Biochemical and functional characterization of skeletal muscle cells differentiated from tonsil-derived mesenchymal stem cells.

INTRODUCTION/AIMS: Human tonsils are a readily accessible source of stem cells for the potential treatment of skeletal muscle disorders. We reported previously that tonsil-derived mesenchymal stem cells (TMSCs) can differentiate into skeletal muscle cells (SKMCs), which renders TMSCs promising candidates for cell therapy for skeletal muscle disorders. However, the functional properties of the myocytes differentiated from mesenchymal stem cells have not been clearly evaluated. In this study we investigated whether myocytes differentiated from TMSCs (skeletal muscle cells derived from tonsil mesenchymal stem cells [TMSC-SKMCs]) exhibit the functional characteristics of SKMCs. METHODS: To test the insulin reactivity of TMSC-SKMCs, the expression of glucose transporter 4 (GLUT4) and phosphatidylinositol 3-kinase/Akt was analyzed after the cells were treated for 30 minutes with 100 nmol/L insulin in normal or high-glucose medium. We also examined whether these cells formed a neuromuscular junction (NMJ) when cocultured with motor neurons, and whether they were stimulated by electrical signals using whole-cell patch clamping. RESULTS: Skeletal muscle cells derived from tonsil mesenchymal stem cells expressed SKMC markers, such as MYOD, MYH3, MYH8, TNNI1, and TTN, at high levels, and exhibited a multinucleated cell morphology and a myotube-like shape. The expression of the acetylcholine receptor and GLUT4 was confirmed in TMSC-SKMCs. In addition, these cells exhibited insulin-mediated glucose uptake, NMJ formation, and transient changes in cell membrane action potential, all of which are representative functions of human SKMCs. DISCUSSION: Tonsil-derived mesenchymal stem cells can be functionally differentiated into SKMCs and may have potential for clinical application for the treatment of skeletal muscle disorders.

New Sources, Differentiation, and Therapeutic Uses of Mesenchymal Stem Cells 2.0.

For the clinical application of mesenchymal stem cells (MSCs), the optimization of biological products (e [...].

Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells.

Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

New Sources, Differentiation, and Therapeutic Uses of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) are multipotent cells derived from various tissues including bone marrow and adipose tissues [...].

Differentiation of Motor Neuron-Like Cells from Tonsil-Derived Mesenchymal Stem Cells and Their Possible Application to Neuromuscular Junction Formation.

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.

Application of differentiated human tonsil-derived stem cells to trembler-J mice.

INTRODUCTION: Mesenchymal stem cells (MSCs) can differentiate into various cell types. METHODS: In this study we investigated the potential of human tonsil-derived MSCs (T-MSCs) for neuromuscular regeneration in trembler-J (Tr-J) mice, a model for Charcot-Marie-Tooth disease type 1A (CMT1A). RESULTS: T-MSCs differentiated toward skeletal myocytes with increased expression of skeletal muscle-related markers (including troponin I type 1, and myogenin), and the formation of myotubes in vitro. In-situ transplantation of T-MSC-derived myocytes (T-MSC myocytes) into the gastrocnemius muscle in Tr-J mice enhanced motor function, with recovery of compound muscle action potential amplitudes. Morphology of the sciatic nerve and skeletal muscle recovered without the formation of teratomas, and the expression levels of nerve growth factor and glial-cell-line-derived neurotrophic factor were increased significantly in T-MSC myocytes compared with T-MSCs in vitro. DISCUSSION: Transplantation of T-MSC myocytes could enable neuromuscular regeneration in patients with CMT1A. Muscle Nerve 57: 478-486, 2018.

Differentiation of Human Tonsil-Derived Mesenchymal Stem Cells into Schwann-Like Cells Improves Neuromuscular Function in a Mouse Model of Charcot-Marie-Tooth Disease Type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited motor and sensory neuropathy, and is caused by duplication of PMP22, alterations of which are a characteristic feature of demyelination. The clinical phenotype of CMT1A is determined by the degree of axonal loss, and patients suffer from progressive muscle weakness and impaired sensation. Therefore, we investigated the potential of Schwann-like cells differentiated from human tonsil-derived stem cells (T-MSCs) for use in neuromuscular regeneration in trembler-J (Tr-J) mice, a model of CMT1A. After differentiation, we confirmed the increased expression of Schwann cell (SC) markers, including glial fibrillary acidic protein (GFAP), nerve growth factor receptor (NGFR), S100 calcium-binding protein B (S100B), glial cell-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF), which suggests the differentiation of T-MSCs into SCs (T-MSC-SCs). To test their functional efficiency, the T-MSC-SCs were transplanted into the caudal thigh muscle of Tr-J mice. Recipients' improved locomotive activity on a rotarod test, and their sciatic function index, which suggests that transplanted T-MSC-SCs ameliorated demyelination and atrophy of nerve and muscle in Tr-J mice. Histological and molecular analyses showed the possibility of in situ remyelination by T-MSC-SCs transplantation. These findings demonstrate that the transplantation of heterologous T-MSC-SCs induced neuromuscular regeneration in mice and suggest they could be useful for the therapeutic treatment of patients with CMT1A disease.

Tonsil-Derived Mesenchymal Stem Cells Differentiate into a Schwann Cell Phenotype and Promote Peripheral Nerve Regeneration.

Schwann cells (SCs), which produce neurotropic factors and adhesive molecules, have been reported previously to contribute to structural support and guidance during axonal regeneration; therefore, they are potentially a crucial target in the restoration of injured nervous tissues. Autologous SC transplantation has been performed and has shown promising clinical results for treating nerve injuries and donor site morbidity, and insufficient production of the cells have been considered as a major issue. Here, we performed differentiation of tonsil-derived mesenchymal stem cells (T-MSCs) into SC-like cells (T-MSC-SCs), to evaluate T-MSC-SCs as an alternative to SCs. Using SC markers such as CAD19, GFAP, MBP, NGFR, S100B, and KROX20 during quantitative real-time PCR we detected the upregulation of NGFR, S100B, and KROX20 and the downregulation of CAD19 and MBP at the fully differentiated stage. Furthermore, we found myelination of axons when differentiated SCs were cocultured with mouse dorsal root ganglion neurons. The application of T-MSC-SCs to a mouse model of sciatic nerve injury produced marked improvements in gait and promoted regeneration of damaged nerves. Thus, the transplantation of human T-MSCs might be suitable for assisting in peripheral nerve regeneration.

Prevention of Staphylococcus aureus infections by glycoprotein vaccines synthesized in Escherichia coli.

BACKGROUND: Staphylococcus aureus is a leading cause of superficial and invasive human disease that is often refractory to antimicrobial therapy. Vaccines have the potential to reduce the morbidity, mortality, and economic impact associated with staphylococcal infections. However, single-component vaccines targeting S. aureus have failed to show efficacy in clinical trials. METHODS: A novel glycoengineering technology for creation of a multicomponent staphylococcal vaccine is described. Genes encoding S. aureus capsular polysaccharide (CP) biosynthesis, PglB (a Campylobacter oligosaccharyl transferase), and a protein carrier (detoxified Pseudomonas aeruginosa exoprotein A or S. aureus α toxin [Hla]) were coexpressed in Escherichia coli. Recombinant proteins N-glycosylated with S. aureus serotype 5 or 8 CPs were purified from E. coli. RESULTS: Rabbits and mice immunized with the glycoprotein vaccines produced antibodies that were active in vitro in functional assays. Active and passive immunization strategies targeting the CPs protected mice against bacteremia, and vaccines targeting Hla protected against lethal pneumonia. The CP-Hla bioconjugate vaccine protected against both bacteremia and lethal pneumonia, providing broad-spectrum efficacy against staphylococcal invasive disease. CONCLUSIONS: Glycoengineering technology, whereby polysaccharide and protein antigens are enzymatically linked in a simple E. coli production system, has broad applicability for use in vaccine development against encapsulated microbial pathogens.

Antibodies to Staphylococcus aureus serotype 8 capsular polysaccharide react with and protect against serotype 5 and 8 isolates.

Most Staphylococcus aureus isolates produce either a serotype 5 (CP5) or 8 (CP8) capsular polysaccharide, and the CP antigens are targets for vaccine development. Since CP5 and CP8 have similar trisaccharide repeating units, it is important to identify an epitope shared by both CP5 and CP8. To characterize cross-reactivity between CP5 and CP8, the immunogenicity of CP5 and CP8 conjugate vaccines in mice and rabbits was evaluated by serological assays. Immune sera were also tested for functional activity by in vitro opsonophagocytic-killing assays and a murine bacteremia model. Antibodies to the CP5-cross-reactive material 197 (CRM197) conjugate vaccine bound only to purified CP5. In contrast, antibodies to the CP8-CRM conjugate vaccine reacted with CP8 and (to a lesser extent) CP5. De-O-acetylation of CP5 increased its reactivity with CP8 antibodies. Moreover, CP8 antibodies bound to Pseudomonas aeruginosa O11 lipopolysaccharide, which has a trisaccharide repeating unit similar to that of the S. aureus CPs. CP8-CRM antibodies mediated in vitro opsonophagocytic killing of S. aureus expressing CP5 or CP8, whereas CP5-CRM antibodies were serotype specific. Passive immunization with antiserum to CP5-CRM or CP8-CRM protected mice against bacteremia induced by a serotype 5 S. aureus isolate, suggesting that CP8-CRM elicits antibodies cross-reactive to CP5. The identification of epitopes shared by CP5 and CP8 may inform the rational design of a vaccine to protect against infections caused by CP5- or CP8-producing strains of S. aureus.

Therapeutic Effect of Schwann Cell-Like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells on Diabetic Neuropathy in db/db Mice.

BACKGROUND: Diabetic neuropathy (DN) is the most common complication of diabetes, and approximately 50% of patients with this disease suffer from peripheral neuropathy. Nerve fiber loss in DN occurs due to myelin defects and is characterized by symptoms of impaired nerve function. Schwann cells (SCs) are the main support cells of the peripheral nervous system and play important roles in several pathways contributing to the pathogenesis and development of DN. We previously reported that human tonsil-derived mesenchymal stem cells differentiated into SCs (TMSC-SCs), named neuronal regeneration-promoting cells (NRPCs), which cells promoted nerve regeneration in animal models with peripheral nerve injury or hereditary peripheral neuropathy. METHODS: In this study, NRPCs were injected into the thigh muscles of BKS-db/db mice, a commonly used type 2 diabetes model, and monitored for 26 weeks. Von Frey test, sensory nerve conduction study, and staining of sural nerve, hind foot pad, dorsal root ganglia (DRG) were performed after NRPCs treatment. RESULTS: Von Frey test results showed that the NRPC treatment group (NRPC group) showed faster responses to less force than the vehicle group. Additionally, remyelination of sural nerve fibers also increased in the NRPC group. After NRPCs treatment, an improvement in response to external stimuli and pain sensation was expected through increased expression of PGP9.5 in the sole and TRPV1 in the DRG. CONCLUSION: The NRPCs treatment may alleviate DN through the remyelination and the recovery of sensory neurons, could provide a better life for patients suffering from complications of this disease.

Human CD4-binding site antibody elicited by polyvalent DNA prime-protein boost vaccine neutralizes cross-clade tier-2-HIV strains.

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.

Novel synthetic (poly)glycerolphosphate-based antistaphylococcal conjugate vaccine.

Staphylococcal infections are a major source of global morbidity and mortality. Currently there exists no antistaphylococcal vaccine in clinical use. Previous animal studies suggested a possible role for purified lipoteichoic acid as a vaccine target for eliciting protective IgG to several Gram-positive pathogens. Since the highly conserved (poly)glycerolphosphate backbone of lipoteichoic acid is a major antigenic target of the humoral immune system during staphylococcal infections, we developed a synthetic method for producing glycerol phosphoramidites to create a covalent 10-mer of (poly)glycerolphosphate for potential use in a conjugate vaccine. We initially demonstrated that intact Staphylococcus aureus elicits murine CD4(+) T cell-dependent (poly)glycerolphosphate-specific IgM and IgG responses in vivo. Naive mice immunized with a covalent conjugate of (poly)glycerolphosphate and tetanus toxoid in alum plus CpG-oligodeoxynucleotides produced high secondary titers of serum (poly)glycerolphosphate-specific IgG. Sera from immunized mice enhanced opsonophagocytic killing of live Staphylococcus aureus in vitro. Mice actively immunized with the (poly)glycerolphosphate conjugate vaccine showed rapid clearance of staphylococcal bacteremia in vivo relative to mice similarly immunized with an irrelevant conjugate vaccine. In contrast to purified, natural lipoteichoic acid, the (poly)glycerolphosphate conjugate vaccine itself exhibited no detectable inflammatory activity. These data suggest that a synthetic (poly)glycerolphosphate-based conjugate vaccine will contribute to active protection against extracellular Gram-positive pathogens expressing this highly conserved backbone structure in their membrane-associated lipoteichoic acid.

Comparison of human first and third trimester placental mesenchymal stem cell.

Placenta mesenchymal stem cells (PMSCs) have the characteristic features of stem cells including renewability in vitro, surface expression, differentiation potency and ability to adhere to the culture surface. PMSCs expressed genes are normally found in the embryonic tissues before the onset of gastrulation, indicating multipotency. However, the stemness can depend on the stages of the placenta from which the cells were isolated. PMSCs were isolated from two different stages of placenta for comparison, that is the first and third trimesters. Both sets had very similar patterns of surface expression as CD44, CD73, CD90 and CD105, and of self renewability in vitro. Expressions of pluripotency-coupled genes were also confirmed in both sets of cells; however, there was a significant difference in the expression levels: fPMSC (mesenchymal stem cells isolated from the first trimester human placenta) being 2-11-fold higher than tPMSC (mesenchymal stem cells isolated from the third trimester human placenta). Possibly due to the difference in the expression levels of the pluripotency-related genes, induction of genes specific to the ectodermal tissues were more prominent in fPMSC than tPMSC after induced differentiation.

Preclinical Efficacy of Peripheral Nerve Regeneration by Schwann Cell-like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells in C22 Mice.

Charcot-Marie-Tooth disease (CMT) is a hereditary disease with heterogeneous phenotypes and genetic causes. CMT type 1A (CMT1A) is a type of disease affecting the peripheral nerves and is caused by the duplication of the peripheral myelin protein 22 (PMP22) gene. Human tonsil-derived mesenchymal stem cells (TMSCs) are useful for stem cell therapy in various diseases and can be differentiated into Schwann cell-like cells (TMSC-SCs). We investigated the potential of TMSC-SCs called neuronal regeneration-promoting cells (NRPCs) for peripheral nerve and muscle regeneration in C22 mice, a model for CMT1A. We transplanted NRPCs manufactured in a good manufacturing practice facility into the bilateral thigh muscles of C22 mice and performed behavior and nerve conduction tests and histological and ultrastructural analyses. Significantly, the motor function was much improved, the ratio of myelinated axons was increased, and the G-ratio was reduced by the transplantation of NRPCs. The sciatic nerve and gastrocnemius muscle regeneration of C22 mice following the transplantation of NRPCs downregulated PMP22 overexpression, which was observed in a dose-dependent manner. These results suggest that NRPCs are feasible for clinical research for the treatment of CMT1A patients. Research applying NRPCs to other peripheral nerve diseases is also needed.

Human CD4-Binding Site Antibody Elicited by Polyvalent DNA Prime-Protein Boost Vaccine Neutralizes Cross-Clade Tier-2-HIV Strains.

The vaccine elicitation of HIV-neutralizing antibodies with tier-2-neutralization breadth has been a challenge. Here, we report the isolation and characteristics of a CD4-binding site specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent gp120 DNA prime-protein boost vaccine. HmAb64 derived from heavy chain variable germline gene IGHV1-18, light chain germline gene IGKV1-39, and had a 3rd heavy chain complementarity determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 21 (10%), including tier-2 neutralization resistant strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 bound to a conformation between prefusion closed and occluded open forms of envelope trimer, using both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4-binding site. A gp120 subunit-based vaccine can thus elicit an antibody capable of tier 2-HIV neutralization.

Transplantation of Differentiated Tonsil-Derived Mesenchymal Stem Cells Ameliorates Murine Duchenne Muscular Dystrophy via Autophagy Activation.

BACKGROUND: Skeletal muscles play many important roles in the human body and any malfunction or disorder of the skeletal muscles can lead to a reduced quality of life. Some skeletal dysfunctions are acquired, such as sarcopenia but others are congenital. Duchenne muscular dystrophy (DMD) is one of the most common forms of hereditary muscular dystrophy and is caused by a deficiency of the protein, Dystrophin. Currently, there is no clear treatment for DMD, there are only methods that can alleviate the symptoms of the disease. Mesenchymal stem cells, including tonsil-derived mesenchymal stem cells (TMSCs) have been shown to differentiate into skeletal muscle cells (TMSC-myocyte) and can be one of the resources for the treatment of DMD. Skeletal muscle cell characteristics of TMSC-myocytes have been confirmed through changes in morphology and expression of skeletal muscle markers such as Myogenin, Myf6, and MYH families after differentiation. MEOTHDS: Based on these characteristics, TMSC-myocytes have been transplanted into mdx mice, a mouse model of DMD, to investigate whether they can help improve the symptoms of DMD. The red fluorescent protein gene was transduced into TMSC (TMSC-R) for tracking transplanted cells. RESULTS: Prior to transplantation (TP), it was confirmed whether TMSC-R-myocytes had the same differentiation potential as TMSC-myocytes. Increased expression of dystrophin and autophagy markers in the TP group compared with the sham group was confirmed in the gastrocnemius muscle 12 weeks after TP. CONCLUSION: These results demonstrate muscle regeneration and functional recovery of mdx via autophagy activation following TMSC-myocyte TP.

Older adults have a low capacity to opsonize pneumococci due to low IgM antibody response to pneumococcal vaccinations.

Since the 23-valent pneumococcal polysaccharide vaccine (PPV23) is less effective for older adults than for young adults, it is important to investigate the immunologic basis for the reduced efficacy of PPV23 among older adults. We determined the effectiveness of PPV23 among young (n = 55) and older (n = 44) adults by measuring the serum IgG, IgM, and IgA concentrations and opsonic capacities against serotypes 14, 18C, and 23F. While young and older adults showed no difference in levels of IgG antibodies against pneumococcal polysaccharide (PPS), older adults had lower IgA and IgM antibody levels than young adults for all three serotypes. In both age groups, anti-PPS IgA or IgM antibody levels were much lower than anti-PPS IgG antibody levels. Young adults showed higher opsonic capacities than older adults for serotypes 14 and 23F. In order to determine the effects of anti-PPS IgA or IgM antibodies on the functional difference between young and older adults, anti-PPS IgA or IgM antibodies were removed from immune sera by affinity chromatography. The difference in opsonic capacity between young and older adults disappeared for serotypes 14 and 23F (but not for serotype 18C) when IgM antibody was removed. However, there was no significant difference between the two age groups when IgA antibody was removed. In conclusion, even though anti-PPS IgG antibody levels are high compared with anti-PPS IgM antibody levels, the low levels of anti-PPS IgM antibody alone can explain the functional difference observed between young and older adults immunized with PPV23 with regard to some pneumococcal serotypes.

A novel histone deacetylase 6 inhibitor improves myelination of Schwann cells in a model of Charcot-Marie-Tooth disease type 1A.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth (CMT) disease is the most common hereditary peripheral neuropathy. CMT type 1A (CMT1A) accounts for approximately 50% of CMT patients and is linked to PMP22 gene duplication. Histone deacetylase-6 (HDAC6) has pleiotropic effects, such as regulating lipid homeostasis and cellular stress. Although HDAC6 has been regarded as a promising drug target for neurodegenerative diseases, its inhibition has not yet been tested in CMT1A. Here we have tested the therapeutic potential of CKD-504, a clinical stage HDAC6 inhibitor, in a mouse model of CMT1A EXPERIMENTAL APPROACH: The potency and selectivity of CKD-504 was evaluated, using a HDAC enzyme panel assay and western blots. The therapeutic potential of CKD-504 was evaluated using behavioural testing and electrophysiological assessments in the C22 mouse model of CMT1A. PMP22 protein expression and aggregation were analysed in mesenchymal stem cell-derived Schwann cells from CMT1A patients and sciatic nerves from C22 mice. KEY RESULTS: The HDAC6 inhibitor, CKD-504, modulated molecular chaperon proteins such as HSP90 and HSP70, which are involved in the folding/refolding of proteins such as PMP22. CKD-504 treatment restored myelination in both mesenchymal stem cell-derived Schwann cells from CMT1A patients and sciatic nerves of C22 mice and improved the axonal integrity of the sciatic nerve, leading to behavioural, electrophysiological, and histological improvements in C22 mice. CONCLUSION AND IMPLICATIONS: A novel HDAC6 inhibitor, CKD-504, has potent therapeutic efficacy for CMT1A.

Identification of a simple chemical structure associated with protective human antibodies against multiple pneumococcal serogroups.

Streptococcus pneumoniae, a major human pathogen, expresses at least 91 serologically distinct carbohydrate capsules. Since pneumococcal vaccines are designed to elicit antibodies against many different capsular polysaccharides (PSs), it is important to identify the epitopes involved in eliciting anti-capsular PS antibodies. We investigated the epitopes recognized by Dob1, which is a hybridoma-secreting human immunoglobulin G2 antibody to the PS of serotype 6B (Y. Sun et al., Infect. Immun. 67:1172-1179, 1999). We found that Dob1 bound synthetic capsular carbohydrates Gal(1-->3)alpha-d-Glcp(1-->3)alpha-l-Rhap(1-->3)Rib-ol and alpha-d-Glcp(1-->3)alpha-l-Rhap(1-->3)Rib-ol but did not bind alpha-l-Rhap(1-->3)Rib-ol. The critical epitope alpha-d-Glcp(1-->3)alpha-l-Rhap is found in the capsular PSs of serotypes 6A, 6B, 6C, and 19A but not in the 19F PS. Consistent with this observation, Dob1 bound to the PSs of serotypes 6A, 6B, 6C, and 19A but did not bind the 19F PS and 23 additional unrelated pneumococcal capsular PSs. Also, Dob1 could opsonize pneumococci expressing serotypes 6A, 6B, 6C, and 19A but did not opsonize 19F pneumococci. In addition, ca. 7% of immune sera (12 of 175 sera) had significant amounts of Dob1-like antibodies, i.e., reacted with 6B and 19A PSs, but not with 19F PS. Humans can produce antibodies to the Dob1 epitope and the antibodies to that epitope cross-react with the four serotypes 6A, 6B, 6C, and 19A that belong to different serogroups. This epitope may be useful for producing a totally synthetic, simple chemical structure that is capable of generating protective antibodies to multiple pneumococcal serogroups.