Characteristics of IgG subclasses and complement deposition in BP230-type bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is the most common autoimmune blistering disease. BP180 is the primary autoantigen of BP, and in a portion of BP cases, BP230 is the only target of autoantibodies. Such BP is called BP230-type BP. BP230-type BP tends to show milder clinical phenotypes than conventional BP, but the reason is unclear. The pathogenic roles of autoantibodies and complement activation have been shown in conventional BP, but the distribution of IgG subclasses and the degree of complement deposition in BP230-type BP remain unclear. OBJECTIVE: To compare the distribution of IgG subclasses and the degree of complement deposition in BP230-type BP with those in conventional BP with autoantibodies to BP180 and BP230 (BP180-BP230-type BP). METHODS: The diagnosis of BP was confirmed by the histopathology of the lesions, the deposition of IgG and complement in the perilesional skin and the presence of circulating autoantibodies to BP180 and BP230. The disease severity was determined by bullous pemphigoid disease area index. The deposition of IgG subclasses and complement deposition were examined by direct immunofluorescence of the perilesional skin in 6 BP230-type BP cases and 11 BP180-BP230-type BP cases. RESULTS: Sixty seven percent of BP230-type BP cases show a mild clinical phenotype. All BP230-type BP cases and 82% of BP180-BP230-type BP cases were found to demonstrate the clear deposition of IgG4 at the basement membrane zone of skin specimens. Notably, the deposition of IgG1 and IgG3 was faint or negative in all of the BP230-type BP cases, whereas they were clearly detected in 91% and 64% of the BP180-BP230-type BP cases, respectively. The deposition of complement C3 tended to be weaker in BP230-type BP than in BP180-BP230-type BP. CONCLUSION: The mild clinical phenotype of BP230-type BP may correlate with the weaker deposition of IgG1, IgG3 and complement in the skin lesions.

Clinical and mutational spectrum of Japanese patients with Charcot-Marie-Tooth disease caused by GDAP1 variants.

BACKGROUND: Mutations in GDAP1 are responsible for heterogeneous clinical and electrophysiological phenotypes of Charcot-Marie-Tooth disease (CMT), with autosomal dominant or recessive inheritance pattern. The aim of this study is to identify the clinical and mutational spectrum of CMT patients with GDAP1 variants in Japan. MATERIALS AND METHODS: From April 2007 to October 2014, using three state-of-art technologies, we conducted gene panel sequencing in a cohort of 1,030 patients with inherited peripheral neuropathies (IPNs), and 398 mutation-negative cases were further analyzed with whole-exome sequencing. RESULTS: We identified GDAP1 variants from 10 patients clinically diagnosed with CMT. The most frequent recessive variant in our cohort (5/10), c.740C>T (p.A247V), was verified to be associated with a founder event. We also detected three novel likely pathogenic variants: c.928C>T (p.R310W) and c.546delA (p.E183Kfs*23) in Case 2 and c.376G>A (p.E126K) in Case 8. Nerve conduction study or sural nerve biopsy of all 10 patients indicated axonal type peripheral neuropathy. CONCLUSION: We identified GDAP1 variants in approximately 1% of our cohort with IPNs, and established a founder mutation in half of these patients. Our study originally described the mutational spectrum and clinical features of GDAP1-related CMT patients in Japan.

The Beneficial Effect of Carboxylated Poly-L-Lysine on Cryosurvival of Vitrified Early Stage Embryos.

BACKGROUND: In the vitrification of embryos, dimethyl sulfoxide (DMSO) is one of the most effective cryoprotectant agents (CPAs), but cytotoxic effects of DMSO on embryos are well known. Carboxylated poly-L-lysine (CPLL) has been identified as an effective cryoprotectant of cultured cell lines and mammalian oocytes. OBJECTIVE: To evaluate the efficacy and safety of CPLL as a CPA for developmental stage embryos. MATERIALS AND METHODS: Mouse 8-cell embryos and blastocysts were vitrified with ethylene glycol (EG), DMSO/EG, or CPLL/EG and the developmental potency assessed in vitro. RESULTS: In 8-cell embryos, there were no differences between the levels of survival and developmental progress into the blastocyst stage in each solution. At the blastocyst stage, the proportion of dead cells was significantly higher in the EG compared with other solutions. In contrast, there were no differences between the DMSO/EG and CPLL/EG. CONCLUSION: These results indicate that CPLL can be used as a replacement for DMSO in the vitrification of mouse embryos.

Sudden onset of sleep due to hypothalamic lesions in neuromyelitis optica spectrum disorder positive for anti-aquaporin-4 antibody.

We report a patient with neuromyelitis optica spectrum disorders who presented with sudden onset of sleep as the sole manifestation. Magnetic resonance imaging investigation revealed lesions in the hypothalamus bilaterally, which vanished completely after methylprednisolone pulse therapy.

TAP-deficient human iPS cell-derived myeloid cell lines as unlimited cell source for dendritic cell-like antigen-presenting cells.

We previously reported a method to generate dendritic cell (DC)-like antigen-presenting cells (APC) from human induced pluripotent stem (iPS) cells. However, the method is relatively complicated and laborious. In the current study, we attempted to establish a method through which we could obtain a large number of functional APC with a simple procedure. We transduced iPS cell-derived CD11b(+) myeloid cells with genes associated with proliferative or anti-senescence effects, enabling the cells to propagate for more than 4 months in a macrophage colony-stimulating factor (M-CSF)-dependent manner while retaining their capacity to differentiate into functional APC. We named these iPS cell-derived proliferating myeloid cells 'iPS-ML', and the iPS-ML-derived APC 'ML-DC'. In addition, we generated TAP2-deficient iPS cell clones by zinc finger nuclease-aided targeted gene disruption. TAP2-deficient iPS cells and iPS-ML avoided recognition by pre-activated allo-reactive CD8(+) T cells. TAP2-deficient ML-DC expressing exogenously introduced HLA-A2 genes stimulated HLA-A2-restricted MART-1-specific CD8(+) T cells obtained from HLA-A2-positive allogeneic donors, resulting in generation of MART-1-specific cytotoxic T lymphocyte (CTL) lines. TAP-deficient iPS-ML introduced with various HLA class I genes may serve as an unlimited source of APC for vaccination therapy. If administered into allogeneic patients, ML-DC with appropriate genetic modifications may survive long enough to stimulate antigen-specific CTL and, after that, be completely eliminated. Based on the present study, we propose an APC-producing system that is simple, safe and applicable to all patients irrespective of their HLA types.

Cryoprotection of fibroblasts by carboxylated poly-L-lysine upon repeated freeze/thaw cycles.

The cryoprotection of carboxylated h-poly-L-lysine (COOH-PLL) was investigated on fibroblasts [L-929 cells and human dermal fibroblasts (HDFs)] during multiple freeze/thaw cycles. COOH-PLL was not toxic to two fibroblast cell types even at 25% (w/v) concentration, whereas dimethylsulphoxide (DMSO) was highly toxic over 3.13% (v/v). When L-929 cells were subjected to 5 freeze/thaw cycles, the media containing 7.5% (w/v) COOH-PLL maintained cell morphology and significantly suppressed growth inhibition as well as cell detachment (P < 0.05). The result was comparable to the media containing 10% (v/v) DMSO. For HDFs, COOH-PLL could effectively retain cell viability and proliferation against 3 freeze/thaw cycles. Cell viability of HDFs was decreased after 5 freeze/thaw cycles, but COOH-PLL exerted better cryoprotection. The cell type might account for the difference in the observations. The data demonstrated that COOH-PLL is a good cryoprotectant for mammalian cells against repeated freeze/thaw cycles, and may be used for cell preservation in fields of cell transplantation, tissue engineering and regenerative medicine.

Selective localization of T helper subsets in labial salivary glands from primary Sjögren's syndrome patients.

The aim of this study was to investigate the initiation and progression of autoimmune damage in the lesions of labial salivary glands (LSGs) from primary Sjögren's syndrome (SS) patients by examining the selective localization of T helper (Th) subsets such as Th1, Th2, Th17 regulatory T cells (T(regs)) and follicular T helper cells (Tfh). The expression of cytokines and transcription factors associated with these Th subsets in the LSGs from 54 SS patients and 16 healthy controls was examined using real-time polymerase chain reaction (PCR) and immunostaining. Additionally, infiltrating lymphocytes without germinal centre (GC(-)) and with GC (GC(+)) in the LSGs specimens from eight SS patients were extracted selectively by laser capture microdissection (LCM). The mRNA expression of these molecules was compared between the two sample groups of GC(-) and GC(+) by real-time PCR. The mRNA expression of cytokines and transcription factors of all T helper (Th) subsets in the LSGs from the SS patients was increased significantly in comparison with controls. In LSGs from the SS patients, Th2 and Tfh was associated closely with strong lymphocytic infiltration; however, Th1, Th17 and T(regs) was not. In the selectively extracted lesions of LSGs, Th1 and Th17-related molecules were detected strongly in the GC(-), while Th2 and Tfh-related molecules were detected in the GC(+). In contrast, no significant association with strong lymphocytic infiltration was observed in T(reg)-related molecules. These results indicate that SS has selective localization of Th subsets such as Th1, Th2, Th17 and Tfh in the LSGs, which is associated closely with disease severity and/or status. SS might be initiated by Th1 and Th17 cells, and then progressed by Th2 and Tfh cells via GC formation.

Generation of dendritic cells and macrophages from human induced pluripotent stem cells aiming at cell therapy.

This report describes generation of dendritic cells (DCs) and macrophages from human induced pluripotent stem (iPS) cells. iPS cell-derived DC (iPS-DC) exhibited the morphology of typical DC and function of T-cell stimulation and antigen presentation. iPS-DC loaded with cytomegalovirus (CMV) peptide induced vigorous expansion of CMV-specific autologous CD8+ T cells. Macrophages (iPS-MP) with activity of zymosan phagocytosis and C5a-induced chemotaxis were also generated from iPS cells. Genetically modified iPS-MPs were generated by the introduction of expression vectors into undifferentiated iPS cells, isolation of transfectant iPS cell clone and subsequent differentiation. By this procedure, we generated iPS-MP expressing a membrane-bound form of single chain antibody (scFv) specific to amyloid ß (Aß), the causal protein of Alzheimer's disease. The scFv-transfectant iPS-MP exhibited efficient Aß-specific phagocytosis activity. iPS-MP expressing CD20-specific scFv engulfed and killed BALL-1 B-cell leukemia cells. Anti-BALL-1 effect of iPS-MP in vivo was demonstrated in a xeno-transplantation model using severe combined immunodeficient mice. In addition, we established a xeno-free culture protocol to generate iPS-DC and iPS-MP. Collectively, we demonstrated the possibility of application of iPS-DC and macrophages to cell therapy.

Deletion of fcgamma receptor IIB renders H-2(b) mice susceptible to collagen-induced arthritis.

Autoimmune diseases, like rheumatoid arthritis, result from a dysregulation of the immune response culminating in hyperactivation of effector cells leading to immune-mediated injury. To maintain an appropriate immune response and prevent the emergence of autoimmune disease, activation signals must be regulated by inhibitory pathways. Biochemical and genetic studies indicate that the type IIB low-affinity receptor for immunoglobulin (Ig)G (FcgammaRIIB) inhibits cellular activation triggered through antibody or immune complexes and may be an important component in preventing the emergence of autoimmunity. To investigate the role of FcgammaRIIB in the development of type II collagen (CII)-induced arthritis (CIA), a model for rheumatoid arthritis in humans, we have examined its contribution in determining the susceptibility to CIA in the nonpermissive H-2(b) haplotype. H-2(b) mice immunized with bovine CII do not develop appreciable disease. In contrast, immunization of the FcgammaRIIB-deficient, H-2(b) mice with bovine CII induced CIA at an incidence of 42.2%. The maximal arthritis index of the FcgammaRIIB-deficient mice developing CIA (6.9 +/- 3.6) was comparable to that of DBA/1 mice (8.6 +/- 1.9), an H-2(q) strain susceptible for CIA induction. IgG1, IgG2a, and IgG2b antibody responses against CII were elevated in the FcgammaRIIB-deficient animals, especially in those mice showing arthritis, but less pronounced than DBA/1 mice. Histological examinations of the arthritic paws from FcgammaRIIB-deficient mice revealed that cartilage was destroyed and bone was focally eroded in association with marked lymphocyte and monocyte/macrophage infiltration, very similar to the pathologic findings observed in DBA/1 mice. These results indicate that a nonpermissive H-2(b) haplotype can be rendered permissive to CIA induction through deletion of FcgammaRIIB, suggesting that FcgammaRIIB plays a critical role in suppressing the induction of CIA.

Antifouling activity of synthetic polymer gels against cyprids of the barnacle (Balanus amphitrite) in vitro.

Barnacle (Balanus amphitrite) settlement on synthetic hydrogels with various chemical structures was tested in laboratory assays. The results demonstrated that cyprids settle less or not at all on hydrogels and PDMS elastomer compared with the polystyrene control. The low settlement on gels is most likely due to the 'easy release' of initially attached cyprids from the gel surfaces. This low adhesion of cyprids is independent of surface hydrophilicity or hydrophobicity, and of surface charge. The results also revealed that hydrogels can be categorized into two groups. One group showed an extremely strong antifouling (AF) performance that was independent of the elasticity (E) or swelling degree (q) of the gels. The second group showed relatively less strong AF performance that was E- or q-dependent. In the latter case, E, rather than the q, may be the more important factor for cyprid settlement.

Dystrophin-related protein is localized to neuromuscular junctions of adult skeletal muscle.

Dystrophin-related protein (DRP) is an autosomal gene product with high homology to dystrophin. We have used highly specific antibodies to the unique C-terminal peptide sequences of DRP and dystrophin to examine the subcellular localization and biochemical properties of DRP in adult skeletal muscle. DRP is enriched in isolated sarcolemma from control and mdx mouse muscle, but is much less abundant than dystrophin. Immunofluorescence microscopy localized DRP almost exclusively to the neuromuscular junction region in rabbit and mouse skeletal muscle, as well as mdx mouse muscle and denervated mouse muscle. DRP is also present in normal size and abundance and localizes to the neuromuscular junction region in muscle from the dystrophic mouse model dy/dy. Thus, DRP is a junction-specific membrane cytoskeletal protein that may play an important role in the organization of the postsynaptic membrane of the neuromuscular junction.

Persistent lymphoedema in Morbihan disease: formation of perilymphatic epithelioid cell granulomas as a possible pathogenesis.

Morbihan disease is a rare complication of rosacea, characterized by persistent lymphoedema on the upper half of the face, occurring during the chronic clinical course of rosacea. This refractory condition has been also designated as 'rosacea lymphoedema' and 'solid persistent facial oedema of rosacea'. We report a patient with Morbihan disease showing persistent lymphoedema on the upper half of the face accompanied by unique histological findings of striking dermal dilated lymphatics and damage of the lymphatics at the site of the adjacent epithelioid cell granulomas, with histiocytes bulging into the lymphatic lumen. The marked epithelioid cell granulomas forming around dermal lymphatic vessels with subsequent lymphatic damage and luminal obstruction by histiocytic infiltration may account for the development of lymphoedema in this patient.

Routine Referral by Urologists Increase Opportunities for Corneal Donation.

BACKGROUND: The levels of corneal donation are insufficient to meet the demand for corneal transplantation in Japan. To overcome this problem, we started to routinely mention the possibility of corneal donation to the families of patients who died in our hospital's Urology Department in February 2008. In this study, we evaluated the effectiveness of this approach. METHODS: We retrospectively reviewed the medical records of the patients who died in the Department of Urology, St. Marianna University School of Medicine Hospital, and analyzed the patients' characteristics and information about corneal donation. RESULTS: In total, 211 patients died in our department between February 2008 and March 2017, and 155 patients were medically suitable corneal donors. We mentioned the possibility of corneal donation to 129 (83.2%) families, and 29 (18.7%) families agreed. Three families subsequently withdrew their consent. Finally, 26 (16.8%) of the families that were approached about corneal donation by urologists agreed to donate their relatives' corneas. Another 2 families voluntarily offered to donate their relatives' corneas. Thus, 28 (18.1%) of 155 medically suitable donors donated their corneas for transplantation. Twenty-six (92.8%) donors were 60 years or older and all donors were affected with malignant genitourinary tumors. Fifty-four (96.4%) corneas were successfully transplanted into recipients. CONCLUSIONS: Even elderly patients who die of solid carcinoma can be an important source of corneal donors. In this study, we showed that routine referral by urologists increased corneal donation. If this approach were adopted by other departments, it might further increase the number of corneal donations.

Page Kidney Following a Nonepisode Protocol Renal Allograft Biopsy: A Case Report.

Page kidney refers to a clinical condition that is characterized by the acute onset of hypertension and renal dysfunction owing to external compression of the kidney by a hematoma, tumor, lymphocele, or urinoma. We report a case in which Page kidney occurred after a nonepisode protocol renal allograft biopsy. A 31-year-old man with end-stage renal disease received a living related kidney transplant from his father. One year later, a nonepisode protocol renal allograft biopsy was performed. A day later, the patient's serum creatinine level increased to 4.23 mg/dL, and a subcapsular renal hematoma was detected using ultrasonography and computed tomography. Page kidney was diagnosed, and immediate surgical removal of the hematoma was performed. Nine days after the operation, the patient's serum creatinine level had improved to 1.89 mg/dL. Page kidney is a serious but treatable complication of renal allograft biopsies, and clinicians should pay attention to such complications, even in the setting of nonepisode protocol renal allograft biopsies.

Deficiency of dystrophin-associated proteins in Duchenne muscular dystrophy patients lacking COOH-terminal domains of dystrophin.

Dystrophin, the protein product of the Duchenne muscular dystrophy (DMD) gene, is a cytoskeletal protein tightly associated with a large oligomeric complex of sarcolemmal glycoproteins including dystroglycan, which provides a linkage to the extracellular matrix component, laminin. In DMD, the absence of dystrophin leads to a drastic reduction in all of the dystrophin-associated proteins, causing the disruption of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix which, in turn, may render muscle cells susceptible to necrosis. The COOH-terminal domains (cysteine-rich and carboxyl-terminal) of dystrophin have been suggested to interact with the sarcolemmal glycoprotein complex. However, truncated dystrophin lacking these domains was reported to be localized to the sarcolemma in four DMD patients recently. Here we report that all of the dystrophin-associated proteins are drastically reduced in the sarcolemma of three DMD patients in whom dystrophin lacking the COOH-terminal domains was properly localized to the sarcolemma. Our results indicate that the COOH-terminal domains of dystrophin are required for the proper interaction of dystrophin with the dystrophin-associated proteins and also support our hypothesis that the loss of the dystrophin-associated proteins in the sarcolemma leads to severe muscular dystrophy even when truncated dystrophin is present in the subsarcolemmal cytoskeleton.

Immunohistochemical analysis of dystrophin-associated proteins in Becker/Duchenne muscular dystrophy with huge in-frame deletions in the NH2-terminal and rod domains of dystrophin.

The absence of dystrophin causes the drastic reduction of the dystrophin-associated proteins (DAPs) in the sarcolemma and the loss of the linkage between the subsarcolemmal cytoskeleton and the extracellular matrix in Duchenne muscular dystrophy (DMD) skeletal muscle. Here, we report a mild reduction of the DAPs in the unique Becker muscular dystrophy patients with huge deletions in the rod domain of dystrophin and a moderate reduction of the DAPs in patients with huge deletions that involve both the NH2-terminal and rod domains of dystrophin. The phenotype of the latter patients was more severe than that of the former. In both cases, however, the reduction in the DAPs was milder than in typical DMD patients or DMD patients lacking the COOH-terminal domains of dystrophin. Our results suggest that (a) the NH2-terminal and rod domains of dystrophin may not be essential for the interaction with the sarcolemmal glycoprotein complex; and (b) defects in the actin binding activity of dystrophin may cause disruption of the anchorage of the dystrophin-glycoprotein complex to the subsarcolemmal cytoskeleton, which may render muscle fibers susceptible to degeneration.

Interleukin-6 is a candidate molecule that transmits inflammatory information to the CNS.

Peripheral inflammation induces reactions within the CNS such as central sensitization, which is involved in the mechanism of inflammatory hyperalgesia. However, the precise mechanism of inflammatory signal transmission from the peripheral inflammatory site to the CNS is not clear. We studied the role of circulating interleukin (IL)-6 as a messenger of inflammatory information from the periphery to the CNS. In the rat model of inflammatory hyperalgesia induced by carrageenan, levels of IL-6 but not IL-1beta or tumor necrosis factor alpha (TNFalpha) were significantly elevated in the circulating blood 3 h after an injection of carrageenan. In addition, injecting carrageenan into the hind paw evoked thermal hyperalgesia and the release of prostaglandin E(2) (PGE(2)) from isolated blood vessels of the CNS ex vivo, as well as the induction of cyclooxygenase (COX)-2 and microsomal prostaglandin E synthase (mPGES)-1 and nuclear translocation of signal transducer and activator of transcription 3 (STAT3) in vascular endothelial cells of the CNS. A prior i.p. injection of IL-6 antiserum (IL-6AS) abolished or attenuated these responses. The present results suggested that circulating IL-6 could act as a messenger of inflammatory information from peripheral inflammatory sites to the CNS and as the afferent circulating signal to the CNS to produce prostaglandins in the vascular endothelial cells of the CNS through a COX-2 dependent pathway.