Interleukin-1 Converting Enzyme Research Articles

Transcriptional responses consistent with perturbation in dermo-epidermal homeostasis in septic sole ulceration

The aim of this study was to evaluate transcriptional changes in sole epidermis and dermis of bovine claws with septic sole ulceration of the lateral claw. Assessment included changes in transcripts orchestrating epidermal homeostatic processes including epidermal proliferation, differentiation, inflammation, and cell signaling. Sole epidermis and dermis was removed from region 4 of lesion-bearing lateral and lesion-free medial claws of pelvic limbs in multiparous, lactating Holstein cows. Control sole epidermis and dermis was obtained from region 4 of lateral claws of normal pelvic limbs. Transcript abundances were evaluated by real-time QPCR and relative expression analyzed by ANOVA. Relative to normal lateral claws, sole epidermis and dermis in ulcer-bearing claws exhibited downregulation of genes associated with growth factors, growth factor receptors, activator protein 1 (AP-1) and proto-oncogene (CMYC) transcription components, cell cycle elements, lateral cell-to-cell signaling elements and structures of early and late keratinocyte differentiation. These changes were accompanied by upregulation of pro-inflammatory transcripts interleukin 1 α (IL1A), interleukin1 β (IL1B), interleukin 1 receptor 1 (IL1R1), inducible nitric oxide synthase (NOS2), the inflammasome components NOD like receptor protein 3 (NLRP3), pyrin and caspase recruitment domain (PYCARD), and caspase-1 interleukin converting enzyme (CASPASE), the matrix metalloproteinases (MMP2 and MMP9), and anti-inflammatory genes interleukin 1 receptor antagonist (IL1RN) and interleukin1 receptor 2 (IL1R2). Transcript abundance varied across epidermis and dermis from the ulcer center, margin and epidermis and dermis adjacent to the lesion. Sole epidermis and dermis of lesion-free medial claws exhibited changes paralleling those in the adjacent lateral claws in an environment lacking inflammatory transcripts and downregulated IL1A, interleukin 18 (IL18), tumor necrosis factor α (TNFA) and NOS2. These data imply perturbations in signal pathways driving epidermal proliferation and differentiation are associated with, but not inevitably linked to epidermis and dermis inflammation. Further work is warranted to better define the role of crushing tissue injury, sepsis, metalloproteinase activity, and inflammation in sole ulceration.

Inflammasome Activation in Human Macrophages: IL-1β Cleavage Detection by Fully Automated Capillary-Based Immunoassay.

Interleukin (IL)-1β is a key mediator of inflammation and activates via pattern recognition receptors (PRR) of the inflammasome family by proteolytic maturation. Proteolysis is driven by proteases such as caspase-1 (also known as IL-1 converting enzyme, ICE) and converts the intact pro-IL-1β ~31kDa pro-peptide into a mature, ~17kDa form that can exit cells through nanomolecular pores or via microvesicles. Whereas pro-IL-1β fails to trigger IL-1 receptor (IL-1R) activation, mature IL-1β, upon release from the cell, triggers pleiotropic downstream effects, establishing an inflammatory state. Hence, being able to detect IL-1β conversion is physiologically relevant for measuring inflammation, but it cannot be easily accomplished by conventional ELISA or flow cytometry as most commercially available antibodies do not discriminate mature and pro-form. Furthermore, unlike for other cytokines, the mere induction and translation of IL1B mRNA cannot serve as a proxy of inflammasome PRR activation. Rather the cleavage of IL-1β needs to be verified. Hence, conventional immunoblotting has emerged as the gold standard for demonstrating inflammasome activation as the difference in molecular weight between pro- and mature form can easily be detected. However, conventional immunoblotting suffers from poor standardization, quantification, and reproducibility, may require sample concentration, and is also not suitable for medium to high throughput. Some of these shortcomings are prohibitive for analysis of human primary samples but can be overcome by fully automated capillary-based immunoassay as we outline here. We here provide a practical guide to quantify pro- vs mature IL-1β directly from unconcentrated supernatants of human monocyte-derived macrophages. The assay may be useful for more standardized and medium-throughput analysis in these cells or other biospecimen.

Cardioprotective Effects of Oroxylum indicum Extract Against Doxorubicin and Cyclophosphamide-Induced Cardiotoxicity.

Administration of Chemotherapeutics, especially doxorubicin (DOX) and cyclophosphamide (CPS), is commonly associated with adverse effects such as myelosuppression and cardiotoxicity. At this time, few approved therapeutic options are currently available for the management of chemotherapy-associated cardiotoxicity. Thus, identification of novel therapeutics with potent cardioprotective properties and minimal adverse effects are pertinent in treating Doxorubicin and Cyclophosphamide-induced cardiotoxicity. Oroxylum indicum extract (OIE, Sabroxy®) is a natural product known to possess several beneficial biological functions including antioxidant, anti-inflammatory and cytoprotective effects. We therefore set to investigate the cardioprotective effects of OIE against Doxorubicin and Cyclophosphamide-induced cardiotoxicity and explore the potential cardioprotective mechanisms involved. Adult male mice were treated with DOX and CPS in combination, OIE alone, or a combination of OIE and DOX & CPS. Swimming test was performed to assess cardiac function. Markers of oxidative stress were assessed by levels of reactive oxygen species (ROS), nitrite, hydrogen peroxide, catalase, and glutathione content. The activity of interleukin converting enzyme and cyclooxygenase was determined as markers of inflammation. Mitochondrial function was assessed by measuring Complex-I activity. Apoptosis was assessed by Caspase-3 and protease activity. Mice treated with DOX and CPS exhibited reduced swim rate, increased oxidative stress, increased inflammation, and apoptosis in the heart tissue. These cardiotoxic effects were significantly reduced by co-administration of OIE. Furthermore, computational molecular docking studies revealed potential binding of DOX and CPS to tyrosine hydroxylase which validated our in vivo findings regarding the inhibition of tyrosine hydroxylase activity. Our current findings indicated that OIE counteracts Doxorubicin and Cyclophosphamide-induced cardiotoxicity-through inhibition of ROS-mediated apoptosis and by blocking the effect on tyrosine hydroxylase. Taken together, our findings suggested that OIE possesses cardioprotective effects to counteract potentially fatal cardiac complications associated with chemotherapy treatment.

Cardioprotective effects of Oroxylum indicum extract against chemotherapeutics‐induced cardiotoxicity

Cancer chemotherapy, especially doxorubicin (DOX) and cyclophosphamide (CPS) is commonly associated with adverse effects such as myelosuppression and cardiotoxicity. At this time, little approved therapeutic options are available for the management of chemotherapy-associated cardiotoxicity. Thus, identification of novel therapeutics, with potent cardioprotective effects and minimal adverse effects are pertinent in chemotherapy-induced cardiotoxicity. Oroxylum indicum extract (OIE, Sabroxy®) is a natural product known to possess several biological functions including antioxidant, anti-inflammatory and cytoprotective effects. This study was designed to examine whether OIE extract confers protection against chemotherapy-induced cardiotoxicity, and explored potential mechanism involved. Adult male mice were treated with DOX and CPS, OIE or a combination of OIE and DOX & CPS. Swimming test was performed to assess cardiac function. Markers of oxidative stress were assessed by levels of reactive oxygen species (ROS), nitrite, hydrogen peroxide, catalase and glutathione content. Activity of interleukin converting enzyme and cyclooxygenase was determined as markers of inflammation. Mitochondrial function was assessed by measuring complex I activity. Apoptosis was assessed by caspase and protease activity. Mice treated with DOX and CPS exhibited reduced swim rate, increased oxidative stress markers, increased markers of inflammation and apoptosis. These effects were prevented by co-administration of OIE. Furthermore, computational molecular docking revealed potential binding of DOX and CPS to tyrosine hydroxylase and OIE as a potential protective agent. These data indicated that OIE counteracts chemotherapy-induced cardiotoxicity through inhibition of ROS-mediated apoptosis. Taken together, our findings suggested that OIE possesses potential as a means to counteract potentially fatal cardiac complications associated with chemotherapy treatment.

Activated human NK and CD8+ T cells express both TNF-related apoptosis-inducing ligand (TRAIL) and TRAIL receptors but are resistant to TRAIL-mediated cytotoxicity

AbstractThe expression of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and TRAIL receptors was investigated in resting and cytokine-activated purified primary human natural killer (NK) and CD8+ T cells. Resting NK and CD8+ T cells expressed the mRNA for all TRAIL receptors, but TRAIL-R4 was the only receptor clearly detectable on the surface of both cell types. NK cells were activated by interleukin 2 (IL-2) or IL-15, whereas CD8+ T cells were activated by phytohemagglutinin (PHA) + IL-2 followed by IL-2 alone for up to 10 days. On activation, both cell types rapidly expressed TRAIL-R2 and TRAIL-R3, whose expression peaked at day 10 of culture. TRAIL-R1, however, was never expressed at any time point examined, whereas the expression of TRAIL-R4, which showed a progressive increase in CD8+ T cells, remained constant in NK cells. Notwithstanding the expression of TRAIL-R2, recombinant TRAIL did not show any cytotoxic activity on either NK or CD8+ T cells. Both resting and activated NK and CD8+ T cells were found to express high levels of the 2 isoforms of c-FLIP (cellular Fas-associated death domain protein [FADD]–like IL-1–converting enzyme [FLICE]–inhibitory protein). Small interference RNA-mediated inhibition of c-FLIP expression in NK cells abrogated their resistance to the apoptotic effect of soluble TRAIL. Thus, once activated the major cytotoxic effector cells are potentially sensitive to TRAIL but are physiologically protected from its apoptotic action by intracellular level of c-FLIP.

IL-1 in osteoarthritis: time for a critical review of the literature.

The concept of interleukin-1 (IL-1) as a target in osteoarthritis (OA) has been an attractive one for many years. It is a highly potent inducer of cartilage degradation, causing the induction of mRNA and controlling the bioavailability of disease-relevant proteases such as ADAMTS5 and MMP13. It drives synovitis and can induce other disease-relevant genes such as nerve growth factor, a key pain sensitiser in OA. However, the quality of evidence for its involvement in disease is modest. Descriptive studies have demonstrated expression of IL-1α and β in OA cartilage and elevated levels in the synovial fluid of some patients. Agnostic transcriptomic and genomic analyses do not identify IL-1 as a key pathway. In vivo models show a conflicting role for this molecule; early studies using therapeutic approaches in large animal models show a benefit, but most murine studies fail to demonstrate protection where the ligands (IL-1α/β), the cytokine activator (IL-1–converting enzyme), or the receptor (IL-1R) have been knocked out. Recently, a number of large double-blind randomised controlled clinical studies targeting IL-1 have failed. Enthusiasm for IL-1 as a target in OA is rapidly dwindling.

Versatile effects of magnesium hydroxide nanoparticles in PLGA scaffold–mediated chondrogenesis

Artificial scaffolds made up of various synthetic biodegradable polymers have been reported to have many advantages including cheap manufacturing, easy scale up, high mechanical strength, convenient manipulation, and molding into an unlimited variety of shapes. However, the synthetic biodegradable polymers still have the insufficiency for cartilage regeneration owing to their acidic degradation products. To reduce acidification by degradation of synthetic polymers, we incorporated magnesium hydroxide (MH) nanoparticles into porous polymer scaffold not only to effectively neutralize the acidic hydrolysate but also to minimize the structural disturbance of scaffolds. The neutralization effect of poly(D,L-lactic-co-glycolic acid; PLGA)/MH scaffold was confirmed with the maintenance of neutral pH, contrary to a PLGA scaffold with low pH. Further, the scaffolds were applied to evaluate the chondrogenic differentiation of the human bone marrow mesenchymal stem cells. In in vitro study, the PLGA/MH scaffold enhanced the chondrogenesis markers and reduced the calcification, compared to the PLGA scaffold. Additionally, the PLGA/MH scaffold reduced the release of inflammatory cytokines, compared to the PLGA scaffold, as the cell death decreased. Moreover, the addition of MH reduced necrotic cell death at the early stage of chondrogenic differentiation. Further, the necrotic cell death by the PLGA scaffold was mediated by cleavage of caspase-1, the so-called interleukin 1-converting enzyme, and MH alleviated it as well as nuclear factor kappa B expression. Furthermore, the PLGA/MH scaffold highly supported chondrogenic healing of rat osteochondral defect sites in in vivo study. Therefore, it was suggested that a synthetic polymer scaffold containing MH could be a novel healing tool to support cartilage regeneration and further treatment of orthopedic patients. Statement of SignificanceSynthetic polymer scaffolds have been widely utilized for tissue regeneration. However, they have a disadvantage of releasing acidic products through degradation. This paper demonstrated a novel type of synthetic polymer scaffold with pH-neutralizing ceramic nanoparticles composed of magnesium hydroxide for cartilage regeneration. This polymer showed pH-neutralization property during polymer degradation and significant enhancement of chondrogenic differentiation of mesenchymal stem cells. It reduced not only chondrogenic calcification but also release of proinflammatory cytokines. Moreover, it has an inhibitory effect on necrotic cell death, particularly caspase-1-mediated necrotic cell death (pyroptosis). In in vivo study, it showed higher healing rate of the damaged cartilage in a rat osteochondral defect model. We expected that this novel type of scaffold can be effectively applied to support cartilage regeneration and further treatment of orthopedic patients.

MOLECULAR DOCKING, PHARMACOPHORE MODELLING, AND ADME-TOXICITY PREDICTION OF CURCUMIN ANALOG COMPOUNDS AS INFLAMMATORY INHIBITOR ON RHEUMATOID ARTHRITIS

Objective: The objective of this research was to examine the activity and cytokine inhibitory mechanism of curcumin analog compound against multiple protein targets in a patient with rheumatoid arthritis (RA) and identify the absorption, distribution, metabolism, excretion and toxicity (ADME-toxicity).Methods: Identification was carried out by in silico through pharmacophore modelling using Ligand Scout, molecular docking using iGemDock in various protein (tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), transcription factors, signalling kinase, and cyclooxygenase enzyme) and identification of ADME-toxicity based on the physicochemical properties of the compound to simulate, predict and analyze interaction between protein and compound.Results: The obtained results indicated that gamavuton (GVT-0) and penta-gamavuton (PGV) possessed high bioavailability with lower toxicity than curcumin. However, GVT-0, a curcumin analog, possessed high and specific inhibitory activity on tumor necrosis factor-α converting enzyme (TACE) and interleukin converting enzyme (ICE)/Caspase-1.Conclusion: GVT-0 as a curcumin derivate possessed the best inhibitory activity against TNF-α converting enzyme and IL-1β converting enzyme which are the main route of inflammatory mediators in rheumatoid arthritis. In addition, GVT-0 influences less in metabolism of CYP450 enzymes, and has low toxicity.

Inflammasome Signalling Affects Anxiety And Depressive-Like Behaviors

The inflammasome dysregulation may be implicated in major depressive disorder (MDD). Inflammasome activation causes the maturation of caspase-1 (Casp1, interleukin converting enzyme), which plays a role in a number of physio/pathological processes both in the CNS and periphery (i.e. immune response, microglia activation, LTP, synaptic platicity, adipocyte differentiation, chronic inflammation). Casp1 causes the activation of interleukin (IL)-1β and IL-18, two pro-inflammatory cytokines involved in neuroimmunomodulation, neuroinflammation, and neurodegeneration. C57BL/6 mice with genetic deficiency were screened for anxiety- and depressive-like behaviors, and locomotion at baseline and after chronic restraint stress (4-6 h/day for 3 weeks). Genetic deficiency of caspase-1 decreased depressive- and anxiety-like behaviors, and increased locomotor activity and skills. Caspase-1 deficiency also prevented the exacerbation of depressive-like behaviors following chronic stress. We suggest that the pathway that links chronic stress to depressive-like behaviors involves the inflammasome. Further work should address whether strategies to block caspase 1 bioactivity may have potential as a novel type of antidepressant treatment.

High Ca2+ Influx During Traumatic Brain Injury Leads to Caspase-1-Dependent Neuroinflammation and Cell Death.

We investigated the hypothesis that high Ca2+ influx during traumatic brain injury induces the activation of the caspase-1 enzyme, which triggers neuroinflammation and cell apoptosis in a cell culture model of neuronal stretch injury and an in vivo model of fluid percussion injury (FPI). We first established that stretch injury causes a rapid increase in the intracellular Ca2+ level, which activates interleukin-converting enzyme caspase-1. The increase in the intracellular Ca2+ level and subsequent caspase-1 activation culminates into neuroinflammation via the maturation of IL-1β. Further, we analyzed caspase-1-mediated apoptosis by TUNEL staining and PARP western blotting. The voltage-gated sodium channel blocker, tetrodotoxin, mitigated the stretch injury-induced neuroinflammation and subsequent apoptosis by blocking Ca2+ influx during the injury. The effect of tetrodotoxin was similar to the caspase-1 inhibitor, zYVAD-fmk, in neuronal culture. To validate the in vitro results, we demonstrated an increase in caspase-1 activity, neuroinflammation and neurodegeneration in fluid percussion-injured animals. Our data suggest that neuronal injury/traumatic brain injury (TBI) can induce a high influx of Ca2+ to the cells that cause neuroinflammation and cell death by activating caspase-1, IL-1β, and intrinsic apoptotic pathways. We conclude that excess IL-1β production and cell death may contribute to neuronal dysfunction and cognitive impairment associated with TBI.

Cloning and expression analysis of two pro-inflammatory cytokines, IL-1β and its receptor, IL-1R2, in the Asian swamp eel Monopterus albus

Interleukin-1β (IL-1β) is the prototypic pro-inflammatory cytokine, whose functions are mediated through interaction with its receptors (IL-1R1 and IL-1R2). Herein, we cloned the full-length cDNA and genomic DNA of IL-1β and IL-1R2 in the Asian swamp eel (Monopterus albus). The eel IL-1β cDNA encodes a putative polypeptide of 246 amino acids. The protein sequence includes a typical IL-1 family signature, but lacked an interleukin-converting enzyme cleavage site. The genomic DNA of eel IL-1β was 2520 bp and comprised five exons and four introns. The eel IL-1R2 cDNA encoded a putative propeptide of 423 amino acid residues, comprising a signal peptide, a transmembrane region and two Ig-like domains in the extracellular region. Similar to other vertebrates, the genomic DNA of the eel IL-1R2 has nine exons and eight introns. Real-time PCR analysis indicated that IL-1β and IL-1R2 were constitutively expressed in all tissues, especially in the liver and immune-related organs. After infection with Aeromonas hydrophila, the transcript levels of IL-1β and IL-1R2 were induced in the head kidney and spleen, reaching their highest levels at 6 h post injection. In vitro, IL-1β and IL-1R2 mRNA levels were also upregulated rapidly at 1h post infection with A. hydrophila. Furthermore, acanthocephalan Pallisentis (Neosentis) celatus could induce the expression of both genes in the head kidney and intestine. In infected intestines, the transcript levels of IL-1β and IL-1R2 were increased by 21.4-fold and 20.8-fold, respectively, relative to the control. The present study indicated that IL-1β and IL-1R2 play an important role in inflammation and host defense, especially in the antiacanthocephalan response.

Alarmin High Mobility Group Box 1 (HMGB1) Activates Alternative NFkB Signaling in Bone Marrow Macrophages in the Leukemia Microenvironment

High mobility group box 1 (HMGB1) is a damage-associated molecular pattern (DAMP) that signals through pattern recognition receptors on monocyte/macrophages to recruit them to sites of tissue injury. In models of sepsis and autoimmunity, HMGB1 can have both immune-activating and regulatory effects depending on the receptor system engaged and the downstream pathway activated. Although chemotherapy-stressed tumor cells are known to release HMGB1, little is known about the effects of tumor-derived HMGB1 on macrophages in the tumor microenvironment.We have previously shown that primary human acute lymphoblastic leukemia cells release high levels of HMGB1 after treatment with anthracycline chemotherapy, and that leukemia-associated HMGB1 induces expression of the IL-1 converting enzyme (caspase-1), consistent with activation of the inflammasome. Paradoxically, inflammatory cytokine production (including IL-1beta) is reduced in this setting. In the present study, we test the hypothesis leukemia-associated HMGB1 activates alternative NFkB signaling through the repressor protein RelB which has been recently shown to silence inflammatory cytokine production via epigenetic effects in sepsis models.To establish proof-of-principle, we first treated primary human bone marrow macrophages (BMM) and THP1 monocytes with 200 ng/ml recombinant HMGB1 (r-HMGB1) in the presence or absence of bacterial lipopolysaccharide (LPS). As predicted, LPS alone increased baseline expression of total NFkB, total p65/RelA and phosphorylated p65/RelA mRNA (20.6 + 7.9-fold by qPCR ddCT, p<0.0001) and protein (measured by Western blot, in BMM and THP1. In contrast, r-HMGB1 treatment produced a significant increase in the expression of RelB mRNA (2.5 + 0.7 fold increase p<0.05) and protein without a significant change in the expression of tNFkB or p65/RelA. Treatment with both r-HMGB1 and LPS maintained this pattern of increased RelB expression without increased expression of p65/RelA, suggesting HMGB1 activity as the dominant effect. Increased RelB expression was unaffected by the addition of an antibody to TLR4, the primary receptor for LPS and a candidate receptor for HMGB1.Next, we determined whether induction of RelB expression also occurs in response to leukemia-derived HMGB1. Immunomagnetically selected ALL cells from the bone marrow of 3 pediatric patients and NALM 6 ALL cells were treated with 20 nM doxorubicin, washed, and placed in culture for 24 hours prior to the addition of BMM in direct coculture. In our model, ALL cells typically release 100-450 ng/ml HMGB1 under these conditions. Again, significantly increased expression of RelB mRNA (6.7 + 2.9 fold, p<0.001) and protein was observed in BMM cultured with doxorubicin-treated ALL cells compared to ALL cells treated with media control, with a corresponding decrease in phospho-IkBa protein expression and unchanged total NFkB, p65/RelA and phosphorylated p65/RelA. Pre-treatment of ALL cells with quercetin, a commercially available flavonoid antioxidant shown to inhibit HMGB1 release, inhibited protein expression of RelB in BMM in a dose-dependent fashion.These data indicate that tumor-derived HMGB1 activates alternative NFkB signaling and is capable of reprogramming the inflammatory response to LPS. Studies are ongoing to the determine downstream effects on macrophage antitumor function to optimize therapeutic reversal of HMGB1 effects and improve the potential for bone marrow macrophages to eradicate residual leukemia that remains after chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Enzymatically inactive procaspase-1 stabilizes the ASC-pyroptosome

Caspase-1 (or interleukin-1 converting enzyme, ICE) plays an important role in mediating proinflammatory innate immune responses, especially by activation of pro-IL-1s within inflammasomes. Some patients with recurrent febrile episodes and systemic inflammation of yet unknown origin harbor CASP1-mutations with incomplete penetrance. These CASP1-variants cause reduced enzymatic activity of procaspase-1 and less IL-1s secretion.

635. Mechanisms of Caspase-1 Mediated Schwannoma Regression

Schwannomas are tumors composed of Schwann-lineage cells that form along peripheral nerves. These tumors can cause pain, sensory/motor dysfunction, and death through compression of peripheral nerves, the spinal cord, and/or the brain stem. Management of these tumors-essentially restricted to operative resection and gamma-knife debulking- is limited in scope and efficacy and has significant associated morbidity. Chemotherapeutic treatment strategies have shown incomplete and transient tumor regression. Treatment of Schwannomas remains a major unmet clinical need.Schwannomas are appealing targets for gene therapy as they: 1) grow slowly, 2) have cellular and genetic homogeneity, and 3) can be readily localized using magnetic resonance and/or ultrasound imaging for direct intratumoral vector injection. Gene-therapy is potentially advantageous compared to resection as gene therapy is minimally invasive, kills tumor cells without damaging tumor-associated nerve, and may allow treatment of lesions not amenable to resection. We have previously published that schwannomas can be effectively treated with a gene therapy strategy that uses an adeno-associated virus (AAV) vector to deliver the apoptotic and inflammatory enzyme ICE (IL1-converting enzyme, aka caspase-1) under the Schwann cell specific promoter, myelin Protein 0 (P0). It efficiently debulkes schwannomas and resolves schwannoma associated pain, without any neuronal toxicity.However, the mechanisms by which AAV-P0-ICE selectively kills schwannomas without causing schwann-cell damage and associated neural demyelination are not clear. Understanding these mechanisms would provide the basis for broader utilization of schwannoma gene therapy and insight into how we might increase the efficacy of our current therapy.Through this project we have explored how the role of the P0 promoter and the inflammasome in regulating selective killing of schwannoma cells. Our results indicate that P0 is a highly stringent promoter as it is not expressed in neurons or even in schwannoma cells grown in vitro. P0 is active only in schwann and schannoma cells in vivo, and lack of P0 activity prevents any caspase-1 induced death in other cell types. Second, our data indicates that caspase-1 killing requires concomitant activation of the inflammasome. This pathway was active in 2 different schwannoma cell lines and may be the differentiating factor that prevents caspase-1 to cause axonal demyelination following AAV-P0-ICE injection into peripheral nerve. Finally, our data indicate that adjuvant treatment with factors that stimulate the inflammasome activity increases the efficacy of caspase-1 mediated schwannoma killing. Taken together, these data strongly support the safety of using the P0 promoter for the development of gene therapies for schwannomas and provide new, more efficient, venues for schwannoma capsase-1 mediated gene therapy.

Kinases: a remote control in inflammasome activity.

Kinases: a remote control in inflammasome activity.

On Terminal Alkynes That Can React with Active-Site Cysteine Nucleophiles in Proteases

Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

The heart on fire: inflammasome and cardiomyopathy

The heart on fire: inflammasome and cardiomyopathy

Diacerein: A potential therapeutic drug for periodontal disease

Periodontal diseases are chronic inflammatory diseases characterized by the destruction of the tooth-supporting structures. They are the most prevalent form of bone pathology in humans and act as a modifying factor of the systemic health of patients. Accumulating evidence has provided insight into mechanisms of periodontal inflammation revealing that oral pathogens induce inflammatory cascades, including a variety of cytokines produced by different cell types, which promotes host-mediated tissue destruction. Cytokine networks established in diseased periodontal tissues are extremely complex, and substances regulating immuno-inflammatory reactions and signaling pathways, in addition to traditional periodontal treatment, could potentially be targeted as an approach for prevention and treatment of periodontal diseases. Diacerein, a purified anthraquinone derivative, was derived originally from plants with profound anti-inflammatory and analgesic activities. Its wide range of biological activities have been applied and discussed for several decades; however, studies of diacerein have mainly concentrated on effects on joint-derived tissues/cells, which suggest a beneficial role in osteoarthritis treatment. Diacerein reduces association of the IL-1 receptor to form heterodimer complexes, repressing IL-1 and its related downstream events and impairing active IL-1 release due to the inhibition of the IL-1-converting enzyme (ICE). To date, there are no reports describing the therapeutic effect of diacerein for treatment of periodontitis. Given the involvement of inflammation and occurrence of tissue destruction in periodontal disease, we propose that diacerein might be a promising biological drug for periodontal disease due to its therapeutic advantages. In addition, we hypothesize that the underlying mechanisms might involve the capacity of diacerein to selectively inhibit signal transduction to affect the cytokine profiles and, consequently, produce the outcome of ameliorating disease breakdown.

Imbalance in Cytokines from Interleukin‐1 Family – Role in Pathogenesis of Endometriosis

To assess whether interleukin (IL)-1beta, IL-18 and interleukin-1 converting enzyme (ICE) are involved in the pathogenesis of endometriosis. Peritoneal fluid (PF) was obtained from 85 women with and without endometriosis. Peritoneal macrophages were cultured and the culture media collected. IL-1beta, IL-18 and ICE levels were measured by the enzyme-linked immunosorbent assay (ELISA). Levels of IL-1beta and ICE in PF of women with endometriosis were higher than those in the control group. However, PF level of IL-18 was significantly lower in the study group than in the controls. Higher secretion of IL-1beta by peritoneal macrophages and lower IL-18 and ICE in endometriosis patients than in control were observed. Following lipopolysaccharide (LPS) stimulation, the macrophages secreted more IL-1beta, IL-18 and ICE in all groups. The results pointed to impairment of the secretion of the IL-1 cytokine family in endometriosis. Invalid IL-1beta and IL-18 maturation by ICE may be an important pathogenic factor in endometriosis.

Molecular cloning and characterization of interleukin-1β in half-smooth tongue sole Cynoglossus semilaevis

As a member of the interleukin (IL)-1 family, IL-1β is a prototypical proinflammatory cytokine, which plays a crucial role in immune responses. Herein, we reported the full length cDNA of IL-1β in half-smooth tongue sole (Cynoglossus semilaevis). The csIL-1β cDNA contained a 130bp 5′ UTR, a 417bp 3′ UTR and a 741bp coding sequence (CDS) that translated into a polypeptide of 246 amino acids. The protein sequence included a typical IL-1 family signature and lacked an interleukin-converting enzyme (ICE) cut site. RT-PCR analysis indicated a broad expression of csIL-1β, especially in immune-related organs. After injection with inactive Vibrio anguillarum, the expression of csIL-1β was induced in the head kidney and spleen and reached the highest level at 8h post injection. Higher expression of csIL-1β was observed at gastrula stage, eye-bud stage and hatching stage and lower level of csIL-1β mRNA was detected at metamorphic stage. The expression of csIL-1β during development suggested IL-1β might be involved not only in immunity but also development. Taken together, the present study indicated that csIL-1β played an important role in immune responses and development like its mammalian counterparts, although species-specific features were present.