Juryun Kim

Metformin downregulates Th17 cells differentiation and attenuates murine autoimmune arthritis.

INTRODUCTION This study was undertaken to determine whether metformin has anti-inflammatory effects in the collagen antibody-induced arthritis (CAIA) murine model. The effect of metformin on Th17 cell differentiation was also investigated. METHODS CAIA mice were treated with 100 and 150 mg/kg i.p. metformin (low- and high-dose groups, respectively). Arthritis activity and histological joint destruction were studied. Flow cytometry was used to (i) determine RORγt-expressing CD4+ percentages in draining axillary lymph nodes (ALNs) from metformin-treated and untreated mice with CAIA, (ii) determine Th17 percentages in splenic CD4+ T cells cultured ex vivo for 3 days in Th17-differentiation-inducing conditions, and (iii) determine the percentages of RORγt+CD4+ T cells when normal splenic T cells from DBA/1 mice were cultured in Th17-differentiation-inducing conditions together with various metformin doses. Western blot analysis was used to assess the intracellular signaling of the metformin-treated splenocytes. RESULTS Metformin attenuated both arthritis scores and bone destruction in CAIA mice, decreased the serum levels of the pro-inflammatory cytokines, TNF-α and IL-1, and reduced the number of RORγt+CD4+ T cells in the ALNs. Splenocytes from metformin-treated CAIA mice differentiated less readily into Th17 cells upon ex vivo stimulation. Metformin treatment of normal cells cultured in Th17-differentiation-inducing conditions decreased the number of RORγt-expressing CD4+ cells in a dose-dependent manner and downregulated STAT3 phosphorylation via the AMPK pathway. CONCLUSIONS Metformin had an anti-inflammatory effect on murine autoimmune arthritis due to the inhibition of Th17 cell differentiation. Metformin may have a possible therapeutic value for treatment of rheumatoid arthritis.

Temporal differential effects of proinflammatory cytokines on osteoclastogenesis

Bone destruction and inflammation are closely linked. Cytokines play an important role in inflammatory bone destruction by upregulating the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL). The direct role of cytokines that act in a non-RANKL-dependent manner has yet to be elucidated. The aim of this study was to investigate the direct osteoclastogenic properties of inflammatory cytokines at different time-points of osteoclastogenesis. Mouse bone marrow macrophages were stimulated with the macrophage colony-stimulating factor (M-CSF) and various concentrations of RANKL. Inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-17 and IL-23, were added to the culture system of osteoclastogenesis. Two time-points of cytokine treatment were set. The ‘early’ effect of each cytokine was investigated at the time of first RANKL treatment, whereas the ‘late’ effect was investigated 48 h after the first RANKL challenge. Osteoclast differentiation and function were assessed using an osteoclast marker [tartrate-resistant acid phosphatase (TRAP)] and by visualization of pit formation. A permissive level of RANKL was required for cytokine-associated osteoclastogenesis in all experiments. In the M-CSF/RANKL monocellular culture system, IL-1β enhanced and IL-6 decreased osteoclast formation in a dose-dependent manner, regardless of temporal differences. Other cytokines showed various responses according to the phase of osteoclast maturation and the concentration of each cytokine and RANKL. Furthermore, luciferase assays showed that both IL-1β and RANKL activated the NF-κB signaling pathway. Collectively, our data revealed that targeting IL-1β may be a promising strategy to inhibit inflammation-associated bone destruction and osteoporosis.

Generation of disease-specific induced pluripotent stem cells from patients with rheumatoid arthritis and osteoarthritis

IntroductionSince the concept of reprogramming mature somatic cells to generate induced pluripotent stem cells (iPSCs) was demonstrated in 2006, iPSCs have become a potential substitute for embryonic stem cells (ESCs) given their pluripotency and “stemness” characteristics, which resemble those of ESCs. We investigated to reprogram fibroblast-like synoviocytes (FLSs) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) to generate iPSCs using a 4-in-1 lentiviral vector system.MethodsA 4-in-1 lentiviral vector containing Oct4, Sox2, Klf4, and c-Myc was transduced into RA and OA FLSs isolated from the synovia of two RA patients and two OA patients. Immunohistochemical staining and real-time PCR studies were performed to demonstrate the pluripotency of iPSCs. Chromosomal abnormalities were determined based on the karyotype. SCID-beige mice were injected with iPSCs and sacrificed to test for teratoma formation.ResultsAfter 14 days of transduction using the 4-in-1 lentiviral vector, RA FLSs and OA FLSs were transformed into spherical shapes that resembled embryonic stem cell colonies. Colonies were picked and cultivated on matrigel plates to produce iPSC lines. Real-time PCR of RA and OA iPSCs detected positive markers of pluripotency. Immunohistochemical staining tests with Nanog, Oct4, Sox2, Tra-1-80, Tra-1-60, and SSEA-4 were also positive. Teratomas that comprised three compartments of ectoderm, mesoderm, and endoderm were formed at the injection sites of iPSCs. Established iPSCs were shown to be compatible by karyotyping. Finally, we confirmed that the patient-derived iPSCs were able to differentiate into osteoblast, which was shown by an osteoimage mineralization assay.ConclusionFLSs derived from RA and OA could be cell resources for iPSC reprogramming. Disease- and patient-specific iPSCs have the potential to be applied in clinical settings as source materials for molecular diagnosis and regenerative therapy.

The Effects of Antihypertensive Drugs on Bone Mineral Density in Ovariectomized Mice

The effects of several antihypertensive drugs on bone mineral density (BMD) and micro-architectural changes in ovariectomized (OVX) mice were investigated. Eight-week-old female C57/BL6 mice were used for this study. Three days after ovariectomy, mice were treated intraperitoneally with nifedipine (15 mg/kg), telmisartan (5 mg/kg), enalapril (20 mg/kg), propranolol (1 mg/kg) or hydrochlorothiazide (12.5 mg/kg) for 35 consecutive days. Uterine atrophy of all mice was confirmed to evaluate estrogen deficiency state. BMD and micro-architectural analyses were performed on tibial proximal ends by micro-computed tomography (micro-CT). When OVX mice with uterine atrophy were compared with mice without atrophy, BMD decreased (P < 0.001). There were significant differences in BMD loss between different antihypertensive drugs (P = 0.005). Enalapril and propranolol increased BMD loss in mice with atrophied uteri compared with control mice. By contrast, thiazide increased BMD in mice with uterine atrophy compared with vehicle-treated mice (P = 0.048). Thiazide (P = 0.032) and telmisartan (P = 0.051) reduced bone loss and bone fraction in mice with uterine atrophy compared with the control. Thiazide affects BMD in OVX mice positively. The reduction in bone loss by thiazide and telmisartan suggest that these drugs may benefit menopausal women with hypertension and osteoporosis.

The antimicrobial peptide human cationic antimicrobial protein‐18/cathelicidin LL‐37 as a putative growth factor for malignant melanoma

Background  Recent evidence suggests cathelicidin LL‐37 to be a growth factor for various human cancers such as lung cancer, ovarian cancer and breast cancer. However, the effect of LL‐37 against malignant skin cancer has not been reported.

A Dual Target-directed Agent against Interleukin-6 Receptor and Tumor Necrosis Factor α ameliorates experimental arthritis.

A considerable proportion of patients with rheumatoid arthritis (RA) do not respond to monospecific agents. The purpose of our study was to generate a hybrid form of biologics, targeting tumor-necrosis factor alpha (TNFα) and interleukin-6 receptor (IL-6R), and determine its anti-arthritic properties in vitro and in vivo. A novel dual target-directed agent (DTA(A7/sTNFR2)) was generated by conjugating soluble TNF receptor 2 (sTNFR2) to the Fc region of A7, a new anti-IL-6R antibody obtained by screening the phage display human antibody library. DTA(A7/sTNFR2) inhibited the proliferation and migration of fibroblast-like synoviocytes from patients with RA (RA-FLS) more efficiently than single target-directed agents. DTA(A7/sTNFR2) also blocked osteoclastogenesis from bone marrow cells. The arthritis severity scores of the experimental arthritis mice with DTA(A7/sTNFR2) tended to be lower than those of mice with IgG, A7, or sTNFR2. Histological data suggested that DTA(A7/sTNFR2) is more efficient than single-target drugs in preventing joint destruction and bone loss. These results were confirmed in vivo using the minicircle system. Taken together, the results show that DTA(A7/sTNFR2) may be a promising therapeutic agent for the treatment of RA.

Immunogenicity of anti-tumour necrosis factor therapy in Korean patients with rheumatoid arthritis and ankylosing spondylitis

The aim of this study was to investigate the prevalence of antidrug antibodies (ADAs) against tumour necrosis factor (TNF) inhibitors in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). ADAs were detected in 18 (9.8%) patients with RA and in 18 (10.2%) patients with AS of the 360 patients. Development of ADAs was significantly associated with treatment failure in RA patients (P=0.003). When classified by drugs, the prevalence of immunogenicity in descending order was 17 (28.8%) patients treated with infliximab, 17 (10.4%) with adalimumab, and 2 (1.4%) with etanercept. After adjustment for disease and duration of anti-TNF therapy, the odds ratio as a reference of adalimumab-treated patients was 9.159 (95% confidence interval [CI] 2.005-41.845) for infliximab and 0.280 (95% CI 0.128-0.611) for etanercept. The immunogenicity of anti-TNF therapy was highest in the infliximab-treated group and significantly lower in the etanercept-treated group.

A New Strategy to Deliver Synthetic Protein Drugs: Self-reproducible Biologics Using Minicircles

Biologics are the most successful drugs used in anticytokine therapy. However, they remain partially unsuccessful because of the elevated cost of their synthesis and purification. Development of novel biologics has also been hampered by the high cost. Biologics are made of protein components; thus, theoretically, they can be produced in vivo. Here we tried to invent a novel strategy to allow the production of synthetic drugs in vivo by the host itself. The recombinant minicircles encoding etanercept or tocilizumab, which are synthesized currently by pharmaceutical companies, were injected intravenously into animal models. Self-reproduced etanercept and tocilizumab were detected in the serum of mice. Moreover, arthritis subsided in mice that were injected with minicircle vectors carrying biologics. Self-reproducible biologics need neither factory facilities for drug production nor clinical processes, such as frequent drug injection. Although this novel strategy is in its very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics.

Etanercept-Synthesising Mesenchymal Stem Cells Efficiently Ameliorate Collagen-Induced Arthritis

Mesenchymal stem cells (MSCs) have multiple properties including anti-inflammatory and immunomodulatory effects in various disease models and clinical treatments. These beneficial effects, however, are sometimes inconsistent and unpredictable. For wider and proper application, scientists sought to improve MSC functions by engineering. We aimed to invent a novel method to produce synthetic biological drugs from engineered MSCs. We investigated the anti-arthritic effect of engineered MSCs in a collagen-induced arthritis (CIA) model. Biologics such as etanercept are the most successful drugs used in anti-cytokine therapy. Biologics are made of protein components, and thus can be theoretically produced from cells including MSCs. MSCs were transfected with recombinant minicircles encoding etanercept (trade name, Enbrel), which is a tumour necrosis factor α blocker currently used to treat rheumatoid arthritis. We confirmed minicircle expression in MSCs in vitro based on GFP. Etanercept production was verified from the conditioned media. We confirmed that self-reproduced etanercept was biologically active in vitro. Arthritis subsided more efficiently in CIA mice injected with mcTNFR2MSCs than in those injected with conventional MSCs or etanercept only. Although this novel strategy is in a very early conceptual stage, it seems to represent a potential alternative method for the delivery of biologics and engineering MSCs.

A case of idiopathic leuconychia totalis and partialis

SIR, Leuconychia is the most common chromatic disorder of the nail and is classified as pseudoleuconychia resulting from nail plate alteration due to an external origin, apparent leuconychia resulting from subungual and nail bed abnormalities, and true leuconychia where nail plate abnormalities originate in the nail matrix. True leuconychia includes four distinct categories according to distribution of white colour: leuconychia punctata, leuconychia striata, leuconychia partialis and leuconychia totalis. Most cases of true leuconychia may be congenital or, if acquired, may be associated with underlying systemic diseases such as typhoid fever, hepatic cirrhosis, ulcerative colitis or leprosy, and may also be associated with local trauma of the nail matrix, use of emetine, cytostatic agents or local exposure to salty solutions. Idiopathic true leuconychia is a very rare condition and only a few previous reports have described the findings in the progression from leuconychia partialis to leuconychia totalis. We report a 26-year-old man who presented with idiopathic leuconychia which demonstrated a progression from leuconychia partialis to combined leuconychia totalis and partialis. He had had asymptomatic white fingernails for 13 years, affecting the digits on both hands with the exception of the left thumb, with no family history of white fingernails (Fig. 1a). On examination, he had no hair, teeth or skin abnormalities. Leuconychia partialis was seen on the right thumb, left ring finger and both little fingers, with distal transverse white bands. The other fingers showed leuconychia totalis except for the left thumb nail, which showed yellow spikes unlike the other white fingernails. No toenail was involved. Pitting, splitting or ridging of the fingernails was not seen, and there was no subungual hyperkeratosis. Texture, shape and hardness were also normal. He denied taking any medication and having any other systemic illness. He also had experienced no specific trauma and no chemical exposures to his fingers or fingernails. According to the patient, he had had this condition for 13 years, during which period the whitening of the fingernails had progressed slowly. Repeated potassium hydroxide preparations and fungal cultures of the white nails were negative except for the left thumb nail. Potassium hydroxide preparation was positive at the left thumb nail and Trichophyton rubrum was cultured. Laboratory studies including full blood count, urinalysis, liver function tests, renal function tests, total protein, albumin, erythrocyte sedimentation rate and C-reactive protein were normal or negative. Microscopic examination of a white nail plate revealed a globular collection of large immature keratohyaline granules (Fig. 1b), whereas biopsy specimens from the nail bed and nail matrix were unremarkable. Interestingly, our patient also had onychomycosis of the left thumb, for which we prescribed itraconazole pulse therapy (two pulses of 400 mg daily for 7 days). Although no treatment is indicated or available for true leuconychia, we gave intralesional injections of corticosteroid (triamcinolone acetonide 5 mg mL at 1–2-week intervals) into the proximal nail fold for cosmetic reasons. Because true leuconychia is thought to be due to abnormal matrix keratinization, with persistence of keratohyaline granules in the nail plate, we considered that corticosteroid treatment might help to make these cells differentiate. After 2 months of treatment, the left thumb nail was somewhat improved, but the other fingernails showed no visible change. Our patient has shown persistence and ⁄or a slight progression in his nail whitening during 2 years of follow-up. b a