An affibody-adalimumab hybrid blocks combined IL-6 and TNF-triggered serum amyloid A secretion in vivo

In inflammatory disease conditions, the regulation of the cytokine system is impaired, leading to tissue damages. Here, we used protein engineering to develop biologicals suitable for blocking a combination of inflammation driving cytokines by a single construct. From a set of interleukin (IL)-6-binding affibody molecules selected by phage display, five variants with a capability of blocking the interaction between complexes of soluble IL-6 receptor α (sIL-6Rα) and IL-6 and the co-receptor gp130 were identified. In cell assays designed to analyze any blocking capacity of the classical or the alternative (trans) signaling IL-6 pathways, one variant, Z_{IL-6_13} with an affinity (K_D) for IL-6 of ∼500 pM, showed the best performance. To construct fusion proteins (“AffiMabs”) with dual cytokine specificities, Z_{IL-6_13} was fused to either the N- or C-terminus of both the heavy and light chains of the anti-tumor necrosis factor (TNF) monoclonal antibody adalimumab (Humira®). One AffiMab construct with Z_{IL-6_13} positioned at the N-terminus of the heavy chain, denoted Z_{IL-6_13}-HC_Ada, was determined to be the most optimal, and it was subsequently evaluated in an acute Serum Amyloid A (SAA) model in mice. Administration of the AffiMab or adalimumab prior to challenge with a mix of IL-6 and TNF reduced the levels of serum SAA in a dose-dependent manner. Interestingly, the highest dose (70 mg/kg body weight) of adalimumab only resulted in a 50% reduction of SAA-levels, whereas the corresponding dose of the Z_{IL-6_13}-HC_Ada AffiMab with combined IL-6/TNF specificity, resulted in SAA levels below the detection limit.     

Flow cytometric analysis of phospho-p38 mitogen-activated kinase (MAPK): p38 MAPK does not mediate the effect of adalimumab on peripheral T cell cytokine production in rheumatoid arthritis

Background: The therapeutic effect of TNFα inhibition in rheumatoid arthritis (RA) is accompanied by an altered peripheral T cell cytokine profile, but the underlying mechanisms are not well known. In CD4+ T cells, TNF signalling includes the p38 MAP kinase (MAPK) pathway, which is also involved in proliferation and production of IL-4 and IFNγ. Methods: Phosphorylation of p38 MAPK was analysed flow cytometrically in peripheral blood mononuclear cells (PBMC) from healthy individuals and RA patients before and after adalimumab therapy. Cytokine production by CD3/CD28-stimulated PBMC was measured in the supernatant. Results: Despite a transient activation of p38 MAPK in response to cellular stress from the cell separation, a significant decrease of spontaneous p38 MAPK phosphorylation was observed after adalimumab, compared to RA patients with active disease. Brief stimulation with TNFα/IL-1β significantly activated p38 MAPK after but not before adalimumab therapy. In CD3/CD28-stimulated PBMC, significantly less p38 MAPK activation and increased IFNγ production were observed after adalimumab therapy. Conclusion: In rheumatoid arthritis, adalimumab therapy decreases the phosphorylation of p38 MAPK except for its response to TNF/IL-1, while enhancing the production of IFNγ. This suggests that p38 MAPK is not directly involved in the effect of TNF inhibition on cytokine production.     

Modification of proinflammatory cytokine production by the antirheumatic agents tenidap and naproxen. A possible correlate with clinical acute phase response.

The cytokines IL-6, IL-1, and TNF play a key role in the pathogenesis of rheumatoid arthritis (RA) and initiate hepatic serum amyloid A (SAA) expression after injury. To provide a possible mechanistic explanation for the previous observation that plasma SAA concentrations decreased during treatment of RA patients with tenidap, but increased during treatment with naproxen, the present study compared the effects of tenidap and naproxen on the two stages of SAA expression: cytokine production by human PBMC and cytokine-stimulated SAA synthesis by human Hep3B hepatoma cells. Tenidap inhibited production of IL-6 greater than TNF greater than IL-1; the effect of naproxen on production of all three cytokines was lesser and least on IL-6. Indeed, an increase in IL-6 production was observed after exposure to naproxen. PBMC beta-2-microglobulin production and total protein synthesis were unaffected at concentrations and times at which effects on cytokine production were observed. Cell density was a significant factor in the extent to which cytokines were stimulated by LPS. Approximately physiologic cell densities, 0.5 to 1 x 10(6) cells/ml, were optimal for stimulation of IL-1-beta and IL-6 production by LPS; however, greater amounts of TNF were produced at lower cell densities. Because neither tenidap nor naproxen inhibited SAA synthesis by cytokine-stimulated Hep3B cells and because they differ most significantly in their effect on IL-6 production, the results support a role for IL-6 in the continued stimulation of SAA production during RA.     

Proinflammatory activity of TWEAK on human dermal fibroblasts and synoviocytes: blocking and enhancing effects of anti-TWEAK monoclonal antibodies

Human tumour necrosis factor (TNF)-like weak inducer of apoptosis (hTWEAK) and two anti-hTWEAK mAbs were tested for their ability to elicit or block inflammatory responses in cultured human dermal fibroblasts and synoviocytes. Incubation with hTWEAK increased the production of prostaglandin E₂, matrix metalloproteinase-1 (MMP-1), IL-6, and the chemokines IL-8, RANTES (regulated on activation, normal T expressed and secreted) and interferon-γ-inducible protein-10 (IP-10) in culture supernatant of fibroblasts and synoviocytes. In combination with TNF or IL-1β, hTWEAK further stimulated the secretion of prostaglandin E₂, MMP-1, IL-6 and IL-8 up to fourfold, and IP-10 and RANTES up to 70-fold compared to TNF or IL-1β alone. An anti-hTWEAK mAb, BCB10, blocked the effects of hTWEAK, whereas hTWEAK crosslinked by the anti-hTWEAK mAb, BEB3, further stimulated the inflammatory response of fibroblasts and synoviocytes. The anti-hTWEAK mAbs were ineffective in blocking or increasing the responses of TNF or IL-1β and blocking anti-TNF mAb was ineffective in preventing the responses to TWEAK. These results were also confirmed at the RNA level for MMP-1, macrophage chemoattractant protein-1, RANTES, macrophage inflammatory protein-1α, IP-10 and IL-8. TWEAK in synergism with IL-1 and TNF may be an additional cytokine that plays a role in destructive chronic arthritic diseases.     

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.     

Compartmentalized Cytokine Responses in Hidradenitis Suppurativa

Background

Favorable treatment outcomes with TNF blockade led us to explore cytokine responses in hidradenitis suppurativa (HS).

Methods

Blood monocytes of 120 patients and 24 healthy volunteers were subtyped by flow cytometry. Isolated blood mononuclear cells (PBMCs) were stimulated for cytokine production; this was repeated in 13 severe patients during treatment with etanercept. Cytokines in pus were measured.

Results

CD14^brightCD16^dim inflammatory monocytes and patrolling monocytes were increased in Hurley III patients. Cytokine production by stimulated PBMCs was low compared to controls but the cytokine gene copies did not differ, indicating post-translational inhibition. The low production of IL-17 was restored, when cells were incubated with adalimumab. In pus, high concentrations of pro-inflammatory cytokines were detected. Based on the patterns, six different cytokine profiles were discerned, which are potentially relevant for the choice of treatment. Clinical improvement with etanercept was predicted by increased production of IL-1β and IL-17 by PBMCs at week 8.

Conclusions

Findings indicate compartmentalized cytokine expression in HS; high in pus but suppressed in PBMCs. This is modulated through blockade of TNF.     

Therapy with interleukin-2 induces the systemic release of phospholipase-A2

Therapy with interleukin-2 (IL-2) induces remissions in some forms of cancer. This treatment however, is accompanied by side-effects which, in part, may be mediated by the formation of eicosanoids and plateletactivating factor. We investigated the systemic release of phospholipase A₂ (PLA₂), a rate-limiting enzyme in the formation of these lipid mediators, in patients receiving IL-2. In a pilot study of 4 patients we observed an increase in PLA₂ activity in serial plasma samples obtained during the first day after a bolus infusion of IL-2, which increase closely correlated with that of antigen levels of secretory phospholipase A₂ (sPLA₂) as measured by enzyme-linked immunosorbent assay (r=0.92;P<0.001). In 20 patients, receiving 12×10⁶–18×10⁶ IU IL-2/m², we then investigated the course of antigenic levels of sPLA₂ in relation to those of the cytokines tumour necrosis factor (TNF) and interleukin-6 (IL-6) (both cytokines may induce sPLA₂ in vivo). From 4 h on, sPLA₂ levels significantly increased, reaching a peak 24 h after the IL-2 infusion. Subsequent IL-2 infusions even induced a further increase of sPLA₂. This increase of sPLA₂ was presumably not due to a direct effect of IL-2 on, for example, hepatocytes, since this cytokine, in contrast to IL-1, IL-6, TNF and interferon , was not able to induce the synthesis of sPLA₂ by Hep G2 cells in vitro. Consistent with this, plasma levels of TNF and IL-6 in the patients rose, reaching peak levels before a zenith of sPLA₂ occurred, i.e at 2 h and 4 h after the start of the IL-2 infusion respectively. sPLA₂ levels significantly correlated with the development of the side-effects increase in body weight (r=0.49;P<0.0001) and decrease in mean arterial blood pressure (r=0.40;P<0.0001). Moreover, maximum sPLA₂ levels induced by IL-2 were higher in patients who had progressive disease after therapy than in patients who had stable disease or a partial response.     

Protective effects of a thromboxane synthetase inhibitor and continuous arteriovenous hemofiltration in rat endotoxic shock

We determined the efficacy of continuous arteriovenous hemofiltration (CAVH) and a thromboxane synthetase inhibitor (TSI) on survival and their effect on TXA₂, PGI₂, TNFα, and IL-1β production in rat endotoxemia. Thirty-six endotoxemic rats were randomized to one of 4 groups: (A) no TSI, sham CAVH; (B) no TSI, CAVH; (C) TSI, sham CAVH; and (D) TSI, CAVH. Either CAVH (Group B) or pretreatment with TSI (Group C) resulted in increased survival time. CAVH did not prevent the rise in TX (Group B). TNFα levels at 2 h after LPS infusion were higher in Group D compared to Group B (26.1 ± 3.7 vs 13.2 ± 4.3 ng/mL, P < 0.05) respectively. IL-1β was detected earlier in Groups C,D when compared to Groups A,B (P < 0.02). TNFα and IL-1β were not ultrafiltered. CAVH and the inhibition of TX synthesis independently improved survival in endotoxemia, however, their beneficial effects were not additive. While TSI may improve survival by blocking TXA₂ production, the salutary effects of CAVH appear to be from removal of an undetermined TXA₂ dependent mediator. TNFα and IL-1β concentrations do not appear to influence survival times in this model.     

Serum Amyloid A Induces Toll-Like Receptor 2-Dependent Inflammatory Cytokine Expression and Atrophy in C2C12 Skeletal Muscle Myotubes

Background

Skeletal muscle wasting is an important comorbidity of Chronic Obstructive Pulmonary Disease (COPD) and is strongly correlated with morbidity and mortality. Patients who experience frequent acute exacerbations of COPD (AECOPD) have more severe muscle wasting and reduced recovery of muscle mass and function after each exacerbation. Serum levels of the pro-inflammatory acute phase protein Serum Amyloid A (SAA) can rise more than 1000-fold in AECOPD and are predictively correlated with exacerbation severity. The direct effects of SAA on skeletal muscle are poorly understood. Here we have examined SAA effects on pro-inflammatory cachectic cytokine expression (IL-6 and TNFα) and atrophy in C2C12 myotubes.

Results

SAA increased IL-6 (31-fold) and TNFα (6.5-fold) mRNA levels compared to control untreated cells after 3h of SAA treatment, and increased secreted IL-6 protein at 24h. OxPAPC, a dual TLR2 and TLR4 inhibitor, reduced the response to SAA by approximately 84% compared to SAA alone, and the TLR2 neutralising antibody T2.5 abolished SAA-induced expression of IL-6, indicating that SAA signalling in C2C12 myotubes is primarily via TLR2. SAA also reduced myotube width by 10–13% and induced a 2.5-fold increase in the expression of the muscle atrophy gene Atrogin-1, suggesting direct effects of SAA on muscle wasting. Blocking of TLR2 inhibited the SAA-induced decrease in myotube width and Atrogin-1 gene expression, indicating that SAA induces atrophy through TLR2.

Conclusions

These data demonstrate that SAA stimulates a robust pro-inflammatory response in skeletal muscle myotubes via the TLR2-dependent release of IL-6 and TNFα. Furthermore, the observed atrophy effects indicate that SAA could also be directly contributing to the wasting and poor recovery of muscle mass. Therapeutic strategies targeting this SAA-TLR2 axis may therefore ameliorate muscle wasting in AECOPD and a range of other inflammatory conditions associated with loss of muscle mass.     

Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10

Background

Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages.

Materials and Methods

Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR.

Results

HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ_IL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6.

Conclusion

HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.     

Serum Amyloid A Complexed with Extracellular Matrix Induces the Secretion of Tumor Necrosis Factor-α by human T-lymphocytes

Summary Serum amyloid A (SAA), an acute-phase reactant, exists naturally as a minor protein in the sera of healthy individuals. However, its levels in sera are increased markedly during various transient and chronic inflammatory diseases, often concomitantly with accumulation at inflicted sites. SAA is synthesized mainly in the liver following the synergistic action of cytokines, mainly tumor necrosis factor-α (TNF-α) and interleukin-1 and-6 (IL-1 and IL-6). It was already shown by us that upon interaction with SAA or amyloid A (AA), the extracellular matrix (ECM) and laminin induced the adhesion of resting human CD4⁺ T-cells in an apparently β₁-integrin-mediated manner. Herein we have shown that the SAA-ECM complex modulates the regulation of cytokine synthesis by human T-lymphocytes. The SAA-ECM complex dramatically enhanced the release of TNF-α by human T-cells in a dose-dependent manner, reaching its maximal effect in the presence of 100 μM recombinant SAA. The SAA domain, responsible for the enhanced release of TNF-α by human T-lymphocytes, is apparently the amyloid A protein (AA, i.e. SAA2-82). Specifically, TNF-α enhanced secretion is mediated through intimate interactions of SAA/AA, with laminin. Thus, the ECM serving as a temporary anchorage site for SAA and AA seems to be involved in regulating TNF-α secretion and the recruitment and accumulation of immunocytes in extravascular, inflammatory compartments.     

Role of bacterial components in macrophage activation by the LAC and MW2 strains of community-associated, methicillin-resistant Staphylococcus aureus

We tested the contribution of four staphylococcal components – PSM-α, PSM-β, δ-toxin, and PVL – in triggering macrophage secretion of tumor necrosis factor (TNF) and interleukins 6 (IL-6) and 12 (IL-12) by two prominent, circulating strains of community-associated, methicillin-resistant Staphylococcus aureus (CA-MRSA): LAC, USA300; MW2, USA400. RAW 264.7 murine macrophages were stimulated with live, antibiotic-exposed bacteria, and cytokine secretion was quantitated in supernatants. Deletion of PSM-α expression in LAC led to >50% reduction in macrophage TNF and IL-6 secretion and a 20% reduction in IL-12 secretion, while PSM-α deletion in MW2 did not significantly reduce macrophage TNF secretion but resulted in a 15–20% reduction in IL-6 and IL-12 secretion. Deletion of δ-toxin in either strain led to more than 50% reduction in macrophage IL-6 secretion and smaller reductions in macrophage TNF and IL-12 secretion (8–25%). Our data implicate both PSM-α and δ-toxin in stimulating macrophage cytokine responses to CA-MRSA bacteria.     

Effect of BCG stimulus on proinflammatory cytokine production in laryngeal cancer

Background  Evaluate the production of TNF and IL-6 in the supernatant of peripheral blood mononuclear cell (PBMC) cultures of patients with supraglottic laryngeal cancer before and after surgical treatment. Materials and methods  Adherent cell cultures were stimulated with LPS and BCG. Fourteen patients with advanced supraglottic laryngeal cancer were studied. Cytokine concentration was determined by ELISA in supernatants of mononuclear cell cultures. Results  In non-stimulated cultures, lower TNF cytokine levels were detected during the late postoperative (LP) period compared to control (P = 0.02). LP TNF and IL-6 levels were high in cultures stimulated with LPS compared with the preoperative period (PREOP) (P = 0.007; P = 0.008, respectively). Stimulation with BCG led to increased levels of TNF and IL-6 during the LP period compared to control (P = 0.001; P = 0.04, respectively). Conclusion  BCG is able to modulate the immune response of patients with advanced supraglottic laryngeal cancer in vitro, increasing the secretion of TNF and IL-6 by macrophages during the postoperative period.     

Inhibition of TNF-induced IL-6 by the TWEAK-Fn14 interaction in rheumatoid arthritis fibroblast like synoviocytes

ObjectivesTNF-like weak inducer of apoptosis (TWEAK), a member of the TNF superfamily, has been shown to increase cytokine production by rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). In this study, we determined the effect of interaction between TWEAK and its receptor fibroblast growth factor-inducible-14 (Fn14) on cytokine expression in RAFLS.MethodsRAFLS were obtained from surgical synovial specimens and used at passage 5–10. Cytokine protein and mRNA expression were measured with ELISA and real time-PCR, respectively. Apoptotic cells were detected by TUNEL assay. RelB activation was detected by Western blot analysis.ResultsTWEAK inhibited IL-6 production from total synovial cells from RA. TWEAK weakly induced FLS IL-6 and IL-8, but in contrast TWEAK dose-dependently inhibited IL-6 and IL-8 production by TNFα-activated FLS. TWEAK did not induce apoptosis in FLS but inhibited proliferation of TNFα-activated FLS. TWEAK induced RelB activation and suppressed IL-6 mRNA expression in TNFα-activated FLS and both of these phenomenon were abolished by inhibition of new protein synthesis with cycloheximide.ConclusionsTWEAK has a previously unsuspected inhibitory effect on cytokine production by TNFα-activated RAFLS. This observation suggests that the effects of TWEAK on cytokine expression varies with the pro-inflammatory context, and that in TNFα-activated states such as RA TWEAK may have a net inhibitory effect.     

Plasma pro- and anti-inflammatory cytokine levels and outcome prediction in unselected critically ill patients

Purpose. To determine the inter-relationships between cytokine levels and physiological scores in predicting outcome in unselected, critically ill patients. Methods. To this end, 127 patients (96 men), having a mean ± SD age of 45 ± 20 years, with a wide range in admission diagnoses (medical, surgical, and multiple trauma patients) were prospectively investigated. Severity of critical illness and organ dysfunction were graded by acute physiology and chronic health evaluation (APACHE II) and sequential organ failure assessment (SOFA) scores, respectively. Blood samples were drawn on admission in the ICU to determine pro- and anti-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, and IL-10. The main outcome measure was 28-day mortality. Results. Overall, 88 patients survived and 39 patients died. Univariate logistic regression analysis showed that SOFA, APACHE II, IL-8, IL-6, and IL-10 on admission in the ICU were related to mortality. Multiple logistic regression analysis in the entire cohort of critically ill patients revealed that SOFA (OR = 1.341, p < 0.001) and IL-6 (OR = 1.075, p = 0.01) constituted independent outcome predictors. receiver operator characteristics curve analysis showed that SOFA, APACHE II, and IL-6 had the highest area under the curve values. IL-6 correlated with APACHE II (r_s = 0.44, p < 0.0001) and SOFA (r_s = 0.40, p < 0.0001) scores. Conclusions. In mixed ICU patients cytokine concentrations on admission in the ICU represent independent outcome predictors in the presence of disease severity scores.     

慢性阻塞性肺疾病急性加重期患者血清SAA水平与肺功能、炎性因子的相关性及其诊断价值分析

目的:探讨慢性阻塞性肺疾病急性加重期(AECOPD)患者血清淀粉样蛋白A(SAA)水平与肺功能及炎性因子的相关性,并分析其诊断价值。方法:选取2013年6月-2018年6月中国人民解放军第970医院收治的204例慢性阻塞性肺疾病(COPD)患者作为研究对象,其中COPD稳定期患者132例作为COPD稳定组,AECOPD患者72例作为AECOPD组。另选取同期于中国人民解放军第970医院进行健康体检的50例健康体检者作为健康组。比较三组受试者血清SAA水平、白细胞介素-8(IL-8)、白细胞介素-6(IL-6)、C反应蛋白(CRP)、降钙素原(PCT)水平及肺功能,采用Pearson相关性分析AECOPD患者血清SAA水平与肺功能及炎性因子的相关性,并分析SAA对AECOPD的诊断价值。结果:AECOPD组患者血清SAA、PCT、CRP、IL-6、IL-8水平较COPD稳定组及健康组升高(P0.05),COPD稳定组患者血清SAA、IL-6、IL-8、PCT、CRP水平均高于健康组,差异有统计学意义(P0.05);AECOPD组患者第一秒用力呼气容积(FEV1)、用力肺活量(FVC)、第一秒用力呼吸容积占用力肺活量的百分比(FEV1/FVC%)、第一秒用力呼气容积占预计值百分比(FEV1%)低于COPD稳定组及健康组,差异有统计学意义(P0.05),COPD稳定组患者FEV1、FVC、FEV1/FVC%、FEV1%低于健康组,差异有统计学意义(P0.05)。Pearson相关性分析显示,AECOPD患者血清SAA水平与IL-8、IL-6、CRP、PCT呈正相关,与FEV1%、FEV1/FVC%、FEV1、FVC呈负相关(P0.05)。受试者工作特征(ROC)曲线结果显示,SAA对AECOPD诊断的敏感度为80.85%,特异度为80.07%,曲线下面积为0.832。结论:AECOPD患者血清SAA水平明显升高,其与患者肺功能及炎症因子存在相关性,具有较高的诊断价值,可用于AECOPD患者病情的评估。     

Synthesis and non-linear optical properties of (η-pentaphenylcyclopentadienyl)dicarbonylruthenium(II) σ-alkenyl complexes

The complexes [Ru{(Z)-HCCHPh}(CO)₂(η⁵-C₅Ph₅)] (1) and [Ru{(Z)-HCCHC₆H₄NO₂}(CO)₂(η⁵-C₅Ph₅)] (2) have been synthesized and their identity confirmed by single-crystal X-ray diffraction studies. Reaction of 2 with PMe₂Ph and Me₃NO in tetrahydrofuran afforded [Ru{(Z)-HCCHC₆H₄NO₂}(CO)(PMe₂Ph)(η⁵-C₅Ph₅)] (3). Cyclic voltammetry confirms the expected increase in ease of oxidation on proceeding from 2 to 1 and from 2 to 3. Hyper-Rayleigh scattering studies at 1064 nm reveal a dramatic increase in quadratic non-linearity on co-ligand replacement of CO by PMe₂Ph, in proceeding from 2 to 3. Z-scan studies at 800 nm are consistent with significant contribution from two-photon states, and with an increase in γ_real on co-ligand replacement of CO by PMe₂Ph in proceeding from 2 to 3.     

Role of transient receptor potential vanilloid 2 in LPS-induced cytokine production in macrophages

There is considerable evidence indicating that intracellular Ca²⁺ participates as a second messenger in TLR4-dependent signaling. However, how intracellular free Ca²⁺ concentrations ([Ca²⁺]_i) is increased in response to LPS and how they affect cytokine production are poorly understood. Here we examined the role of transient receptor potential (TRP), a major Ca²⁺ permeation pathway in non-excitable cells, in the LPS-induced cytokine production in macrophages. Pharmacologic experiments suggested that TRPV family members, but neither TRPC nor TRPM family members, are involved in the LPS-induced TNFα and IL-6 production in RAW264 macrophages. RT-PCR and immunoblot analyses showed that TRPV2 is the sole member of TRPV family expressed in macrophages. ShRNA against TRPV2 inhibited the LPS-induced TNFα and IL-6 production as well as IκBα degradation. Experiments using BAPTA/AM and EGTA, and Ca²⁺ imaging suggested that the LPS-induced increase in [Ca²⁺]_i involves both the TRPV2-mediated intracellular and extracellular Ca²⁺ mobilizations. BAPTA/AM abolished LPS-induced TNFα and IL-6 production, while EGTA only partially suppressed LPS-induced IL-6 production, but not TNFα production. These data indicate that TRPV2 is involved in the LPS-induced Ca²⁺ mobilization from intracellular Ca²⁺ store and extracellular Ca²⁺. In addition to Ca²⁺ mobilization through the IP₃-receptor, TRPV2-mediated intracellular Ca²⁺ mobilization is involved in NFκB-dependent TNFα and IL-6 expression, while extracellular Ca²⁺ entry is involved in NFκB-independent IL-6 production.     

Blockade of Toll-like receptor 2 prevents spontaneous cytokine release from rheumatoid arthritis ex vivo synovial explant cultures

Introduction

The aim of this study was to examine the effect of blocking Toll-like receptor 2 (TLR2) in rheumatoid arthritis (RA) synovial cells.

Methods

RA synovial tissue biopsies, obtained under direct visualization at arthroscopy, were established as synovial explant cultures ex vivo or snap frozen for immunohistology. Mononuclear cell cultures were isolated from peripheral blood and synovial fluid of RA patients. Cultures were incubated with the TLR1/2 ligand, Pam3CSK4 (200 ng, 1 and 10 μg/ml), an anti-TLR2 antibody (OPN301, 1 μg/ml) or an immunoglobulin G (IgG) (1 μg/ml) matched control. The comparative effect of OPN301 and adalimumab (anti-tumour necrosis factor alpha) on spontaneous release of proinflammatory cytokines from RA synovial explants was determined using quantitative cytokine MSD multiplex assays or ELISA. OPN301 penetration into RA synovial tissue explants cultures was assessed by immunohistology.

Results

Pam3CSK4 significantly upregulated interleukin (IL)-6 and IL-8 in RA peripheral blood mononuclear cells (PBMCs), RA synovial fluid mononuclear cells (SFMCs) and RA synovial explant cultures (P < 0.05). OPN301 significantly decreased Pam3CSK4-induced cytokine production of tumour necrosis factor alpha (TNF-α), IL-1β, IL-6, interferon (IFN)-γ and IL-8 compared to IgG control in RA PBMCs and SFMCs cultures (all P < 0.05). OPN301 penetration of RA synovial tissue cultures was detected in the lining layer and perivascular regions. OPN301 significantly decreased spontaneous cytokine production of TNF-α, IL-1β, IFN-γ and IL-8 from RA synovial tissue explant cultures (all P < 0.05). Importantly, the inhibitory effect of OPN on spontaneous cytokine secretion was comparable to inhibition by anti-TNFα monoclonal antibody adalimumab.

Conclusions

These findings further support targeting TLR2 as a potential therapeutic agent for the treatment of RA.