Pharmacodynamic biomarkers of long-term interferon beta-1a therapy in REFLEX and REFLEXION.

This post-hoc analysis evaluated candidate biomarkers of long-term efficacy of subcutaneous interferon beta-1a (sc IFN ß-1a) in REFLEX/REFLEXION studies of clinically isolated syndrome. Samples from 507 REFLEX and 287 REFLEXION study participants were analyzed. All investigated biomarkers were significantly upregulated 1.5-4-fold in response to sc IFN ß-1a treatment versus baseline (p ≤ 0.008). The validity of MX1, 2'5'OAS, and IL-1RA as biomarkers of response to sc IFN ß-1a was confirmed in this large patient cohort, with biomarkers consistently upregulated in a dose-dependent manner. Neopterin, TRAIL, and IP-10 were confirmed as biomarkers associated with long-term sc IFN ß-1a treatment efficacy over 5 years.

Recombinant Production and One-Step Purification of IL-1Ra in Escherichia coli and Evaluation of its IL-1 Antagonizing Efficacy.

BACKGROUND: Anakinra (Kineret®), an IL-1 receptor antagonist, is the first FDA-approved biologic drug for antagonizing IL-1 in patients with Rheumatoid arthritis. The less expensive production of this drug might help reduce the final therapeutic costs. OBJECTIVES: To evaluate the possibility of producing biologically active recombinant IL-1Ra by a single-step purification procedure mediated by a self-cleavable intein. METHODS: Soluble expression of the rIL-1Ra was performed in E. coli BL21 (DE3) infusion to intein1 of pTWIN-1 vector and its cleavage induction using an elution buffer (pH 6.8) at room temperature. Evaluation of the antagonizing efficacy of this protein in various concentrations was performed on A375 and HEK293 cells treated by a constant concentration of IL-1ß (2 ng/mL). RESULTS: IPTG induction of E. coli BL21 (DE3) transformed with the recombinant pTWIN-1, revealed a band approximately in 45 kDa, which is related to the intein1-rIL-1Ra fusion protein in the SDS-PAGE. Moreover, protein purification was confirmed by observing a band in 18 kDa. Finally, the percentage of inhibition effects of rIL-1Ra and Kineret® against IL-1ß was not statistically significant in IL-1-responsive A375 cells. The inhibition percentage was calculated as 86% in cells treated with 15µg/mL of rIL-1Ra, which was 96% for the inhibitory effects of the standard drug. CONCLUSION: In this study, biologically active soluble rIL1-Ra was successfully produced with high purity through a one-step procedure. This method can reduce the cost and time of production for this protein and might be applicable other biological products.

Microbial therapeutics for acute colitis based on genetically modified Lactococcus lactis hypersecreting IL-1Ra in mice.

The increased incidence of inflammatory bowel disease (IBD) in Western and rapidly Westernizing developing countries poses a global pandemic threat. The development of affordable drugs for treating IBD worldwide is thus a priority. Genetically modified lactic acid bacteria (gmLAB) as microbial therapeutics are inexpensive protein producers suitable for use as carriers of protein to the intestinal mucosa. Here, we successfully constructed gmLAB hypersecreting interleukin 1 receptor antagonist (IL-1Ra). Oral administration of these gmLAB suppressed body weight reduction and exacerbation of the disease activity index score in mice with acute colitis and decreased the number of CD4+ IL-17A+ cells in the mesenteric lymph nodes. These data suggest that the gmLAB deliver IL-1Ra to the colon, where it inhibits IL-1 signaling. We thus developed a novel IBD therapeutic that blocks IL-1 signaling using a gmLAB protein delivery system. This system could be an inexpensive oral microbial therapeutic.

Emerging Approaches for Regulation and Control of CAR T Cells: A Mini Review.

Chimeric antigen receptor (CAR) T cells have emerged as a promising treatment for patients with advanced B-cell cancers. However, widespread application of the therapy is currently limited by potentially life-threatening toxicities due to a lack of control of the highly potent transfused cells. Researchers have therefore developed several regulatory mechanisms in order to control CAR T cells in vivo. Clinical adoption of these control systems will depend on several factors, including the need for temporal and spatial control, the immunogenicity of the requisite components as well as whether the system allows reversible control or induces permanent elimination. Here we describe currently available and emerging control methods and review their function, advantages, and limitations.

Evaluation of Tear Protein Markers in Dry Eye Disease with Different Lymphotoxin-Alpha Expression Levels.

PURPOSE: To compare tear protein markers between normal subjects and patients with dry eye (DE) and high and low lymphotoxin-alpha (LT-α) levels. DESIGN: Prospective cross-sectional study. METHODS: Patients with DE were divided into low (≤700 pg/mL) and high (>700 pg/mL) LT-α groups. Twelve protein markers were measured by microsphere-based immunoassay and ocular surface parameters were determined in right eyes (33 high LT-α DE, 27 low LT-α DE, and 20 control eyes) and left eyes (21 high LT-α DE, 39 low LT-α DE, and 20 control eyes). RESULTS: In both eyes, tumor necrosis factor-α (TNF-α), interleukin (IL)-10, IL-1ß, IL-1 receptor antagonist (IL-1Ra), IL-17A, and IL-12/23 p40 levels in high LT-α DE were significantly higher (P < .01) than in low LT-α DE. Significant correlations identified in high LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = 0.43, P = .013), IL-1ß (R = 0.48, P = .005), and IL-12/23 p40 (R = 0.50, P = .003), IL-12/23 p40 with ocular surface disease index (R = 0.35, P = .049), and epidermal growth factor with corneal fluorescein staining score (R = -0.36, P = .038). Significant correlations in low LT-α DE were: Standard Patient Evaluation Eye Dryness with IL-10 (R = -0.39, P = .046), TNF-α (R = -0.39, P = .047), and IL-17A (R = -0.48, P = .013), ocular surface disease index with TNF-α (R = -0.47, P = .017) and IL-17A (R = -0.46, P = .018), and IL-6 with tear breakup time (R = -0.40, P = .044). Lastly, IL-1Ra levels significantly increased in DE patients, positively correlated with temporal conjunctival hyperemia index, and negatively correlated with Schirmer I test (P < .05). CONCLUSIONS: Our study identified tear IL-1Ra level as a potential biomarker to replace the Schirmer I test. Multiple tear protein marker levels increased in high LT-α DE, indicating that high LT-α DE might have a different pathogenesis.

Crystallized but not soluble uric acid elicits pro-inflammatory response in short-term whole blood cultures from healthy men.

Several epidemiological studies have pointed at serum uric acid (SUA) as an independent risk factor for mortality, diabetes, hypertension, cardiovascular and kidney disease; however, no clear pathogenic pathway is established. Uric acid (UA) crystals show pro-inflammatory properties and can thus create or contribute to the state of chronic low-grade inflammation, a widely accepted pathogenic mechanism in several of the above-mentioned pathologies. On the other hand, soluble uric acid possesses antioxidant properties that might attenuate inflammatory responses. We aimed to explore the net effects of experimentally rising SUA in human whole blood cultures on several mediators of inflammation. Production of TNF-α, IL-1ß, IL-1RA, MCP-1 and IL-8 was assessed upon addition of 200 µM UA, 500 µM UA or monosodium urate (MSU) crystals in the presence or absence of 5 ng/ml lipopolysaccharide (LPS). RT-qPCR and multiplex bead based immunoassay were used to measure mRNA expression and cytokine release at 2 and 4 h of culture, respectively. 14C labeled UA was used to assess intracellular uptake of UA. We show that crystallized, but not soluble, UA induces production of pro-inflammatory mediators in human whole blood. Soluble UA is internalized in blood cells but does not potentiate or reduce LPS-induced release of cytokines.

A Synthetic Gene Circuit for Self-Regulating Delivery of Biologic Drugs in Engineered Tissues.

IMPACT STATEMENT: We engineered a synthetic transcription system based on nuclear factor kappa-light-chain-enhancer of activated B cells signaling that can attenuate the effects of the inflammatory cytokine interleukin (IL)-1α in a self-regulating manner. This system responds in a time- and dose-dependent manner to rapidly produce therapeutic levels of IL-1 receptor antagonist (IL-1Ra). The use of lentiviral gene therapy allows this system to be utilized through different transduction methods and in different cell types for a variety of applications. Broadly, this approach may be applicable in developing autoregulated biologic systems for tissue engineering and drug delivery in a range of disease applications.

IL1RN mediates the suppressive effect of methionine deprivation on glioma proliferation.

Metabolic abnormality is one of the hallmarks of cancer cells, and limiting material supply is a potential breakthrough approach for cancer treatment. Increasing researchers have been involved in the study of glioma cell metabolism reprogramming since the significance of IDH1 was confirmed in glioma. However, the molecular mechanisms underlying metabolic reprogramming induced by methionine deprivation regulates glioma cell proliferation remain unclear. Here we demonstrated that methionine deprivation inhibited glioma cell proliferation via downregulating interleukin 1 receptor antagonist (IL1RN) both in vitro and in vivo, methionine deprivation or knocking down IL1RN induced glioma cell cycle arrest. Moreover, we confirmed that IL1RN is a tumor associated gene and its expression is negatively correlated with the survival time of glioma patients. Altogether these results demonstrate a strong rationale insight that targeting amino acid metabolism such as methionine deprivation/IL1RN related gene therapy may offer novel direction for glioma treatment.

Combinatorial Prg4 and Il-1ra Gene Therapy Protects Against Hyperalgesia and Cartilage Degeneration in Post-Traumatic Osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of synovial joints characterized by progressive loss of articular cartilage, subchondral bone remodeling, and intra-articular inflammation with synovitis that results in chronic pain and motor impairment. Despite the economic and health impacts, current medical therapies are targeted at symptomatic relief of OA and fail to alter its progression. Given the complexity of OA pathogenesis, we hypothesized that a combinatorial gene therapy approach, designed to inhibit inflammation with interleukin-1 receptor antagonist (IL-1Ra) while promoting chondroprotection using lubricin (PRG4), would improve preservation of the joint compared to monotherapy alone. Employing two surgical techniques to model mild, moderate and severe posttraumatic OA, we found that combined delivery of helper-dependent adenoviruses (HDVs), expressing IL-1Ra and PRG4, preserved articular cartilage better than either monotherapy in both models as demonstrated by preservation of articular cartilage volume and surface area. This improved protection was associated with increased expression of proanabolic and cartilage matrix genes together with decreased expression of catabolic genes and inflammatory mediators. In addition to improvements in joint tissues, this combinatorial gene therapy prolonged protection against thermal hyperalgesia compared to either monotherapy. Taken together, our results show that a combinatorial strategy is superior to monotherapeutic approaches for treatment of posttraumatic OA.

Patients with type 1 diabetes mellitus have impaired IL-1ß production in response to Mycobacterium tuberculosis.

Patients with diabetes mellitus have an increased risk of developing tuberculosis. Although the underlying mechanism is unclear, evidence suggests a role for chronic hyperglycaemia. We examined the influence of hyperglycaemia on Mycobacterium tuberculosis-induced cytokine responses in patients with type 1 diabetes mellitus (T1D). Peripheral blood mononuclear cells (PBMCs) from 24 male T1D patients with sub-optimal glucose control [HbA1c > 7.0% (53 mmol/L)] and from 24 age-matched male healthy controls were stimulated with M. tuberculosis lysate. Cytokine analysis, assessment of aerobic glycolysis, receptor recognition and serum cross-over experiments were performed to explore the mechanistic differences. PBMCs from T1D patients produced less bioactive interleukin (IL)-1ß in response to M. tuberculosis. IL-6 and interferon (IFN)-γ production trended towards a decrease, whilst other cytokines such as tumour necrosis factor (TNF)-α, IL-17 and IL-1Ra were normal. The decrease in cytokine production was not correlated to HbA1c or plasma glucose levels. Cross-over serum experiments did not alter the cytokine profile of T1D or control patients, arguing for an intrinsic cellular defect. Cellular metabolism and the expression of M. tuberculosis-related pattern recognition receptors (PRRs) such as TLR2, TLR4 and NOD2 did not differ between T1D patients and healthy controls. Compared to matched controls, T1D patients have a reduced capacity to produce pro-inflammatory cytokines in response to M. tuberculosis. The impaired IL-1ß production in T1D patients may contribute to the increased susceptibility to tuberculosis. This effect appears not to be related to prevailing glucose levels but to an intrinsic cellular deficit.

Expression of negative immune regulatory molecules, pro-inflammatory chemokine and cytokines in immunopathology of ECM developing mice.

The pathological events in human cerebral malaria are mimicked in the experimental cerebral malaria (ECM) in Plasmodium berghei ANKA (PBA)-infected C57BL/6 mice. Although previously implied in ECM, the kinetics of cytokines and chemokines expression-an essential functional feature for defining causality in ECM development-remained untested. Herein, we characterized the immunopathological changes and the expression of negative immune regulatory molecules, cytokines and chemokines through asymptomatic (3days after infection, 3dpi), symptomatic (5dpi) and ECM (7dpi) stages in PBA-infected C57BL/6 mice. Parasitized RBCs were first detected in brain on 3dpi, edema and tissue alterations on 5dpi, and hemorrhages in different areas of brain on 7dpi. Increased cerebellar PD-1, CTLA-4 and LAG-3 expression and reduced hippocampal CXCL-4 expression on 3dpi were the first observed immunological changes. The negative immune regulatory molecules (PD-L1, CTLA-4), cytokines (TNF-α, sFAS-L), and chemokines (CXCL-10, MIP-1ß) transcript levels varied in different brain areas in symptomatic and ECM phases. By 5dpi, TNF-α, CXCL10 and MIP-1ß significantly increased in all brain parts studied; IL-1RA in whole brain, whereas CXCL4 reduced in hippocampus and cerebrum. By 7dpi, the hippocampal PD-1, CXCL4 and CTLA-4 expression decreased but the cerebral, cerebellar and hippocampal PD-L1 expression were elevated. TNF-α, CXCL10, MIP-1ß, PD-1, CTLA-4 and PD-L1 expression were up-regulated in different brain areas. The TNFR2, IFN-gamma receptor, Lymphotoxin-ß receptor and sFAS-L transcripts significantly increased in brain in ECM. Our data characterize key dynamic immunopathological changes in brain to imply relationship to ECM development.

Interleukin-1 receptor antagonist: an early immunomodulatory cytokine induced by porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection poorly induces pro-inflammatory cytokines (IL-1, IL-6 and TNF-α) and type I IFN production during the early phase of infection. Our microarray analysis indicated strong upregulation of the IL1RA gene in type 2 PRRSV -infected monocyte-derived dendritic cells. Interleukin-1 receptor antagonist (IL-1Ra) is an early inhibitory cytokine that suppresses pro-inflammatory cytokines and T-lymphocyte responses. To investigate the induction of IL-1Ra by PRRSV, monocyte-derived dendritic cells were cultured with type 2 PRRSV or other swine viruses. PRRSV increased both IL1RA gene expression and IL-1Ra protein production in the culture. The enhanced production of IL-1Ra was further confirmed in PRRSV-cultured PBMC and PRRSV-exposed pigs by flow cytometry. Myeloid cell population appeared to be the major IL-1Ra producer both in vitro and in vivo. In contrast to the type 2 PRRSV, the highly pathogenic (HP)- PRRSV did not upregulate IL1RA gene expression in vitro. To determine the kinetics of PRRSV-induced IL1RA gene expression in relation to other pro-inflammatory cytokine genes, PRRSV-negative pigs were vaccinated with a commercially available type 2 modified-live PRRS vaccine or intranasally inoculated with HP-PRRSV. In modified-live PRRS vaccine pigs, upregulation of IL1RA, but not IL1B and IFNA, gene expression was observed from 2 days post- vaccination. Consistent with the in vitro findings, upregulation of IL1RA gene expression was not observed in the HP-PRRSV-infected pigs throughout the experiment. This study identified IL-1Ra as an early immunomodulatory mediator that could be involved in the immunopathogenesis of PRRSV infections.

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.

Aspergillus Cell Wall Chitin Induces Anti- and Proinflammatory Cytokines in Human PBMCs via the Fc-γ Receptor/Syk/PI3K Pathway.

UNLABELLED: Chitin is an important cell wall component of Aspergillus fumigatus conidia, of which hundreds are inhaled on a daily basis. Previous studies have shown that chitin has both anti- and proinflammatory properties; however the exact mechanisms determining the inflammatory signature of chitin are poorly understood, especially in human immune cells. Human peripheral blood mononuclear cells were isolated from healthy volunteers and stimulated with chitin from Aspergillus fumigatus Transcription and production of the proinflammatory cytokine interleukin-1ß (IL-1ß) and the anti-inflammatory cytokine IL-1 receptor antagonist (IL-1Ra) were measured from the cell culture supernatant by quantitative PCR (qPCR) or enzyme-linked immunosorbent assay (ELISA), respectively. Chitin induced an anti-inflammatory signature characterized by the production of IL-1Ra in the presence of human serum, which was abrogated in immunoglobulin-depleted serum. Fc-γ-receptor-dependent recognition and phagocytosis of IgG-opsonized chitin was identified as a novel IL-1Ra-inducing mechanism by chitin. IL-1Ra production induced by chitin was dependent on Syk kinase and phosphatidylinositol 3-kinase (PI3K) activation. In contrast, costimulation of chitin with the pattern recognition receptor (PRR) ligands lipopolysaccharide, Pam3Cys, or muramyl dipeptide, but not ß-glucan, had synergistic effects on the induction of proinflammatory cytokines by human peripheral blood mononuclear cells (PBMCs). In conclusion, chitin can have both pro- and anti-inflammatory properties, depending on the presence of pathogen-associated molecular patterns and immunoglobulins, thus explaining the various inflammatory signatures reported for chitin. IMPORTANCE: Invasive aspergillosis and allergic aspergillosis are increasing health care problems. Patients get infected by inhalation of the airborne spores of Aspergillus fumigatus A profound knowledge of how Aspergillus and its cell wall components are recognized by the host cell and which type of immune response it induces is necessary to develop target-specific treatment options with less severe side effects than the treatment options to date. There is controversy in the literature about the receptor for chitin in human cells. We identified the Fc-γ receptor and Syk/PI3K pathway via which chitin can induce anti-inflammatory immune responses by inducing IL-1 receptor antagonist in the presence of human immunoglobulins but also proinflammatory responses in the presence of bacterial components. This explains why Aspergillus does not induce strong inflammation just by inhalation and rather fulfills an immune-dampening function. While in a lung coinfected with bacteria, Aspergillus augments immune responses by shifting toward a proinflammatory reaction.

Alpha-1-anti-trypsin-Fc fusion protein ameliorates gouty arthritis by reducing release and extracellular processing of IL-1ß and by the induction of endogenous IL-1Ra.

OBJECTIVES: In the present study, we generated a new protein, recombinant human alpha-1-anti-trypsin (AAT)-IgG1 Fc fusion protein (AAT-Fc), and evaluated its properties to suppress inflammation and interleukin (IL)-1ß in a mouse model of gouty arthritis. METHODS: A combination of monosodium urate (MSU) crystals and the fatty acid C16.0 (MSU/C16.0) was injected intra-articularly into the knee to induce gouty arthritis. Joint swelling, synovial cytokine production and histopathology were determined after 4 h. AAT-Fc was evaluated for inhibition of MSU/C16.0-induced IL-1ß release from human blood monocytes and for inhibition of extracellular IL-1ß precursor processing. RESULTS: AAT-Fc markedly suppressed MSU/C16.0-induced joint inflammation by 85-91% (p<0.001). Ex vivo production of IL-1ß and IL-6 from cultured synovia were similarly reduced (63% and 65%, respectively). The efficacy of 2.0 mg/kg AAT-Fc in reducing inflammation was comparable to 80 mg/kg of plasma-derived AAT. Injection of AAT-Fc into mice increased circulating levels of endogenous IL-1 receptor antagonist by fourfold. We also observed that joint swelling was reduced by 80%, cellular infiltration by 95% and synovial production of IL-1ß by 60% in transgenic mice expressing low levels of human AAT. In vitro, AAT-Fc reduced MSU/C16.0-induced release of IL-1ß from human blood monocytes and inhibited proteinase-3-mediated extracellular processing of the IL-1ß precursor into active IL-1ß. CONCLUSIONS: A single low dose of AAT-Fc is highly effective in reducing joint inflammation in this model of acute gouty arthritis. Considering the long-term safety of plasma-derived AAT use in humans, subcutaneous AAT-Fc emerges as a promising therapy for gout attacks.

Arthritic and non-arthritic synovial fluids modulate IL10 and IL1RA gene expression in differentially activated primary human monocytes.

OBJECTIVE: Synovitis with an increased presence of macrophages is observed in osteoarthritis (OA) and rheumatoid arthritis (RA). Given the important role of macrophages in arthritis, we investigated the influence of OA and RA synovial fluid (SF) on primary human monocytes (Mo), their lineage precursors. METHOD: Adherent monocytes without any stimulation (Mo(-)) or stimulated with IFN-γ and TNF-α (Mo(IFN-γ/TNF-α)) or IL-4 (Mo(IL-4)) were exposed to SF from 6 donors without any known joint disease (SF-Ctrl), 10 OA donors (SF-OA), and 10 RA donors (SF-RA). The transcriptional expression of IL6, IL1B, TNFA, IL10, CCL18, CD206, and IL1RA was analyzed. RESULTS: Mo(-) exposed to SF-RA had a lower expression of IL10 and a higher expression of IL1RA than when exposed to SF-Ctrl. Mo(IL-4) exposed to SF-RA had a lower expression of IL10 and CCL18 than when exposed to SF-Ctrl and Mo(IFN-γ/TNF-α) were not affected by SF-RA. Mo exposed to SF-OA also expressed less IL10, but only upon stimulation with IL-4, and expressed more IL1RA than when exposed to SF-Ctrl in any condition. CONCLUSION: A lower expression of IL10 may be regarded as a response to less inflammatory conditions since IL10 expression is higher in response to IFN-γ/TNF-α stimulation, probably as a feedback mechanism. Therefore, the lower expression of IL10 and the higher expression of IL1RA in Mo exposed to arthritic than to non-arthritic SF suggest that arthritic SF is mainly reducing the inflammatory responses in Mo. This may mimic the response of monocytes/macrophages recruited to the joint, where feedback mechanisms counteract pro-inflammatory processes.

Down-regulation of Immune-related Genes by PSCA in Gallbladder Cancer Cells Implanted into Mice.

BACKGROUND/AIM: In previous work, we found that prostate stem cell antigen (PSCA) gene, encoding a glycosylphosphatidylinositol-anchored protein, is a presumable tumor suppressor in gastric cancer and gallbladder cancer (GBC). The introduction of PSCA cDNA into GBC cell lines significantly suppressed tumorigenecity of cells in mice. The PSCA protein is thought to be involved in some form of intracellular signaling that remains to be elucidated. MATERIALS AND METHODS: Using microarrays, we conducted gene-expression profiling on tumors generated by a GBC cell line TGBC-1TKB, with and without expression of PSCA, which was implanted into mice. Genes whose expression was down-regulated by PSCA were selected, and their down-regulation was confirmed by real-time PCR. RESULTS: We identified several immune-related genes down-regulated by PSCA, including interleukin 8 (IL8), IL1 receptor antagonist (IL1RN) and S100 calcium-binding proteins A8 (S100A8) and A9 (S100A9). CONCLUSION: PSCA signaling may suppress tumor growth in vivo by modulating immunological characteristics of GBC cells.

[PRODUCTION OF CERTAIN PRO- AND ANTI-INFLAMMATORY CYTOKINES DURING STIMULATION BY LIVE AND INACTIVATED LEPTOSPIRA PATHOGENS IN THE MODEL OF HUMAN WHOLE BLOOD].

AIM: Study of the ability of clinical isolates of leptospira to cause production of certain pro- and antiinflammatory cytokines in the model of human whole blood. MATERIALS AND METHODS: Leptospira interrogans strain was taken for the experiment. Cytokine content was determined by a method based on xMAP technology using a standard panel, composed of 9 analytes: TNF-α, MCP-1, IL-8, IL-4, IL-6, IL-10, IL-IRa, IL- 12 (p70), IFN-γ. RESULTS: An optimal concentration of L. interrogans was selected for stimulation of human whole blood--1 x 10(6) leptospirae/ml. For the first time in the model of human whole blood it was determined, that at early stages of incubation IFN-γ, IL-12(p70), IL-4 and IL-1Ra are more actively produced; at later stages (6 hour incubation)--IL-8 and TNF-α. CONCLUSION: A differential pattern of cytokine production stimulation was shown in the model of human whole blood by live and inactivated leptospirae.

Mutant p53 gains new function in promoting inflammatory signals by repression of the secreted interleukin-1 receptor antagonist.

The TP53 tumor-suppressor gene is frequently mutated in human cancer. Missense mutations can add novel functions (gain-of-function, GOF) that promote tumor malignancy. Here we report that mutant (mut) p53 promotes tumor malignancy by suppressing the expression of a natural occurring anti-inflammatory cytokine, the secreted interleukin-1 receptor antagonist (sIL-1Ra, IL1RN). We show that mutp53 but not wild-type (wt) p53 suppresses the sIL-1Ra production in conditioned media of cancer cells. Moreover, mutp53, but not wtp53, binds physically the sIL-1Ra promoter and the protein-protein interaction with the transcriptional co-repressor MAFF (v-MAF musculoaponeurotic fibrosarcoma oncogene family, protein F) is required for mutp53-induced sIL-1Ra suppression. Remarkably, when exposed to IL-1 beta (IL-1ß) inflammatory stimuli, mutp53 sustains a ready-to-be-activated in vitro and in vivo cancer cells' response through the sIL-1Ra repression. Taken together, these results identify sIL-1Ra as a novel mutp53 target gene, whose suppression might be required to generate a chronic pro-inflammatory tumor microenvironment through which mutp53 promotes tumor malignancy.

Ex vivo gene delivery to synovium using foamy viral vectors.

BACKGROUND: Gene transfer technologies have the potential to fundamentally improve current therapies for arthritic conditions, although this is essentially dependent on safe and efficient vector systems. The foamy virus (FV)-based vectors have many safety features that favour their use in the treatment of arthritis. In the present study, we investigated the use of safe prototype foamy viral vectors (FVV) for indirect gene delivery to articular tissues. METHODS: We generated recombinant FVV encoding enhanced green fluorescent protein (EGFP) or human interleukin 1 receptor antagonist protein (IL1RA) cDNA under the control of the spleen focus forming virus U3 promoter and explored their transgene expression profile following ex vivo gene delivery to knee joints of Wistar and athymic nude rats. RESULTS: FVV efficiently transduced primary rat synovial fibroblasts using the EGFP and the IL1RA transgene in vitro. FVV-mediated IL1RA expression was functional in blocking IL1 effects in vitro. After the transplantation of FVV transduced synovial fibroblasts, the intra-articular transgene expression in Wistar rats was initially high and declined after approximately 3 weeks for both transgenes. By contrast, FVV-mediated expression of EGFP and IL1RA persisted for at least 12 weeks at high levels in immunocompromised nude rats. FVV-meditated gene delivery was well tolerated by all animals without extra-articular transgene expression, arguing for the safety of this approach. CONCLUSIONS: Our results indicate that FVV are capable of efficient ex vivo gene transfer to synovium and merit further investigation as a means to provide long-term intra-articular transgene expression for arthritis treatment.