Metallothionein 3 promotes osteoclast differentiation and survival by regulating the intracellular Zn2+ concentration and NRF2 pathway.

In osteoclastogenesis, the metabolism of metal ions plays an essential role in controlling reactive oxygen species (ROS) production, mitochondrial biogenesis, and survival, and differentiation. However, the mechanism regulating metal ions during osteoclast differentiation remains unclear. The metal-binding protein metallothionein (MT) detoxifies heavy metals, maintains metal ion homeostasis, especially zinc, and manages cellular redox levels. We carried out tests using murine osteoclast precursors to examine the function of MT in osteoclastogenesis and evaluated their potential as targets for future osteoporosis treatments. MT genes were significantly upregulated upon differentiation from osteoclast precursors to mature osteoclasts in response to receptor activators of nuclear factor-κB (NF-κB) ligand (RANKL) stimulation, and MT3 expression was particularly pronounced in mature osteoclasts among MT genes. The knockdown of MT3 in osteoclast precursors demonstrated a remarkable inhibition of differentiation into mature osteoclasts. In preosteoclasts, MT3 knockdown suppressed the activity of mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways upon RANKL stimulation, leading to affect cell survival through elevated cleaved Caspase 3 and poly (ADP-ribose) polymerase (PARP) levels. Additionally, ROS levels were decreased, and nuclear factor erythroid 2-related factor 2 (NRF2) (a suppressor of ROS) and the downstream antioxidant proteins, such as catalase (CAT) and heme oxygenase 1 (HO-1), were more highly expressed in the MT3 preosteoclast knockdowns. mitochondrial ROS, which is involved in mitochondrial biogenesis and the production of reactive oxygen species, were similarly decreased because cAMP response element-binding (CREB) and peroxisome proliferator-activated receptor γ coactivator 1ß (PGC-1ß) were less activated due to MT3 depletion. Thus, by modulating ROS through the NRF2 pathway, MT3 plays a crucial role in regulating osteoclast differentiation and survival, acting as a metabolic modulator of intracellular zinc ions.

Factors affecting patient satisfaction related to cost and treatment effectiveness in rheumatoid arthritis: results from the multicenter observational cohort study, FRANK Registry.

BACKGROUND: To further improve rheumatoid arthritis (RA) treatment, it is necessary to understand each RA patient's satisfaction and to identify the factors affecting their satisfaction. Despite the rise in medical costs for RA, little is known about the factors that influence patient satisfaction with the cost of treatment in RA patients. METHODS: This is a multicenter observational study of Japanese RA patients from the FRANK Registry with data analyzed from March 2017 to August 2020. We collected data on demographic characteristics, clinical data, quality of life which was evaluated using the EuroQol 5-dimensional questionnaire (EQ5D), and patient satisfaction. The four categories of patient satisfaction were evaluated individually (i.e., cost, treatment efficacy, activities of daily living [ADL], and global treatment satisfaction). We analyzed the factors that affected each patient's satisfaction, such as age, sex, EQ5D, disease duration, disease activity, and treatment. RESULTS: This study included 2235 RA outpatients (406 males, 1829 females). In RA patients, "very satisfied" and "satisfied" were given for nearly half of each satisfaction aspect (cost 49%; efficacy 72%; ADL 58%; global treatment 66%) at the time of the initial registration. To investigate the factors influencing each satisfaction, multivariate analysis has revealed that the use of b/tsDMARDs increased satisfaction of treatment effect (odds ratio [OR] 0.66) and ADL (OR 0.78) but decreased cost satisfaction (OR 2.21). Age (50-64 years; OR 0.91; 65-74 years, 0.55: ≥ 75 years, 0.35), female (OR 0.81), and history of musculoskeletal surgery (OR 0.60) all increased cost satisfaction. Patients with lower disease activity and higher EQ5D scores had higher levels of satisfaction in all areas. CONCLUSIONS: In this study, patient satisfaction in terms of cost, treatment effect, ADL, and overall treatment was generally higher, but some patients were dissatisfied. The cost of satisfaction increased with age and a history of musculoskeletal surgery, while it decreased with a lower EQ5D score and the use of b/tsDMARDs.

Predictive factors of non-treatment and non-persistence to osteoporosis medication after fragility hip fractures at 3 years after discharge: a multicentre, prospective cohort study in the northern Kyushu district of Japan.

We examined osteoporosis medication use and factors affecting persistence in 497 patients with fragility hip fractures. Only 25.5% of patients received continuous medication for 3 years, and 44.1% of patients received no treatment. Low Barthel index at discharge was a risk factor for both non-treatment and non-persistence to osteoporosis medication. PURPOSE: Fragility hip fractures (FHF) caused by osteoporosis decrease the quality of life and worsen life expectancy. Use of osteoporosis medication may be an efficient method in the prevention of secondary FHF. However, previous studies have reported low rates of osteoporosis medication and persistence after FHF. This study aimed to evaluate osteoporosis medication use and factors affecting persistence in patients with FHF in the northern Kyushu area of Japan. METHODS: A total of 497 FHF patients aged ≥ 60 years with a 3-year follow-up were included. We prospectively collected data from questionnaires sent every 6 months regarding compliance with osteoporosis medication. We compared baseline characteristics among three groups: no treatment (NT), no persistence (NP), and persistence (P), and conducted multivariable regression models to determine covariates associated with non-treatment (NT vs. NP/P) and non-persistence (NP vs. P). RESULTS: There were 219 (44.1%), 151 (30.4%), and 127 (25.5%) patients in the NT, NP, and P groups, respectively. Factors associated with non-treatment were male sex, chronic kidney disease, no previous osteoporosis treatment, and low Barthel index (BI) at discharge. The only factor associated with non-persistence was a low BI at discharge. Factors associated with a low BI at discharge were male sex, older age, trochanteric fracture, and surgical delay. CONCLUSION: Low BI at discharge is a risk factor for both non-treatment and non-persistence to osteoporosis medication. Therefore, appropriate interventions to improve BI may result in persistence to osteoporosis medication.

Autoreactivity of Peripheral Helper T Cells in the Joints of Rheumatoid Arthritis.

Autoreactive CD4 T cells are thought to play pivotal roles in the pathogenesis of rheumatoid arthritis (RA). Recently, a subset of CD4 T cells that express high levels of programmed death-1 (PD-1) but are distinct from follicular helper T cells have been identified in the joints of RA patients and named peripheral helper T (Tph) cells. Because PD-1 is expressed on T cells chronically stimulated with the Ags, we tested a hypothesis that Tph cells are the pathogenic autoreactive CD4 T cells in RA. We found that human Tph cells in RA joints produce proinflammatory effector cytokines, including IFN-γ, TNF-α, and GM-CSF, in addition to B cell-helping cytokines, such as IL-21 and CXCL13. Flow cytometric analysis showed different bias of TCR Vß usage between PD-1high Tph cells and PD-1low/neg CD4 T cells, including Th1 cells, in the joint or memory CD4 T cells in the peripheral blood, whereas there was little difference between the latter two subsets. In line with this, deep sequencing of TCR demonstrated an overlap of expanded clones between peripheral blood memory CD4 T cells and PD-1low/neg CD4 T cells but not Tph cells in the joint. Interestingly, Tph cells preferentially exhibited autologous MLR in vitro, which required recognition of self-MHC class II and was pronounced by blocking PD-1 signaling. Taken together, these results suggest that Tph cells are the pathogenic autoreactive CD4 T cells in RA, which expand locally in the joints and are regulated by PD-1 signaling.

Does the introduction of newborn hearing screening improve vocabulary development in hearing-impaired children? A population-based study in Japan.

OBJECTIVE: Permanent hearing impairment has a life-long impact on children and its early identification is important for language development. A newborn hearing screening (NHS) program has started in Okayama Prefecture, Japan, in 1999 to detect hearing impairment immediately after birth. We aim to examine the effect of this screening program on vocabulary development in pre-school children in a before and after comparative study design. METHODS: A total of 107 5-year-old children who graduated from Okayama Kanariya Gakuen (an auditory center for hearing-impaired children) between 1998 and 2011 were enrolled in this study. The pre-NHS group (n=40) was defined as those who graduated between 1998 and 2003, while the post-NHS group (n=67) was defined as those who graduated between 2004 and 2011. The primary outcome was receptive vocabulary, which was assessed by the Picture Vocabulary Test [score <18 (low) vs. score ≥18 (high)]. The secondary outcome was productive vocabulary, or the number of productive words, which was assessed by an original checklist [<1773 words (low) vs. ≥1773 (high)]. We calculated odds ratios and 95% confidence intervals for vocabulary development and compared both groups. RESULTS: The adjusted Picture Vocabulary Test score and number of productive words were significantly higher (p<0.01) in the post-NHS group than the pre-NHS group. Odds ratios were 2.63 (95% confidence interval: 1.17-5.89) for receptive vocabulary and 4.17 (95% confidence interval: 1.69-10.29) for productive vocabulary. CONCLUSIONS: The introduction of NHS in Okayama Prefecture significantly improved both receptive and productive vocabulary development in hearing-impaired children.

Development of an optical fiber SPR sensor for living cell activation.

Surface plasmon resonance (SPR) sensors provide a useful means to study the interactions of biological molecules and the reaction of living cells on a sensor chip. However, conventional SPR sensors are bulky, expensive and complicated to use as common diagnostic equipment. In this study, we developed a relatively small and simple SPR system, using optical fibers of 250 microm diameter to detect the activation of living cells attached to the fiber tip. For this system, the core of 200 microm diameter with 1cm length of an optical fiber was coated by gold film with 50 nm thickness to cause plasmon resonance. The light provided by a white LED and attenuated due to a SPR phenomenon in the sensor part was detected and analyzed using a spectrum detector. The difference in solvents with various refractive indexes and protein-bindings to the sensor tip was detected with sufficient sensitivity. Moreover, it detected a sustained increase of AR in a real-time manner, when RBL-2H3 mast cells were fixed onto the fiber tip and stimulated by an antigen. This small fiber SPR system might serve as a useful tool for various clinical examinations either within or outside the body.

Applying surface plasmon resonance to monitor the IgE-mediated activation of human basophils.

BACKGROUND: The histamine releasing test which detects histamine released from basophils in vitro is safe, sensitive and widely used for clinical examination in the field of allergy. However, basophils of certain individuals do not release histamine, because of dysfunctions in their intracellular signal transduction (non-responder). To overcome potential shortcomings of the histamine releasing test, we applied surface plasmon resonance (SPR) to detect the activation of basophils. METHODS: Basophils of patients with allergy, and those of non-allergic volunteers were isolated from peripheral blood. A batch of basophils obtained from a healthy volunteer was treated with lactic acid and IgE of a patient with atopic dermatitis in order to replace their endogenous IgE. They were fixed on the sensor chip of the SPR apparatus, pretreated with or without various inhibitors for intracellular signal transduction, and exposed to the antigens or anti-IgE antibody. RESULTS: When basophils were sensitized with antigen specific IgE, they immediately caused the increase of resonance angle (AR) in response to either anti-IgE antibody or corresponding antigens, even when they did not release histamine. Moreover, the dose dependent reactions of basophils were reflected by the increase of AR as well as the release of histamine. The increase of AR in response to anti-IgE antibody was reduced by pre-treatment of basophils with inhibitors for intracellular signal transduction, but not more than the level for histamine release. CONCLUSIONS: SPR biosensors may be superior to the histamine release test for studying functions of human basophils including those not releasing histamine.

Surface plasmon resonance biosensor detects the downstream events of active PKCbeta in antigen-stimulated mast cells.

Surface plasmon resonance (SPR) biosensors detect large changes of angle of resonance (AR) when RBL-2H3 mast cells are cultured on a sensor chip and stimulated with antigen. However, the detail of molecular events that are responsible for such large changes of AR remained unknown. In this study, we investigated the relationship between intracellular signaling events induced by antigen and the change of AR, by genetic manipulation of intracellular signaling molecules; spleen tyrosine kinase (Syk), src-like adaptor protein (SLAP), linker for activation of T cells (LAT), growth-factor-receptor-bound protein 2 (Grb2), Grb2-related adaptor protein (Gads), and isotypes of protein kinase C (PKC). RBL-2H3 mast cells overexpressing dominant-negative Syk or SLAP, which both interfere with active Syk, exhibited only minimal increase of AR in response to antigen stimulation. Likewise, the interference of the activation of LAT and Gads, by expressing dominant-negative LAT and Gads, respectively, resulted in nearly complete suppression of the antigen-induced increase of AR. The cells overexpressing PKCs, apart from PKCbeta, showed a reduced extent of increase of AR in response to antigen stimulation. Moreover, the introduction of the small interfering RNA targeted against PKCbeta suppressed the antigen-induced increase of AR. These results indicate that the activation of Syk, LAT, Gads, and subsequent PKCbeta is indispensable for the antigen-induced increase of AR of mast cells detected by SPR biosensors.

Living cell positioning on the surface of gold film for SPR analysis.

Living cell reactions are detected as changes of the angle of resonance (AR) for surface plasmon resonance (SPR). Since SPR reflects the events in the field of evanescence, cells need to be fixed on the sensor chip. In this study, we developed methods to fix living cells on a gold surface and to recover adherent cells from the culture dish, preserving their functions to be analyzed by SPR. Human basophils and B cells were fixed to the sensor chip by a biocompatible anchor for cell membranes (alpha-succinimidyloxysuccinyl omega-oleyloxy polyoxyethylene), aminoalkanethiol (cyteamine, 8-amino octanethiol) or an amino-reactive cross-linker (dithiobis [succinimidylpropionate]). They showed an increase of AR in response to various stimuli. RBL-2H3 cells, which firmly adhered to the culture dish, were cultured/recovered with HydroCell/simple pipetting, with RepCell/pipetting at 4 degrees C, or on normal plastic culture dishes with trypsinization or by scraping at 4 degrees C, respectively. The exocytosis of RBL-2H3 cells was largely impaired by scraping, but only slightly by the treatment with pipetting on HydroCell, on RepCell, or with trypsin. The membrane ruffling of the cells prepared by the last three treatments induced by antigens appeared the same. However, the change of AR with cells prepared by trypsin and those by scraping at 4 degrees C were lower than those by HydroCell or RepCell, suggesting that trypsin may harm molecules involved in cellular reactions. Thus, the methods of cell fixation and removal with HydroCell or RepCell should enable us to analyze various reactions in either adherent or non-adherent cells by SPR.

The SPR signal in living cells reflects changes other than the area of adhesion and the formation of cell constructions.

Surface plasmon resonance (SPR) sensors detected large angle of resonance (AR) changes, when RBL-2H3 rat mast cells were cultured and activated on a sensor chip. Here, we demonstrated that PAM212 mouse keratinocytes also showed a large change in AR, when EGF-stimulated. We explored these changes due to intracellular reactions, through the relationship between the AR and the area of cell adhesion, using confocal microscopy for RBL-2H3 cells and PAM212 cells. The effect of Mycalolide B and Toxin B, inhibitors for cell motility, on AR was observed using RBL-2H3 cells. Measuring AR in the presence of various numbers of non-stimulated cells demonstrated that AR and cell density were proportional. However, the AR increase in response to antigen was 35% higher than that expected by solely an increase of the cell adhesion area. Moreover, the AR with PAM212 cells decreased following a transient increase in response to EGF, whilst the area of cell adhesion remained at an increased level. Furthermore, the treatment of RBL-2H3 cells with either Mycalolide B or Toxin B slightly inhibited, but never abolished the AR increase induced by antigen. These treatments abolished all morphological changes, including ruffling and the increase of cell adhesion area observed by light microscopy. These results suggest that AR changes reflect intracellular events rather than changes in the size of the area to which cells adhere.

Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor.

Surface plasmon resonance (SPR)-based sensors have been used to detect the binding between interactive molecules. We applied the SPR technology to the analysis of interactions between living cells and molecules reactive to the cells, using mast cells and mast cell-reactive antigens. The exposure of dinitrophenol-human serum albumin (DNP-HSA), an antigen that stimulates mast cells, to IgE-sensitized mast cells induced a robust and long-lasting SPR signal in a dose-dependent manner. The maximal increase in SPR signal induced by 100 ng/ml DNP-HSA was 0.200 +/- 0.120 angle (mean +/- SD, n = 37), about 1000 times larger than the theoretically expected increase for the simple binding of DNP-HSA to Fc(epsilon)RI, the high-affinity IgE receptor. A small, but similarly prolonged signal was observed when the cells were stimulated by an agonist of the adenosine A3 receptor. The signal induced by DNP-HSA was abolished by genistein, and partially inhibited by phorbol 12-myristate 13-acetate and wortmannin. Interestingly, the signal induced by DNP-HSA was only weakly inhibited by DNP-lysine, suggesting that DNP-lysine manifests its action not by inhibiting, but by modulating the crosslinking of Fc(epsilon)RI. We concluded that SPR sensors can detect biologically significant signals in a real-time manner from the interactions between cells and molecules reactive to the cells.