly decreased phorbol 12- myristate 13-acetate (PMA)-induced T-cell proliferation. T-MSC from three independent donors formed chimerism in STR analysis. CONCLUSIONS: Our results demonstrate for the first time that T-MSC are a potentially good source for MSC banking.

Tonsil-derived mesenchymal stem cells (T-MSCs) prevent Th17-mediated autoimmune response via regulation of the programmed death-1/programmed death ligand-1 (PD-1/PD-L1) pathway.

Our knowledge of the immunomodulatory role of mesenchymal stem cells (MSCs) in both the innate and adaptive immune systems has dramatically expanded, providing great promise for treating various autoimmune diseases. However, the contribution of MSCs to Th17-dominant immune disease, such as psoriasis and its underlying mechanism remains elusive. In this study, we demonstrated that human palatine tonsil-derived MSCs (T-MSCs) constitutively express both the membrane-bound and soluble forms of programmed death-ligand 1 (PD-L1), which enables T-MSCs to be distinguished from MSCs originating from other organs (i.e. bone marrow or adipose tissue). We also found that T-MSC-derived PD-L1 effectively represses Th17 differentiation via both cell-to-cell contact and a paracrine effect. Further, T-MSCs increase programmed death-1 (PD-1) expression on T-cells by secreting IFN-ß, which may enhance engagement with PD-L1. Finally, transplantation of T-MSCs into imiquimod-induced psoriatic skin inflammation in mice significantly abrogated disease symptoms, mainly by blunting the Th17 response in a PD-L1-dependent manner. This study suggests that T-MSCs might be a promising cell source to treat autoimmune diseases such as psoriasis, via its unique immunoregulatory features. Copyright © 2017 John Wiley & Sons, Ltd.

Heat shock protein 90 is involved in IL-17-mediated skin inflammation following thermal stimulation.

The pathogenesis of inflammatory skin diseases involves interactions between immune cells and keratinocytes, including the T helper 17 (Th17)-mediated immune response. Several chemokines [chemokine (C-X-C motif) ligand (CXCL)1, CXCL5 and CXCL8] and antimicrobial peptides [ß-defensin 1 (BD1), LL-37, S100A8 and S100A9] were transcriptionally upregulated in the keratinocyte cell line HaCaT upon stimulation with interleukin (IL)-17. Balneotherapy, the treatment of disease by bathing, is an alternative therapy that has frequently been used for the treatment of inflammatory skin diseases. Immersion in pools of thermal mineral water is often considered to have chemical, thermal, mechanical and immunomodulatory benefits. We examined the effect of thermal treatment on IL-17-mediated inflammation in a model of skin disease. As Act1 is required for IL-17 signaling and is a client protein of heat shock protein 90 (HSP90), we evaluated the effect of HSP90 inhibition on IL-17-mediated cytokine and antimicrobial peptide expression in keratinocytes following heat treatment. We found that after thermal stimulation, Act1 binding to HSP90α was significantly increased in the presence of IL-17 (100 ng/ml) and 17-N-allylamino-17-demethoxygeldanamycin (17-AAG, 1 µM). Antimicrobial peptide and chemokine expression generally increased after heat treatment; Act1 knockdown and 17­AAG reversed this effect. These observations demonstrate the possible immunomodulatory effect of heat on keratinocytes during the progression of IL-17-mediated inflammatory skin diseases.

HDAC6 Inhibitors Rescued the Defective Axonal Mitochondrial Movement in Motor Neurons Derived from the Induced Pluripotent Stem Cells of Peripheral Neuropathy Patients with HSPB1 Mutation.

The Charcot-Marie-Tooth disease 2F (CMT2F) and distal hereditary motor neuropathy 2B (dHMN2B) are caused by autosomal dominantly inherited mutations of the heat shock 27 kDa protein 1 (HSPB1) gene and there are no specific therapies available yet. Here, we assessed the potential therapeutic effect of HDAC6 inhibitors on peripheral neuropathy with HSPB1 mutation using in vitro model of motor neurons derived from induced pluripotent stem cells (iPSCs) of CMT2F and dHMN2B patients. The absolute velocity of mitochondrial movements and the percentage of moving mitochondria in axons were lower both in CMT2F-motor neurons and in dHMN2B-motor neurons than those in controls, and the severity of the defective mitochondrial movement was different between the two disease models. CMT2F-motor neurons and dHMN2B-motor neurons also showed reduced α-tubulin acetylation compared with controls. The newly developed HDAC6 inhibitors, CHEMICAL X4 and CHEMICAL X9, increased acetylation of α-tubulin and reversed axonal movement defects of mitochondria in CMT2F-motor neurons and dHMN2B-motor neurons. Our results suggest that the neurons derived from patient-specific iPSCs can be used in drug screening including HDAC6 inhibitors targeting peripheral neuropathy.

SET binding factor 1 (SBF1) mutation causes Charcot-Marie-Tooth disease type 4B3.

OBJECTIVE: To identify the genetic cause of an autosomal recessive demyelinating Charcot-Marie-Tooth disease type 4B (CMT4B) family. METHODS: We enrolled 14 members of a Korean family in which 3 individuals had demyelinating CMT4B phenotype and obtained distal sural nerve biopsies from all affected participants. We conducted exome sequencing on 6 samples (3 affected and 3 unaffected individuals). RESULTS: One pair of heterozygous missense mutations in the SET binding factor 1 (SBF1) gene (22q13.33), also called MTMR5, was identified as the underlying cause of the CMT4B family illness. Clinical phenotypes of affected study participants with CMT4B were similar, to some extent, to patients with CMT4B1 and CMT4B2. We found a similar loss of large myelinated fibers and focally folded myelin sheaths in our patients, but the actual number of myelinated fibers was different from CMT4B1 and CMT4B2. CONCLUSIONS: We suggest that the compound heterozygous mutations in SBF1 are the underlying causes of a novel CMT4B subtype, designated as CMT4B3. We believe that this study will lead to mechanistic studies to discover the function of SBF1 and to the development of molecular diagnostics for CMT disease.

Resistance to thyroid hormone with missense mutation (V349M) in the thyroid hormone receptor beta gene.

Resistance to thyroid hormone (RTH) is a syndrome characterized by reduced sensitivity to the thyroid hormone. It is generally caused by mutations in the thyroid hormone receptor beta (TR beta) gene. On the basis of its clinical features, two different forms of this syndrome have been described: generalized resistance and pituitary resistance. A total of 122 TR beta gene mutations have been identified thus far. A 38-year-old woman presented with intermittent palpitation. Thyroid function tests revealed elevated levels of free T4 and TSH. TSH a-subunit levels were 0.41 mlU/mL, and magnetic resonance images of the sellar region evidenced no abnormal findings. The TSH response to TRH stimulation was found to be normal. The sequence analysis of the TR beta gene verified a missense mutation in exon 11, and the observed amino acid alteration was a substitution of a valine for a methionine at codon 349. We report the first case of a woman with RTH, which was found to be caused by a missense mutation (V349M) in the TR beta gene.