Programmed Cell Death in Immune Defense: Knowledge and Presumptions.

Cell-culture studies are our main source of knowledge of the various forms of programmed cell death. Yet genetic perturbations of death-protein function in animal models are almost the only source of our knowledge of the physiological roles of these programs. Shortcomings in the state of knowledge acquired by these two experimental approaches are exemplified in this Perspective by reference to research on the contribution of apoptosis to lymphocyte development, a subject on which there is already much knowledge, and on the role of necroptosis in inflammation, about which information is just beginning to emerge. To address these shortcomings, there is need to find ways to verify the notions obtained through the current experimental approaches by directly monitoring death programs within specific cells in vivo.

Cornus officinalis Seed Extract Inhibits AIM2-Inflammasome Activation and Attenuates Imiquimod-Induced Psoriasis-like Skin Inflammation.

The AIM2 inflammasome is an innate immune system component that defends against cytosolic bacteria and DNA viruses, but its aberrant activation can lead to the progression of various inflammatory diseases, including psoriasis. However, there have been few reports of specific inhibitors of AIM2 inflammasome activation. In this study, we aimed to investigate the inhibitory activity of ethanolic extracts of seeds of Cornus officinalis (CO), a herb and food plant used in traditional medicine, on AIM2-inflammasome activation. We found that CO inhibited the release of IL-1ß induced by dsDNA in both BMDMs and HaCaT cells, but that it showed no effect on the release of IL-1ß induced by NLRP3 inflammasome triggers, such as nigericin and silica, or the NLRC4 inflammasome trigger flagellin. Furthermore, we demonstrated that CO inhibited the cleavage of caspase-1, an inflammasome activation marker, and an upstream event, the translocation and speck formation of ASC. In addition, further experiments and mechanistic investigations revealed that CO can inhibit AIM2 speck formation induced by dsDNA in AIM2-overexpressing HEK293T cells. To verify the correlation in vivo, we investigated the efficacy of CO in an imiquimod (IMQ)-induced psoriasis model, which has reported associations with the AIM2 inflammasome. We found that topical application of CO alleviated psoriasis-like symptoms, such as erythema, scaling, and epidermal thickening, in a dose-dependent manner. Moreover, CO also significantly decreased IMQ-induced expression of AIM2 inflammasome components, including AIM2, ASC, and caspase-1, and led to the elevation of serum IL-17A. In conclusion, our results suggest that CO may be a valuable candidate for the discovery of AIM2 inhibitors and the regulation of AIM2-related diseases.

Novel Activity of ODZ10117, a STAT3 Inhibitor, for Regulation of NLRP3 Inflammasome Activation.

The NLRP3 inflammasome serves as a host defense mechanism against various pathogens, but there is growing evidence linking its activation in sterile condition to diverse inflammatory diseases. Therefore, the identification of specific inhibitors that target NLRP3 inflammasome activation is meaningful and important for novel therapies for NLRP3 inflammasome-associated diseases. In this study, we identified a chemical compound, namely ODZ10117 (ODZ), that showed NLRP3 inflammasome-targeting anti-inflammatory effects during the screening of a chemical library for anti-inflammatory activity. Although ODZ was initially discovered as a STAT3 inhibitor, here we found it also has inhibitory activity on NLRP3 inflammasome activation. ODZ inhibited the cleavage of caspase-1 and IL-1ß-induced canonical NLRP3 inflammasome triggers, but had no effect on those induced by AIM2 or NLRC4 triggers. Mechanistically, ODZ impairs NLRP3 inflammasome activation through the inhibition of NLRP3-NEK7 interaction that is required for inflammasome formation. Moreover, the results obtained from the in silico docking experiment suggested that ODZ targets NLRP3 protein, which provides evidence for the specificity of ODZ to the NLRP3 inflammasome. Furthermore, ODZ administration significantly reduced MSU-induced IL-1ß release and the mortality rate of mice with LPS-induced sepsis. Collectively, these results demonstrate a novel effect of ODZ10117 in regulating NLRP3 inflammasome activation both in vitro and in vivo, making it a promising candidate for the treatment of NLRP3-inflammasome-associated immune disorders and cancer.

Caspase-8 blocks kinase RIPK3-mediated activation of the NLRP3 inflammasome.

Caspase-8 deficiency in certain cells prompts chronic inflammation. One mechanism suggested to account for this inflammation is enhanced signaling for necrotic cell death, mediated by the protein kinases RIPK1 and RIPK3 that caspase-8 can cleave. We describe an activity of caspase-8 in dendritic cells that controls the initiation of inflammation in another way. Caspase-8 deficiency in these cells facilitated lipopolysaccharide-induced assembly and function of the NLRP3 inflammasome. This effect depended on the functions of RIPK1 and RIPK3, as well as of MLKL and PGAM5, two signaling proteins recently shown to contribute to RIPK3-mediated induction of necrosis. However, although enhancement of inflammasome assembly in the caspase-8-deficient cells shares proximal signaling events with the induction of necrosis, it occurred independently of cell death. These findings provide new insight into potentially pathological inflammatory processes to which RIPK1- and RIPK3-mediated signaling contributes.

Down-regulation of pro-necroptotic molecules blunts necroptosis during myogenesis.

Cell death and differentiation are closely related at the molecular level. Differentiation of skeletal muscle cells attenuates susceptibility to apoptosis. Necroptosis has recently been recognized as a form of regulated cell death but its role in myogenesis has not been studied. This study aimed to compare the sensitivity to TNF-induced necroptosis in skeletal muscle at the undifferentiated (myoblasts) and differentiated (myotubes) stages. Surprisingly, our results showed that TNF-induced necroptosis was blunted during myoblast differentiation. Moreover, our data revealed that the key molecules involved in necroptosis, including receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL), were significantly down-regulated during myogenic differentiation, resulting in suppression of necroptosis signal transduction in differentiated myotubes. In addition, RIPK1, RIPK3, and MLKL expression levels were significantly lower in the skeletal muscle of adult mice than in newborn mice, suggesting that the susceptibility to necroptosis might be attenuated in differentiated muscle tissue. In conclusion, this study revealed that expression of key molecules involved in necroptosis is down-regulated during muscle differentiation, which results in the differentiation of muscles becoming insensitive to necroptotic cell death.

Immunostimulatory Activity of Polysaccharides Extracted from Celosia cristata Flowers.

Macrophages play a major role in innate immune responses by producing a variety of immune mediators and cytokines. The stimulation of macrophages by natural products may lead to an enhanced innate immune system. This study evaluated the immunostimulatory effects of a polysaccharide-rich crude fraction of Celosia cristata L. flowers (CCP) on murine macrophages. CCP treatment induced the production of inducible nitric oxide synthase, cyclooxygenase-2, and cytokines by macrophages. Mechanistically, the activation of mitogen-activated protein kinases, NF-κB and toll-like receptor 4 were found to be associated with the stimulatory functions of CCP. CCP was found to be primarily composed of galacturonic acid and glucose in addition to small amounts of arabinose and galactose. This study demonstrated that CCP may enhance the innate immune responses and potentially improve the immune functions in the body.

Regulation of Caspase-8 Activity at the Crossroads of Pro-Inflammation and Anti-Inflammation.

Caspase-8 has been classified as an apoptotic caspase, and its initial definition was an initiator of extrinsic cell death. During the past decade, the concept of caspase-8 functioning has been changed by findings of its additional roles in diverse biological processes. Although caspase-8 was not originally thought to be involved in the inflammation process, many recent works have determined that caspase-8 plays an important role in the regulatory functions of inflammatory processes. In this review, we describe the recent advances in knowledge regarding the manner in which caspase-8 modulates the inflammatory responses concerning inflammasome activation, cell death, and cytokine induction.

Heparin and Its Derivatives: Challenges and Advances in Therapeutic Biomolecules.

Heparin has been extensively studied as a safe medicine and biomolecule over the past few decades. Heparin derivatives, including low-molecular-weight heparins (LMWH) and heparin pentasaccharide, are effective anticoagulants currently used in clinical settings. They have also been studied as functional biomolecules or biomaterials for various therapeutic uses to treat diseases. Heparin, which has a similar molecular structure to heparan sulfate, can be used as a remarkable biomedicine due to its uniquely high safety and biocompatibility. In particular, it has recently drawn attention for use in drug-delivery systems, biomaterial-based tissue engineering, nanoformulations, and new drug-development systems through molecular formulas. A variety of new heparin-based biomolecules and conjugates have been developed in recent years and are currently being evaluated for use in clinical applications. This article reviews heparin derivatives recently studied in the field of drug development for the treatment of various diseases.

NLRP3 Ubiquitination-A New Approach to Target NLRP3 Inflammasome Activation.

In response to diverse pathogenic and danger signals, the cytosolic activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing (3)) inflammasome complex is a critical event in the maturation and release of some inflammatory cytokines in the state of an inflammatory response. After activation of the NLRP3 inflammasome, a series of cellular events occurs, including caspase 1-mediated proteolytic cleavage and maturation of the IL-1ß and IL-18, followed by pyroptotic cell death. Therefore, the NLRP3 inflammasome has become a prime target for the resolution of many inflammatory disorders. Since NLRP3 inflammasome activation can be triggered by a wide range of stimuli and the activation process occurs in a complex, it is difficult to target the NLRP3 inflammasome. During the activation process, various post-translational modifications (PTM) of the NLRP3 protein are required to form a complex with other components. The regulation of ubiquitination and deubiquitination of NLRP3 has emerged as a potential therapeutic target for NLRP3 inflammasome-associated inflammatory disorders. In this review, we discuss the ubiquitination and deubiquitination system for NLRP3 inflammasome activation and the inhibitors that can be used as potential therapeutic agents to modulate the activation of the NLRP3 inflammasome.

The Role of Tumor Necrosis Factor Alpha (TNF-α) in Autoimmune Disease and Current TNF-α Inhibitors in Therapeutics.

Tumor necrosis factor alpha (TNF-α) was initially recognized as a factor that causes the necrosis of tumors, but it has been recently identified to have additional important functions as a pathological component of autoimmune diseases. TNF-α binds to two different receptors, which initiate signal transduction pathways. These pathways lead to various cellular responses, including cell survival, differentiation, and proliferation. However, the inappropriate or excessive activation of TNF-α signaling is associated with chronic inflammation and can eventually lead to the development of pathological complications such as autoimmune diseases. Understanding of the TNF-α signaling mechanism has been expanded and applied for the treatment of immune diseases, which has resulted in the development of effective therapeutic tools, including TNF-α inhibitors. Currently, clinically approved TNF-α inhibitors have shown noticeable potency in a variety of autoimmune diseases, and novel TNF-α signaling inhibitors are being clinically evaluated. In this review, we briefly introduce the impact of TNF-α signaling on autoimmune diseases and its inhibitors, which are used as therapeutic agents against autoimmune diseases.

Mitigating Effect of Lindera obtusiloba Blume Extract on Neuroinflammation in Microglial Cells and Scopolamine-Induced Amnesia in Mice.

Lindera obtusiloba Blume (family, Lauraceae), native to Northeast Asia, has been used traditionally in the treatment of trauma and neuralgia. In this study, we investigated the neuroinflammatory effect of methanol extract of L. obtusiloba stem (LOS-ME) in a scopolamine-induced amnesia model and lipopolysaccharide (LPS)-stimulated BV2 microglia cells. LOS-ME downregulated the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, inflammatory cytokines, and inhibited the phosphorylation of nuclear factor kappa-B (NF-ĸB) and extracellular signal-regulated kinase (ERK) in LPS-stimulated BV2 cells. Male C57/BL6 mice were orally administered 20 and 200 mg/kg of LOS-ME for one week, and 2 mg/kg of scopolamine was administered intraperitoneally on the 8th day. In vivo behavioral experiments (Y-maze and Morris water maze test) confirmed that LOS-ME alleviated cognitive impairments induced by scopolamine and the amount of iNOS expression decreased in the hippocampus of the mouse brain. Microglial hyper-activation was also reduced by LOS-ME pretreatment. These findings suggest that LOS-ME might have potential in the treatment for cognitive improvement by regulating neuroinflammation.

RIG-I RNA helicase activation of IRF3 transcription factor is negatively regulated by caspase-8-mediated cleavage of the RIP1 protein.

Excessive responses to pattern-recognition receptors are prevented by regulatory mechanisms that affect the amounts and activities of the downstream signaling proteins. We report that activation of the transcription factor IRF3 by the ribonucleic acid sensor RIG-I was restricted by caspase-8-mediated cleavage of the RIP1 protein, which resulted in conversion of RIP1 from a signaling enhancer to a signaling inhibitor. The proteins RIP1 and caspase-8 were recruited to the RIG-I complex after viral infection and served antagonistic regulatory roles. Conjugation of ubiquitin chains to RIP1 facilitated assembly of the RIG-I complex, resulting in enhanced phosphorylation of IRF3. However, the ubiquitination of RIP1 also rendered it susceptible to caspase-8-mediated cleavage that yielded an inhibitory RIP1 fragment. The dependence of RIP1 cleavage on the same molecular change as that facilitating RIG-I signaling allows for RIG-I signaling to be restricted in its duration without compromising its initial activation.

Cinobufagin Suppresses Melanoma Cell Growth by Inhibiting LEF1.

Constitutive activation of the ß-catenin dependent canonical Wnt signaling pathway, which enhances tumor growth and progression in multiple types of cancer, is commonly observed in melanoma. LEF1 activates ß-catenin/TCF4 transcriptional activity, promoting tumor growth and progression. Although several reports have shown that LEF1 is highly expressed in melanoma, the functional role of LEF1 in melanoma growth is not fully understood. While A375, A2058, and G361 melanoma cells exhibit abnormally high LEF1 expression, lung cancer cells express lower LEF1 levels. A luciferase assay-based high throughput screening (HTS) with a natural compound library showed that cinobufagin suppressed ß-catenin/TCF4 transcriptional activity by inhibiting LEF1 expression. Cinobufagin decreases LEF1 expression in a dose-dependent manner and Wnt/ß-catenin target genes such as Axin-2, cyclin D1, and c-Myc in melanoma cell lines. Cinobufagin sensitively attenuates cell viability and induces apoptosis in LEF1 expressing melanoma cells compared to LEF1-low expressing lung cancer cells. In addition, ectopic LEF1 expression is sufficient to attenuate cinobufagin-induced apoptosis and cell growth retardation in melanoma cells. Thus, we suggest that cinobufagin is a potential anti-melanoma drug that suppresses tumor-promoting Wnt/ß-catenin signaling via LEF1 inhibition.

Artemisia Extract Suppresses NLRP3 and AIM2 Inflammasome Activation by Inhibition of ASC Phosphorylation.

Artemisia princeps var. orientalis (Asteraceae, A. princeps) is a well-known traditional medicinal herb used for treating various inflammatory disorders in Korea, Japan, China, and other Asian countries. In the present study, we investigated the effects of A. princeps extract (APO) on interleukin- (IL-) 1ß regulation and inflammasome activation in bone marrow-derived macrophages (BMDMs) and monosodium urate- (MSU-) induced peritonitis mouse model in vivo. The APO treatment to BMDMs primed with lipopolysaccharide (LPS) attenuated the NLRP3 and AIM2 inflammasome activation induced by danger signals, such as ATP, nigericin, silica crystals, and poly (dA:dT), respectively. Mechanistic study revealed that APO suppressed the ASC oligomerization and speck formation, which are required for inflammasome activation. APO treatment also reduced the ASC phosphorylation induced by the combination of LPS and a tyrosine phosphatase inhibitor. In vivo evaluation revealed that intraperitoneal administration of APO reduced IL-1ß levels, significantly (p < 0.05) and dose dependently, in the MSU-induced peritonitis mouse model. In conclusion, our study is the first to report that the extract of A. princeps inhibits inflammasome activation through the modulation of ASC phosphorylation. Therefore, APO might be developed as therapeutic potential in the treatment of inflammasome-mediated inflammatory disorders, such as gouty arthritis.

Identification and Characterization of NTB451 as a Potential Inhibitor of Necroptosis.

Necroptosis, or caspase-independent programmed cell death, is known to be involved in various pathological conditions, such as ischemia/reperfusion injury, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. Although several inhibitors of necroptosis have been identified, none of them are currently in clinical use. In the present study, we identified a new compound, 4-({[5-(4-aminophenyl)-4-ethyl-4H-1,2,4-triazol-3-yl]sulfanyl}methyl)-N-(1,3-thiazol-2-yl) benzamide (NTB451), with significant inhibitory activity on the necroptosis induced by various triggers, such as tumor necrosis factor-α (TNF-α) and toll-like receptor (TLR) agonists. Mechanistic studies revealed that NTB451 inhibited phosphorylation and oligomerization of mixed lineage kinase domain like (MLKL), and this activity was linked to its inhibitory effect on the formation of the receptor interacting serine/threonine-protein kinase 1 (RIPK1)-RIPK3 complex. Small interfering RNA (siRNA)-mediated RIPK1 knockdown, drug affinity responsive target stability assay, and molecular dynamics (MD) simulation study illustrated that RIPK1 is a specific target of NTB451. Moreover, MD simulation showed a direct interaction of NTB451 and RIPK1. Further experiments to ensure that the inhibitory effect of NTB451 was restricted to necroptosis and NTB451 had no effect on nuclear factor-κB (NF-κB) activation or apoptotic cell death upon triggering with TNF-α were also performed. Considering the data obtained, our study confirmed the potential of NTB451 as a new necroptosis inhibitor, suggesting its therapeutic implications for pathological conditions induced by necroptotic cell death.

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1.

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1ß, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1ß but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1ß activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/ß. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.

Development of an interleukin (IL)-33 sandwich ELISA kit specific for mature IL-33.

Interleukin (IL)-33 is an inflammatory cytokine and belongs to the IL-1 family of cytokines. There are eleven members of the IL-1 family of cytokines and all have important roles in host defense against infections. Their levels are increased during infection and in various auto-inflammatory diseases. IL-33 is also associated with autoimmune diseases such as asthma, atopic dermatitis, rheumatoid arthritis, and atherosclerosis. IL-33 receptors consist of IL-1R4 and IL-1R3 to induce both Th1 and Th2 type immune response. Here we present the development of monoclonal antibodies (mAbs) against human mature IL-33. Recombinant human mature IL-33 protein was expressed in E. coli and purified by multi-step affinity chromatography. The human IL-33 activity was examined in HMC-1 and Raw 264.7 cells. Mice were immunized with the biologically active mature IL-33 to generate mAb against IL-33. The anti-IL-33 mAb (clone/4) was used as a capture antibody for a sandwich enzyme-linked immunosorbent assay (ELISA). This assay detects mature IL-33 with a high sensitivity (80 pg/mL) but does not recognize the biologically inactive precursor IL-33. This article describes the methods for a newly developed IL-33 ELISA kit that is specific for mature IL-33 and may be used to analyze bioactive mature IL-33 in various immunological diseases.

A novel synthetic derivative of melatonin, 5-hydroxy-2'-isobutyl-streptochlorin (HIS), inhibits inflammatory responses via regulation of TRIF-dependent signaling and inflammasome activation.

Melatonin is substantially reported to possess anti-inflammatory properties. In the present study, we synthesized a novel melatonin derivative, 5-hydroxy-2'-isobutyl-streptochlorin (HIS), which displayed superior anti-inflammatory properties to its parent compound. Further, we explored its underlying mechanisms in cellular and experimental animal models. Lipopolysaccharide was used to induce in vitro inflammatory responses in RAW 264.7 macrophages. LPS-primed macrophages were pulsed with biologically unrelated toxic molecules to evaluate the role of HIS on inflammasome activation. In vivo verifications were carried out using acute lung injury (ALI) and Escherichia coli-induced septic shock mouse models. HIS inhibited the production of proinflammatory mediators and cytokines such as nitric oxide, cyclooxygenase 2, IL-1ß, IL-6 and TNF-α in LPS-stimulated RAW 264.7 macrophages. HIS suppressed the infiltration of immune cells into the lung and the production of pro-inflammatory cytokines such as IL-6 and TNF-α in broncho-alveolar lavage fluid in the ALI mouse model. Mechanistic studies revealed that the inhibitory effects of HIS were mediated through the regulation of the TIR domain-containing, adaptor-inducing, interferon-ß (TRIF)-dependent signaling pathway from toll-like receptors. Further, HIS attenuated IL-1ß secretion via the inhibition of NLRP3 inflammasome activation independent of mitochondrial ROS production. Furthermore, HIS suppressed IL-1ß, IL-6 and interferon-ß production in peritoneal lavage in the Escherichia coli-induced sepsis mouse model. In conclusion, HIS exerted potent anti-inflammatory effects via the regulation of TRIF-dependent signaling and inflammasome activation. Notably, the superior anti-inflammatory properties of this derivative compared with its parent compound could be a promising lead for treating various inflammatory-mediated diseases.

Anti-inflammatory effect of Streptochlorin via TRIF-dependent signaling pathways in cellular and mouse models.

Streptochlorin, a small compound derived from marine actinomycete, has been shown to have anti-angiogenic, anti-tumor, and anti-allergic activities. However, the anti-inflammatory effects and underlying mechanisms have not yet been reported. In the present study, we investigated the effect of streptochlorin on lipopolysaccharide (LPS)-induced inflammatory responses in vitro and in vivo. Streptochlorin attenuated the production of proinflammatory mediators such as nitric oxide, cyclooxygenase-2, pro-interleukin (IL)-1ß, and IL-6 in LPS-stimulated RAW264.7 cells through inhibition of the Toll/interleukin-1 receptor (TIR)-domain-containing adapter-inducing interferon-ß (TRIF)-dependent signaling pathway. Furthermore, streptochlorin suppressed the infiltration of immune cells such as neutrophils into the lung and proinflammatory cytokine production such as IL-6 and TNF-α in broncho-alveolar lavage fluid (BALF) in the LPS-induced acute lung injury (ALI) mouse model. Streptochlorin has potent anti-inflammatory effects through regulating TRIF-dependent signaling pathways, suggesting that streptochlorin may provide a valuable therapeutic strategy in treating various inflammatory diseases.

Anti-inflammatory effect of emodin via attenuation of NLRP3 inflammasome activation.

Emodin, an active constituent of oriental herbs, is widely used to treat allergy, inflammation, and other symptoms. This study provides the scientific basis for the anti-inflammasome effects of emodin on both in vitro and in vivo experimental models. Bone marrow-derived macrophages were used to study the effects of emodin on inflammasome activation by using inflammasome inducers such as ATP, nigericin, and silica crystals. The lipopolysaccharide (LPS)-induced endotoxin shock model was employed to study the effect of emodin on in vivo efficacy. Emodin treatment attenuated interleukin (IL)-1ß secretion via the inhibition of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation induced by ATP, nigericin, and silica crystals. Further, emodin ameliorated the severity of NLRP3 inflammasome-mediated symptoms in LPS-induced endotoxin mouse models. This study is the first to reveal mechanism-based evidence, especially with respect to regulation of inflammasome activation, substantiating traditional claims of emodin in the treatment of inflammation-related disorders.