Therapeutic potential of CKD-506, a novel selective histone deacetylase 6 inhibitor, in a murine model of rheumatoid arthritis.

OBJECTIVES: Histone deacetylase (HDAC) 6 promotes inflammation. We investigated the anti-arthritic effects of CKD-506, a novel HDAC6 inhibitor, in vitro and in a murine model of arthritis as a novel treatment option for rheumatoid arthritis (RA). METHODS: HDAC6 was overexpressed in mouse peritoneal macrophages and RAW 264.7 cells, and the effects of a HDAC6 inhibitor CKD-506 on cytokine production and activity of NF-κB and AP-1 signaling were examined. Peripheral blood mononuclear cells (PBMCs) from RA patients and fibroblast-like synoviocytes (FLS) were activated in the presence of CKD-506. Next, regulatory T cells (Tregs) were induced from RA patients and co-cultured with healthy effector T cells (Teffs) and cell proliferation was analyzed by flow cytometry. Finally, the effects of the inhibitor on the severity of arthritis were assessed in a murine model of adjuvant-induced arthritis (AIA). RESULTS: Overexpression of HDAC6 induced macrophages to produce TNF-α and IL-6. The inhibitory effect of CKD-506 was mediated via blockade of NF-κB and AP-1 activation. HDAC6 inhibition reduced TNF-α and IL-6 production by activated RA PBMCs. CKD-506 inhibited production of MMP-1, MMP-3, IL-6, and IL-8 by activated FLS. In addition, CKD-506 inhibited proliferation of Teffs directly and indirectly by improving iTreg function. In AIA rats, oral CKD-506 improved clinical arthritis in a dose-dependent manner. A combination of sub-therapeutic CKD-506 and methotrexate exerted a synergistic effect. CONCLUSION: The novel HDAC6 inhibitor CKD-506 suppresses inflammatory responses by monocytes/macrophages, improves Treg function, and ameliorates arthritis severity in a murine model of RA. Thus, CKD-506 might be a novel and effective treatment option for RA.

Therapeutic effect of a novel histone deacetylase 6 inhibitor, CKD-L, on collagen-induced arthritis in vivo and regulatory T cells in rheumatoid arthritis in vitro.

BACKGROUND: Histone deacetylase (HDAC) inhibitor has recently been reported to have a therapeutic effect as an anti-inflammatory agent in collagen-induced arthritis (CIA). We investigated the therapeutic effect of a new selective HDAC6 inhibitor, CKD-L, compared to ITF 2357 or Tubastatin A on CIA and regulatory T (Treg) cells in patients with rheumatoid arthritis (RA). METHODS: CIA was induced by bovine type II collagen (CII) in DBA/1 J mice. Mice were treated with HDAC inhibitor for 18 days. Arthritis score was assessed and histological analysis was performed by hematoxylin and eosin (H&E) stain. Cytotoxic T-lymphocyte associated protein (CTLA)-4 expression in induced Treg cells was analyzed and suppression assay was analyzed using Treg cells and effector T (Teff) cells isolated from naive C57BL/6 mice by flow cytometry. Cytokines were analyzed in peripheral blood mononuclear cells (PBMC) of five patients with RA by enzyme-linked immunosorbent assay (ELISA) and real-time polymerase chain reaction (PCR). Tumor necrosis factor (TNF) was analyzed using PMA- activated THP-1 cells by ELISA. Suppression assay was analyzed using Treg cells and Teff cells isolated from RA patients by flow cytometry. RESULTS: In the CIA model, CKD-L and Tubastatin A significantly decreased the arthritis score. CKD-L increased CTLA-4 expression in Foxp3+ T cells and inhibited the proliferation of Teff cells in the suppression assay. In RA PBMC, CKD-L significantly inhibited TNF and interleukin (IL)-1ß, and increased IL-10. CKD-L and Tubastatin A inhibited TNF secretion from PMA-activated THP-1 cells. CKD-L and ITF 2357 inhibited the proliferation of Teff cells in RA patients in the suppression assay. Tubastatin A had no effect on inhibition of proliferation. CONCLUSION: CKD-L decreased the arthritis score in CIA, reduced the expression of TNF and IL-1ß, and increased the expression of IL-10 in PBMC from RA patients. CKD-L increased CTLA-4 expression and the suppressive function of Treg cells. These results suggest that CKD-L may have a beneficial effect in the treatment of RA.

Cyclewise Operation of Printed MoS2 Transistor Biosensors for Rapid Biomolecule Quantification at Femtomolar Levels.

Field-effect transistors made from MoS2 and other emerging layered semiconductors have been demonstrated to be able to serve as ultrasensitive biosensors. However, such nanoelectronic sensors still suffer seriously from a series of challenges associated with the poor compatibility between electronic structures and liquid analytes. These challenges hinder the practical biosensing applications that demand rapid, low-noise, highly specific biomolecule quantification at femtomolar levels. To address such challenges, we study a cyclewise process for operating MoS2 transistor biosensors, in which a series of reagent fluids are delivered to the sensor in a time-sequenced manner and periodically set the sensor into four assay-cycle stages, including incubation, flushing, drying, and electrical measurement. Running multiple cycles of such an assay can acquire a time-dependent sensor response signal quantifying the reaction kinetics of analyte-receptor binding. This cyclewise detection approach can avoid the liquid-solution-induced electrochemical damage, screening, and nonspecific adsorption to the sensor and therefore improves the transistor sensor's durability, sensitivity, specificity, and signal-to-noise ratio. These advantages in combination with the inherent high sensitivity of MoS2 biosensors allow for rapid biomolecule quantification at femtomolar levels. We have demonstrated the cyclewise quantification of Interleukin-1ß in pure and complex solutions (e.g., serum and saliva) with a detection limit of ∼1 fM and a total detection time ∼23 min. This work leverages the superior properties of layered semiconductors for biosensing applications and advances the techniques toward realizing fast real-time immunoassay for low-abundance biomolecule detection.

Biotunable Nanoplasmonic Filter on Few-Layer MoS2 for Rapid and Highly Sensitive Cytokine Optoelectronic Immunosensing.

Monitoring of the time-varying immune status of a diseased host often requires rapid and sensitive detection of cytokines. Metallic nanoparticle-based localized surface plasmon resonance (LSPR) biosensors hold promise to meet this clinical need by permitting label-free detection of target biomolecules. These biosensors, however, continue to suffer from relatively low sensitivity as compared to conventional immunoassay methods that involve labeling processes. Their response speeds also need to be further improved to enable rapid cytokine quantification for critical care in a timely manner. In this paper, we report an immunobiosensing device integrating a biotunable nanoplasmonic optical filter and a highly sensitive few-layer molybdenum disulfide (MoS2) photoconductive component, which can serve as a generic device platform to meet the need of rapid cytokine detection with high sensitivity. The nanoplasmonic filter consists of anticytokine antibody-conjugated gold nanoparticles on a SiO2 thin layer that is placed 170 µm above a few-layer MoS2 photoconductive flake device. The principle of the biosensor operation is based on tuning the delivery of incident light to the few-layer MoS2 photoconductive flake thorough the nanoplasmonic filter by means of biomolecular surface binding-induced LSPR shifts. The tuning is dependent on cytokine concentration on the nanoplasmonic filter and optoelectronically detected by the few-layer MoS2 device. Using the developed optoelectronic biosensor, we have demonstrated label-free detection of IL-1ß, a pro-inflammatory cytokine, with a detection limit as low as 250 fg/mL (14 fM), a large dynamic range of 106, and a short assay time of 10 min. The presented biosensing approach could be further developed and generalized for point-of-care diagnosis, wearable bio/chemical sensing, and environmental monitoring.

Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure.

Immunomodulatory drugs-agents regulating the immune response-are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 µL), short assay time (∼30 min), heightened sensitivity (∼20-30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment.

Multiple MoS2 Transistors for Sensing Molecule Interaction Kinetics.

Atomically layered transition metal dichalcogenides (TMDCs) exhibit a significant potential to enable next-generation low-cost transistor biosensors that permit single-molecule-level quantification of biomolecules. To realize such potential biosensing capability, device-oriented research is needed for calibrating the sensor responses to enable the quantification of the affinities/kinetics of biomolecule interactions. In this work, we demonstrated MoS2-based transistor biosensors capable of detecting tumor necrosis factor--alpha (TNF-α) with a detection limit as low as 60 fM. Such a detection limit was achieved in both linear and subthreshold regimes of MoS2 transistors. In both regimes, all sets of transistors exhibited consistent calibrated responses with respect to TNF-α concentration, and they resulted in a standard curve, from which the equilibrium constant of the antibody-(TNF-α) pair was extracted to be KD = 369 ± 48 fM. Based on this calibrated sensor model, the time-dependent binding kinetics was also measured and the association/dissociation rates of the antibody-(TNF-α) pair were extracted to be (5.03 ± 0.16) × 10(8) M(-1) s(-1) and (1.97 ± 0.08) × 10(-4) s(-1), respectively. This work advanced the critical device physics for leveraging the excellent electronic/structural properties of TMDCs in biosensing applications as well as the research capability in analyzing the biomolecule interactions with fM-level sensitivities.

Integrated nanoplasmonic sensing for cellular functional immunoanalysis using human blood.

Localized surface plasmon resonance (LSPR) nanoplasmonic effects allow for label-free, real-time detection of biomolecule binding events on a nanostructured metallic surface with simple optics and sensing tunability. Despite numerous reports on LSPR bionanosensing in the past, no study thus far has applied the technique for a cytokine secretion assay using clinically relevant immune cells from human blood. Cytokine secretion assays, a technique to quantify intercellular-signaling proteins secreted by blood immune cells, allow determination of the functional response of the donor's immune cells, thus providing valuable information about the immune status of the donor. However, implementation of LSPR bionanosensing in cellular functional immunoanalysis based on a cytokine secretion assay poses major challenges primarily owing to its limited sensitivity and a lack of sufficient sample handling capability. In this paper, we have developed a label-free LSPR biosensing technique to detect cell-secreted tumor necrosis factor (TNF)-α cytokines in clinical blood samples. Our approach integrates LSPR bionanosensors in an optofluidic platform that permits trapping and stimulation of target immune cells in a microfluidic chamber with optical access for subsequent cytokine detection. The on-chip spatial confinement of the cells is the key to rapidly increasing a cytokine concentration high enough for detection by the LSPR setup, thereby allowing the assay time and sample volume to be significantly reduced. We have successfully applied this approach first to THP-1 cells and then later to CD45 cells isolated directly from human blood. Our LSPR optofluidics device allows for detection of TNF-α secreted from cells as few as 1000, which translates into a nearly 100 times decrease in sample volume than conventional cytokine secretion assay techniques require. We achieved cellular functional immunoanalysis with a minimal blood sample volume (3 µL) and a total assay time 3 times shorter than that of the conventional enzyme-linked immunosorbent assay (ELISA).

Optofluidic detection for cellular phenotyping.

Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidics, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection.

An integrated microfluidic platform for in situ cellular cytokine secretion immunophenotyping.

Rapid, quantitative detection of cell-secreted biomarker proteins with a low sample volume holds great promise to advance cellular immunophenotyping techniques for personalized diagnosis and treatment of infectious diseases. Here we achieved such an assay with the THP-1 human acute moncytic leukemia cell line (a model for human monocyte) using a highly integrated microfluidic platform incorporating a no-wash bead-based chemiluminescence immunodetection scheme. Our microfluidic device allowed us to stimulate cells with lipopolysaccharide (LPS), which is an endotoxin causing septic shock due to severely pronounced immune response of the human body, under a well-controlled on-chip environment. Tumor necrosis factor-alpha (TNF-α) secreted from stimulated THP-1 cells was subsequently measured within the device with no flushing process required. Our study achieved high-sensitivity cellular immunophenotyping with 20-fold fewer cells than current cell-stimulation assay. The total assay time was also 7 times shorter than that of a conventional enzyme-linked immunosorbent assay (ELISA). Our strategy of monitoring immune cell functions in situ using a microfluidic platform could impact future medical treatments of acute infectious diseases and immune disorders by enabling a rapid, sample-efficient cellular immunophenotyping analysis.

Serum elastin-derived peptides and anti-elastin antibody in patients with systemic sclerosis.

The elastin metabolism in systemic sclerosis (SSc) has been known to be abnormal. The authors investigated relationship between the clinical manifestations of systemic sclerosis (SSc) and serum levels of soluble elastin-derived peptide (S-EDP) and anti-elastin antibodies. Serum samples were obtained from 79 patients with SSc and 79 age- and sex-matched healthy controls. Concentrations of serum S-EDP and anti-elastin antibodies were measured by ELISA. The serum concentrations of S-EDP in SSc patients were significantly higher than in healthy controls (median, 144.44 ng/mL vs 79.59 ng/mL, P < 0.001). Serum EDP concentrations were found to be correlated with disease duration in SSc (P = 0.002) and particularly in diffuse cutaneous SSc (P = 0.005). Levels of anti-elastin antibodies were found to be more elevated in SSc patients than in healthy controls (median, 0.222 U vs 0.191 U, P = 0.049), more increased in diffuse cutaneous SSc than limited cutaneous SSc (median, 0.368 U vs 0.204 U, P = 0.031). In addition, levels of anti-elastin antibodies were also found to be negatively associated with presence of anti-centromere antibody (P = 0.023). The S-EDP levels were not found to be correlated with levels of anti-elastin antibodies. The increased S-EDP and anti-elastin antibody levels and association with clinical and laboratory characteristics may reflect the abnormal metabolism in SSc.