Elevated IL-1ß and Comparable IL-1 Receptor Antagonist Levels Are Characteristic Features of L-PRP in Female College Athletes Compared to Male Professional Soccer Players.

Autologous platelet-rich plasma (PRP) therapy has been becoming popular for the treatment of musculotendinous injuries among athletes. However, for individual and practical variations, clinical success is hardly predictable. To overcome this difficulty, we have been exploring possible criterion candidates for monitoring its clinical effectiveness. In this study, we focused on sex-based differences in young elite athletes and compared the biochemical compositions of their PRP. Leukocyte-rich PRP (L-PRP) was manually prepared from blood samples collected from male professional soccer players (mPSPs) (n = 25) and female college athletes (fCAs) (n = 36). Platelet-derived growth factor-BB (PDGF-BB), transforming-growth factor-ß1 (TGFß1), platelet factor-4 (PF4), interleukin-1ß (IL-1ß), and IL-1 receptor antagonist (IL-1RA) were quantified using an enzyme-linked immunosorbent assay. The levels of PDGF-BB, TGFß1, and PF4 in L-PRP were significantly higher in mPSPs than in fCAs. Conversely, IL-1ß and IL-1RA were detected at significantly and slightly higher levels, respectively, in fCAs than in mPSPs. Our findings suggest that, even though L-PRP from fCAs may have lower potential to induce cell growth and differentiation than that of mPSPs, due to the latter's higher capacity to control inflammation, it does not necessarily imply that PRP treatment in fCAs is less effective. Thus, these cytokine levels should be checked before PRP therapy.

Expression and characterization of recombinant IL-1Ra in Aspergillus oryzae as a system.

BACKGROUND: The interleukin-1 receptor antagonist (IL-1Ra) is a crucial molecule that counteracts the effects of interleukin-1 (IL-1) by binding to its receptor. A high concentration of IL-1Ra is required for complete inhibition of IL-1 activity. However, the currently available Escherichia coli-expressed IL-1Ra (E. coli IL-1Ra, Anakinra) has a limited half-life. This study aims to produce a cost-effective, functional IL-1Ra on an industrial scale by expressing it in the pyrG auxotroph Aspergillus oryzae. RESULTS: We purified A. oryzae-expressed IL-1Ra (Asp. IL-1Ra) using ion exchange and size exclusion chromatography (53 mg/L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that Asp. IL-1Ra is N-glycosylated and approximately 17 kDa in size. We conducted a comparative study of the bioactivity, binding kinetics, and half-life between Asp. IL-1Ra and E. coli IL-1Ra. Asp. IL-1Ra showed good bioactivity even at a low concentration of 0.5 nM. The in vitro half-life of Asp. IL-1Ra was determined for different time points (0, 24, 48, 72, and 96 h) and showed higher stability than E. coli IL-1Ra, despite exhibiting a 100-fold lower binding affinity (2 nM). CONCLUSION: This study reports the production of a functional Asp. IL-1Ra with advantageous stability, without extensive downstream processing. To our knowledge, this is the first report of a recombinant functional and stable IL-1Ra expressed in A. oryzae. Our results suggest that Asp. IL-1Ra has potential for industrial-scale production as a cost-effective alternative to E. coli IL-1Ra.

Engineering non-covalently assembled protein nanoparticles for long-acting gouty arthritis therapy.

As an incurable metabolic disease, gouty arthritis (GA) requires long-term treatment with frequent drug administration several times per day. Compared to non-specific small organic medications, interleukin-1ß (IL-1ß) blocking therapies, such as IL-1 receptor antagonist (IL-1Ra), show great therapeutic potential in clinical trials of GA. However, IL-1Ra application is starkly limited due to its short half-life and poor bioavailability. Herein, we demonstrate a new type of nanotherapeutic formulation via noncovalent assembly of an engineered IL-1Ra chimera protein. PEGylation was employed to induce such assembly by exploiting electrostatic complexation and hydrophobic interactions. The engineered protein nanoparticles had a combination of biocompatibility, improved bioavailability and therapeutic performance. It showed extraordinary long-term anti-inflammatory effect and robust bio-efficacy for GA therapy in acute GA rat models. Strikingly, this nanoprotein system possesses an ultralong half-life of 27 hours and a bioavailability 7 times higher than that of pristine IL-1Ra, thus extending the dosing interval from several hours to more than 3 days. Therefore, our noncovalent assembly strategy via an engineered chimeric protein empowers the construction of potent delivery nanosystems for efficient GA treatment, and this might be adapted for other therapeutics to form long-acting formulations.

Anti-inflammatory effect of IL-1ra-loaded dextran/PLGA microspheres on Porphyromonas gingivalis lipopolysaccharide-stimulated macrophages in vitro and in vivo in a rat model of periodontitis.

Periodontitis is a multifactorial chronic infectious disease leading to a host immune response involving inflammatory cytokines, especially IL-1ß, which is the main reason for further developing this disease. IL-1 receptor antagonist (IL-1ra) binds IL-1 receptor, inhibiting IL-1ß signaling and reducing the levels of other cytokines closely related to periodontitis, such as IL-6 and TNF-α. Therefore, the use of IL-1ra to inhibit periodontitis development in a system, ensuring its sustained release, might be an effective way to combat this disease. Hence, in this study, a novel IL-1ra-loaded dextran/PLGA microsphere was developed to allow the sustained release of IL-1ra and enhance the anti-inflammatory properties. Therefore, this study's purposes were to develop a novel periodontal treatment for inhibition and treatment of periodontitis and evaluate the sustained-release effect and anti-inflammatory properties of IL-1ra-loaded dextran/PLGA microspheres in vitro by cell experiments and in vivo by animal experiments. The results showed that IL-1ra-loaded dextran/PLGA microspheres were non-toxic both in vitro and in vivo and could be used as a safe and effective treatment. In addition, these microspheres could significantly prolong the half-life of IL-1ra drug, exerting a useful anti-inflammatory effect in macrophages stimulated with P. gingivalis lipopolysaccharide and in rats with periodontitis. In conclusion, IL-1ra-loaded dextran/PLGA microsphere might be a useful tool to combat periodontal disease.

Interleukin-1 receptor antagonist, interleukin-2 receptor alpha subunit and amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: To clarify the causal associations of interleukin-1 receptor antagonist (IL-1ra) and interleukin-2 receptor alpha subunit (IL-2rα) with the risk of amyotrophic lateral sclerosis (ALS). METHODS: A two-sample Mendelian randomization study design was employed. Single-nucleotide polymorphisms associated with IL-1ra (n = 2) and IL-2rα (n = 1) at the genome-wide significance level were used as unbiased instrumental variables. Summary-level data for ALS were obtained from Project MinE, an international collaboration consortium with 12 577 ALS cases and 23 475 controls of European descent. RESULTS: Genetic predisposition to higher levels of IL-1ra was significantly associated with lower odds of ALS. For a 1-SD increase of circulating IL-1ra levels, the odds ratio of ALS was 0.64 (95% confidence intervals, 0.46-0.88; P = 0.005). There was a borderline inverse association between IL-2rα levels and ALS (odds ratio, 0.91; 95% confidence intervals, 0.83-1.00; P = 0.058). CONCLUSIONS: Interleukin-1 receptor antagonist levels were inversely associated with ALS, suggesting that interleukin-1 inhibitors may lower the risk of this always fatal disease. The role of IL-2rα levels in ALS needs further verification in causal inference studies with larger sample sizes.

Nanobubbles in Reconstituted Lyophilized Formulations: Interaction With Proteins and Mechanism of Formation.

Reconstitution of lyophilized disaccharide formulations results in the formation of nanosized air bubbles that persist in suspension for weeks. If proteins are present, interactions with nanobubbles may cause loss of monomeric protein and formation of subvisible particles. The goals of this work are to determine the mechanism(s) by which nanobubbles form in reconstituted lyophilized formulations and to develop strategies for reducing nanobubble generation. We hypothesize that nanobubbles are created from nanosized gas pockets within lyophilized solids, which become bubbles when the surrounding matrix is dissolved away during reconstitution. Nanosized voids may originate from small ice crystals formed within the concentrated liquid during freezing that subsequently sublime during drying. Nanobubble concentrations are correlated with the extent of mannitol crystallization during freezing. Nanosized ice crystals, induced by the release of water during mannitol crystallization, were responsible for nanobubble formation. The presence of trehalose or sucrose, in formulations with low mannitol concentrations, inhibited excipient crystallization during lyophilization and reduced nanobubble levels following reconstitution. Our results show a correlation between nanobubble formation and concentrations of insoluble IL-1ra aggregates, suggesting that minimizing nanobubble generation may be an effective strategy for reducing protein aggregation following reconstitution.

Preparation and Evaluation of IL-1ra-Loaded Dextran/PLGA Microspheres for Inhibiting Periodontal Inflammation In Vitro.

Periodontitis is a chronic infectious disease, the course and progression of which are determined by the interaction between microorganisms and the host. Interleukin 1ß plays an important role in the destruction of periodontal tissues. Interleukin 1 receptor antagonist (IL-1ra) can inhibit the biological activity of IL-1ß without triggering any intracellular signaling. This study aimed to prepare IL-1ra-loaded dextran/PLGA microspheres and evaluate the physical and chemical characteristics and anti-inflammatory properties. Results suggested that the microspheres can be easily prepared into a drug carrier with good biocompatibility and can effectively inhibit the gene expression of pro-inflammatory factors induced by IL-1ß in human gingival fibroblasts. Hence, the microspheres are excellent candidate for periodontitis treatment.

PASylation of IL-1 receptor antagonist (IL-1Ra) retains IL-1 blockade and extends its duration in mouse urate crystal-induced peritonitis.

Interleukin-1 (IL-1) is a key mediator of inflammation and immunity. Naturally-occurring IL-1 receptor antagonist (IL-1Ra) binds and blocks the IL-1 receptor-1 (IL-1R1), preventing signaling. Anakinra, a recombinant form of IL-1Ra, is used to treat a spectrum of inflammatory diseases. However, anakinra is rapidly cleared from the body and requires daily administration. To create a longer-lasting alternative, PASylated IL-1Ra (PAS-IL-1Ra) has been generated by in-frame fusion of a long, defined-length, N-terminal Pro/Ala/Ser (PAS) random-coil polypeptide with IL-1Ra. Here, we compared the efficacy of two PAS-IL-1Ra molecules, PAS600-IL-1Ra and PAS800-IL-1Ra (carrying 600 and 800 PAS residues, respectively), with that of anakinra in mice. PAS600-IL-1Ra displayed markedly extended blood plasma levels 3 days post-administration, whereas anakinra was undetectable after 24 h. We also studied PAS600-IL-1Ra and PAS800-IL-1Ra for efficacy in monosodium urate (MSU) crystal-induced peritonitis. 5 days post-administration, PAS800-IL-1Ra significantly reduced leukocyte influx and inflammatory markers in MSU-induced peritonitis, whereas equimolar anakinra administered 24 h before MSU challenge was ineffective. The 6-h pretreatment with equimolar anakinra or PAS800-IL-1Ra before MSU challenge similarly reduced inflammatory markers. In cultured A549 lung carcinoma cells, anakinra, PAS600-IL-1Ra, and PAS800-IL-Ra reduced IL-1α-induced IL-6 and IL-8 levels with comparable potency. In human peripheral blood mononuclear cells, these molecules suppressed Candida albicans-induced production of the cancer-promoting cytokine IL-22. Surface plasmon resonance analyses revealed significant binding between PAS-IL-1Ra and IL-1R1, although with a slightly lower affinity than anakinra. These results validate PAS-IL-1Ra as an active IL-1 antagonist with marked in vivo potency and a significantly extended half-life compared with anakinra.

Injectable biomaterials for delivery of interleukin-1 receptor antagonist: Toward improving its therapeutic effect.

Interleukin-1 receptor antagonist (IL-1Ra) is a naturally occurring anti-inflammatory cytokine that inhibits IL-1 activity and has been proposed to treat a wide variety of systemic and local inflammatory pathologies for multiple decades. However, the short half-life and high concentration required to inhibit IL-1 activity has limited its use in clinical applications. Many strategies have been developed with the goal of improving the therapeutic efficacy of IL-1Ra for a variety of pathologies, including fusing IL-1Ra to protein/peptide/polymer partners, releasing IL-1Ra from injectable polymer or mineral particles, and release of IL-1Ra from injectable coacervates and gels. This literature review examines injectable biomaterials engineered to improve IL-1Ra delivery, both locally and systemically, to increase its efficacy and ease of use in clinic. STATEMENT OF SIGNIFICANCE: Interleukin-1 receptor antagonist (IL-1Ra) is a therapeutic protein with the potential to treat numerous inflammatory conditions and diseases. However, its short biological half-life and high therapeutic concentration may limit its utility in all but a few clinical scenarios. In this review, we present the biomaterial based delivery strategies which have been explored to deliver IL-1Ra to improve its efficacy and applicability to treat inflammation.

How Can Interleukin-1 Receptor Antagonist Modulate Distinct Cell Death Pathways?

Multiple mechanisms of cell death exist (apoptosis, necroptosis, pyroptosis) and the subtle balance of several distinct proteins and inhibitors tightly regulates the cell fate toward one or the other pathway. Here, by combining coimmunoprecipitation, enzyme assays, and molecular simulations, we ascribe a new role, within this entangled regulatory network, to the interleukin-1 receptor antagonist (IL-1Ra). Our study enlightens that IL-1Ra, which usually inhibits the inflammatory effects of IL-1α/ß by binding to IL-1 receptor, under advanced pathological states prevents apoptosis and/or necroptosis by noncompetitively inhibiting the activity of caspase-8 and -9. Consensus docking, followed by cumulative 10 µs of molecular dynamics simulations unprecedentedly reveal that IL-1Ra binds both caspases at their dimeric interface, preventing, in this manner, the formation of their catalytically/signaling active form. The resulting IL-1Ra/caspase-8(9) adducts are stabilized by hydrophobic and by few key hydrogen bonding interactions, formed by residues fully conserved across distinct caspases (-3, -6, -7, -8, and -9), and closely resemble the binding mode of the caspases inhibitors XIAP (X-linked inhibitor of apoptosis) and c-FLIP (cellular FLICE-like inhibitory protein). Tight regulation of the different forms of cell death has a major impact on distinct human illnesses (i.e., cancer, neurodegeneration, ischemic injury, atherosclerosis, viral/bacterial infections, and immune reaction). Hence, our study, pinpointing IL-1Ra as new actor of the intricate cell death regulatory network and gaining an atomic-scale understanding of its mechanism may open new avenues toward innovative therapeutic strategies to tackle major human diseases.

IL-1RA VNTR and IL-1α 4845G>T polymorphisms and risk of idiopathic male infertility in Iranian men: A case-control study and an in silico analysis.

This study aimed to investigate the association of IL-1RA VNTR and IL-1α 4845G>T polymorphisms with idiopathic male infertility followed by an in silico analysis. In a case-control study, we collected blood samples from 230 infertile and 230 healthy men. Genotyping of IL-1RA VNTR was performed by PCR whereas IL-1α 4845G>T was genotyped by polymerase chain reaction-restriction fragment length polymorphism. An in silico approach was employed for the detection of IL-1RA VNTR and IL-1α 4845G>T effects on some molecular aspects of IL-1RA and IL-1α respectively. The result of our genetic association study for IL-1α 4845G>T revealed that there was a significant association between GT genotype, TT genotype, T allele and idiopathic male infertility. Although there was no significant association between IL-1RA VNTR and male infertility in the overall analysis. However, subgroup analysis revealed that the subjects with VNTR 4R/5R genotype were at a higher risk of oligozoospermia. Furthermore, 4845TT genotype, and 4845T allele were associated with oligozoospermia, asthenozoospermia and nonobstructive azoospermia. Bioinformatics analysis showed that IL-1RA VNTR may affect the splicing pattern of IL-1RA. Moreover, IL-1α 4845G>T has a significant effect on RNA structure and protein function. Based on our findings, both IL-1RA VNTR and IL-1α 4845G>T polymorphisms could be considered as potential biomarkers for screening of susceptible individuals.

Protein-protein interactions controlling interfacial aggregation of rhIL-1ra are not described by simple colloid models.

We investigated the effects of protein-protein interaction strength on interfacial viscoelastic properties and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) at silicone oil-water interfaces. Osmotic second virial coefficients determined by static light scattering were used to quantify protein-protein interactions in bulk solution. Attractive protein-protein interactions dominated at low ionic strengths and their magnitude decreased with increasing ionic strength, in contrast to repulsive interactions that would be expected based on uniformly charged sphere models. Interfacial shear rheometry was used to characterize rhIL-1ra interfacial layers. More attractive protein-protein interactions in bulk solution correlated with stronger interfacial gels. Thioflavin-T fluorescence measurements indicated that the intermolecular ß-sheet content of rhIL-1ra incubated in the presence of silicone oil-water interfaces correlated with gel strength. Siliconized syringes were used to probe the effects of mechanical perturbation of the interfacial gel layers. When rhIL-1ra solutions in siliconized glass syringes were subjected to end-over-end rotation, monomeric rhIL-1ra was lost from solution, and particles containing aggregated protein were released into the bulk aqueous phase. The loss of monomeric rhIL-1ra in response to mechanical perturbation was highest under the conditions where the strongest gels were observed. Aggregation of rhIL-1ra was strictly interface-induced and growth of aggregates in the bulk solution was not observed, even in the presence of particles released from silicone oil-water interfaces.

The polypeptide biophysics of proline/alanine-rich sequences (PAS): Recombinant biopolymers with PEG-like properties.

PAS polypeptides comprise long repetitive sequences of the small L-amino acids proline, alanine and/or serine that were developed to expand the hydrodynamic volume of conjugated pharmaceuticals and prolong their plasma half-life by retarding kidney filtration. Here, we have characterized the polymer properties both of the free polypeptides and in fusion with the biopharmaceutical IL-1Ra. Data from size exclusion chromatography, dynamic light scattering, circular dichroism spectroscopy and quantification of hydrodynamic and polar properties demonstrate that the biosynthetic PAS polypeptides exhibit random coil behavior in aqueous solution astonishingly similar to the chemical polymer poly-ethylene glycol (PEG). The solvent-exposed PAS peptide groups, in the absence of secondary structure, account for strong hydrophilicity, with negligible contribution by the Ser side chains. Notably, PAS polypeptides exceed PEG of comparable molecular mass in hydrophilicity and hydrodynamic volume while exhibiting lower viscosity. Their uniform monodisperse composition as genetically encoded polymers and their biological nature, offering biodegradability, render PAS polypeptides a promising PEG mimetic for biopharmaceutical applications.

Inflammatory 'double hit' model of temporomandibular joint disorder with elevated CCL2, CXCL9, CXCL10, RANTES and behavioural hypersensitivity in TNFR1/R2-/- mice.

BACKGROUND: Patients with temporomandibular joint disorders (TMD), reactive arthritis and rheumatoid arthritis often have combined etiology of hereditary and microenvironmental factors contributing to joint pain. Multiple clinical and animal studies indicate 'double-hit' inflammatory insults can cause chronic inflammation. The first inflammatory insult primes the immune system and subsequent insults elicit amplified responses. The present 'double hit' study produced a chronic orofacial pain model in mice with genetic deletion of both TNFα receptors (TNFR1/R2-/-), investigating the main nociceptive signalling pathways in comparisons to wild type mice. METHODS: An initial inflammatory insult was given unilaterally into the temporomandibular joint (TMJ). Secondary hypersensitivity was tested on the skin over the TMJ throughout the experiment. Three weeks later after complete reversal of hypersensitivity, a second inflammatory insult was imposed on the colon. Pharmacological interventions were tested for efficacy after week 10 when hypersensitivity was chronic in TNFR1/R2-/- mice. Serum cytokines were analysed at Days 1, 14, and Week 18. RESULTS: The double hit insult produced chronic hypersensitivity continuing through the 4-month experimental timeline in the absence of TNFα signalling. P2X7 and NMDA receptor antagonists temporarily attenuated chronic hypersensitivity. Serum cytokine/chemokine analysis on Day 14 when CFA induced hypersensitivity was resolved identified increased levels of pro-inflammatory cytokines CCL2, CXCL9, CXCL10, RANTES and decreased levels of anti-inflammatory cytokines IL-1ra and IL-4 in TNFR1/R2-/- compared to WT mice. CONCLUSIONS: These data suggest a causal feed-forward signalling cascade of these little studied cytokines have the potential to cause recrudescence in this orofacial inflammatory pain model in the absence of TNFα signalling. SIGNIFICANCE: Using a mouse model of chronic inflammatory temporomandibular joint disorder, we determined that absence of functional TNFR1/R2 induces aberrant inflammatory signalling caused by other increased pro-inflammatory and decreased anti-inflammatory cytokines that could serve as blood biomarkers and may predict disease progression.

Evaluating the autoinduction expression system and one-step purification for high-level expression and purification of gallbladder-derived rhIL-1Ra.

Recent advancement in fermentation technologies resulted in the increased yields of recombinant proteins of biopharmaceutical and medicinal importance. Consequently, there is an important task to develop simple and easily scalable methods that can facilitate the production of high-quality recombinant protein. Most of the recent reports described the expression of recombinant human IL-1 receptor antagonist (rhIL-1Ra) in Escherichia coli using isopropyl-ß-d-thiogalacto pyranoside (IPTG), a nonmetabolizable and expensive compound, as an expression inducer. In this study, we describe the expression and one-step purification of gallbladder-derived rhIL-1Ra by autoinduction in E. coli. This method includes special media that automatically induce the target protein expression from T7 promoter and allow the production of the target protein in high yield than the conventional IPTG induction method. In addition to fermentation process improvements, one-step purification strategy is essential to make the process economical. We developed a single-step cation exchange chromatography and obtained 300 mg/L of rhIL-1Ra with 98% purity. Purified protein was characterized by SDS-PAGE and Ion exchange HPLC (IEX-HPLC). The described method can be used to scale up the production of rhIL-1Ra and other recombinant proteins.

Effects of different orthodontic retention protocols on the periodontal health of mandibular incisors.

OBJECTIVES: To test the following two hypotheses: 1) different types of retainers result in distinct levels of biomarkers in gingival crevicular fluid (GCF) and 2) the retainer bonded to all mandibular anterior teeth induces more detrimental outcomes to the periodontium. SETTING AND SAMPLE POPULATION: The Department of Orthodontics at the University of Florida. The population consisted of individuals in the retention phase of orthodontic treatment. MATERIAL AND METHODS: This was a cross-sectional study that enrolled 36 individuals. Subjects in group 1 had retainers bonded to the mandibular canines only. Group 2 consisted of individuals having retainers bonded to all mandibular anterior teeth. Group 3 included patients using mandibular removable retainers. After clinical examination, GCF was collected from the mandibular incisor and biomarker levels were compared between the groups. RESULTS: Plaque accumulation and gingivitis differed significantly among groups, with the highest median values in group 2 subjects. Pairwise comparison of the groups with respect to gingivitis showed significant differences between groups 1 and 2. Significant differences among groups were detected for RANKL, OPG, OPN, M-CSF, MMP-3, and MMP-9. The ratio RANKL/OPG was significantly higher in group 2 subjects, with pairwise comparisons indicating that groups 1 and 2 differed from group 3. CONCLUSION: An association was found between orthodontic retention groups and GCF biomarker levels, which should be further explored in longitudinal studies. The presence of retainers bonded to all anterior teeth seems to increase plaque accumulation and gingivitis.

Particle Formation and Aggregation of a Therapeutic Protein in Nanobubble Suspensions.

The generation of nanobubbles following reconstitution of lyophilized trehalose formulations has recently been reported. Here, we characterize particle formation and aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in reconstituted formulations of lyophilized trehalose. Particle characterization methods including resonant mass measurement and nanoparticle tracking analysis were used to count and size particles generated upon reconstitution of lyophilized trehalose formulations. In addition, accelerated degradation studies were conducted to monitor rhIL-1ra aggregation in solutions containing various concentrations of suspended nanobubbles. Reconstitution of lyophilized trehalose formulations with solutions containing rhIL-1ra reduced nanobubble concentrations and generated negatively buoyant particles attributed to aggregated rhIL-1ra. Furthermore, levels of rhIL-1ra aggregation following incubation in aqueous solution correlated with concentrations of suspended nanobubbles. The results of this study suggest that nanobubbles may be a contributor to protein aggregation and particle formation in reconstituted, lyophilized therapeutic protein formulations.

Role of interleukin-1 receptor 1/MyD88 signalling in the development and progression of pulmonary hypertension.

Pulmonary artery smooth muscle cell (PA-SMC) proliferation and inflammation are key components of pulmonary arterial hypertension (PAH). Interleukin (IL)-1ß binds to IL-1 receptor (R)1, thereby recruiting the molecular adaptor myeloid differentiation primary response protein 88 (MyD88) (involved in IL-1R1 and Toll-like receptor signal transduction) and inducing IL-1, IL-6 and tumour necrosis factor-α synthesis through nuclear factor-κB activation.We investigated the IL-1R1/MyD88 pathway in the pathogenesis of pulmonary hypertension.Marked IL-1R1 and MyD88 expression with predominant PA-SMC immunostaining was found in lungs from patients with idiopathic PAH, mice with hypoxia-induced pulmonary hypertension and SM22-5-HTT(+) mice. Elevations in lung IL-1ß, IL-1R1, MyD88 and IL-6 preceded pulmonary hypertension in hypoxic mice. IL-1R1(-/-), MyD88(-/-) and control mice given the IL-1R1 antagonist anakinra were protected similarly against hypoxic pulmonary hypertension and perivascular macrophage recruitment. Anakinra reversed pulmonary hypertension partially in SM22-5-HTT(+) mice and markedly in monocrotaline-treated rats. IL-1ß-mediated stimulation of mouse PA-SMC growth was abolished by anakinra and absent in IL-1R1(-/-) and MyD88(-/-) mice. Gene deletion confined to the myeloid lineage (M.lys-Cre MyD88(fl/fl) mice) decreased pulmonary hypertension severity versus controls, suggesting IL-1ß-mediated effects on PA-SMCs and macrophages. The growth-promoting effect of media conditioned by M1 or M2 macrophages from M.lys-Cre MyD88(fl/fl) mice was attenuated.Pulmonary vessel remodelling and inflammation during pulmonary hypertension require IL-1R1/MyD88 signalling. Targeting the IL-1ß/IL-1R1 pathway may hold promise for treating human PAH.

Immunoassay analysis of proteins in gingival crevicular fluid samples from resorbing teeth.

OBJECTIVE: To carry out an immunoassay analysis of biomarkers expressed in gingival crevicular fluid (GCF) with the main goal of finding a useful diagnostic pattern to distinguish between resorbing deciduous teeth and nonresorbing controls. MATERIALS AND METHODS: A split-mouth design was used in this study with a total of 22 GCF samples collected from 11 patients in the mixed dentition. For each child, one deciduous molar with radiographic evidence of root resorption was used as the test tooth whereas the contralateral first permanent molar with formed roots was used as the control tooth. Samples were processed with immunoassays using a panel of selected biomarkers including interleukin-1 beta (IL-1b), interleukin-1 receptor antagonist (IL-1RA), nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), matrix metalloproteinase-9 (MMP-9), and dentin sialoprotein (DSP). RESULTS: There were no statistically significant differences in levels of IL-1b, OPG, and MMP-9 between test and control sites (P > .05). IL-1RA was the only biomarker to show a significant down-regulation (P  =  .04) in GCF samples collected from resorbing teeth. RANKL data showed a heavily skewed distribution and was deemed unreliable. Only one deciduous GCF sample had detectable levels of DSP; therefore, no further statistical calculation was applicable because of the limited amount of data for this biomarker. CONCLUSIONS: This study indicated that IL1-RA is down-regulated in GCF from resorbing primary molars, thus suggesting this cytokine as a potential analyte to be included in a panel that can discriminate between resorbing and nonresorbing teeth.

Sustained intra-articular delivery of IL-1RA from a thermally-responsive elastin-like polypeptide as a therapy for post-traumatic arthritis.

Post-traumatic arthritis (PTA) is a rapidly progressive form of arthritis that develops due to joint injury, including articular fracture. Current treatments are limited to surgical restoration and stabilization of the joint; however, evidence suggests that PTA progression is mediated by the upregulation of pro-inflammatory cytokines, such as interleukin-1 (IL-1) or tumor necrosis factor-α (TNF-α). Although these cytokines provide potential therapeutic targets for PTA, intra-articular injections of anti-cytokine therapies have proven difficult due to rapid clearance from the joint space. In this study, we examined the ability of a cross-linked elastin-like polypeptide (xELP) drug depot to provide sustained intra-articular delivery of IL-1 and TNF-α inhibitors as a beneficial therapy. Mice sustained a closed intra-articular tibial plateau fracture; treatment groups received a single intra-articular injection of drug encapsulated in xELP. Arthritic changes were assessed 4 and 8 weeks after fracture. Inhibition of IL-1 significantly reduced the severity of cartilage degeneration and synovitis. Inhibition of TNF-α alone or with IL-1 led to deleterious effects in bone morphology, articular cartilage degeneration, and synovitis. These findings suggest that IL-1 plays a critical role in the pathogenesis of PTA following articular fracture, and sustained intra-articular cytokine inhibition may provide a therapeutic approach for reducing or preventing joint degeneration following trauma.