Mutation in cytochrome b gene of mitochondrial DNA in a family with fibromyalgia is associated with NLRP3-inflammasome activation.

BACKGROUND: Fibromyalgia (FM) is a worldwide diffuse musculoskeletal chronic pain condition that affects up to 5% of the general population. Many symptoms associated with mitochondrial diseases are reported in patients with FM such as exercise intolerance, fatigue, myopathy and mitochondrial dysfunction. In this study, we report a mutation in cytochrome b gene of mitochondrial DNA (mtDNA) in a family with FM with inflammasome complex activation. METHODS: mtDNA from blood cells of five patients with FM were sequenced. We clinically and genetically characterised a patient with FM and family with a new mutation in mtCYB. Mitochondrial mutation phenotypes were determined in skin fibroblasts and transmitochondrial cybrids. RESULTS: After mtDNA sequence in patients with FM, we found a mitochondrial homoplasmic mutation m.15804T>C in the mtCYB gene in a patient and family, which was maternally transmitted. Mutation was observed in several tissues and skin fibroblasts showed a very significant mitochondrial dysfunction and oxidative stress. Increased NLRP3-inflammasome complex activation was observed in blood cells from patient and family. CONCLUSIONS: We propose further studies on mtDNA sequence analysis in patients with FM with evidences for maternal inheritance. The presence of similar symptoms in mitochondrial myopathies could unmask mitochondrial diseases among patients with FM. On the other hand, the inflammasome complex activation by mitochondrial dysfunction could be implicated in the pathophysiology of mitochondrial diseases.

Impact of stereochemistry on the biological activity of novel oleandomycin derivatives.

A set of 8-methylene-, 8-methyl-, and 8-methyl-9-dihydro-oleandomycin derivatives having different combinations of stereochemistries at positions C-8 and/or C-9 have been prepared in a chemoselective and stereoselective manner and tested in vitro for antibacterial activity and inhibition of IL-6 production. Configurations of the stereocenters at C-8 and C-9 were determined using 2D NMR techniques. We have shown that change of stereochemistry at these positions can exert a major influence on antibacterial activity as well as IL-6 inhibition, providing novel macrolide derivatives with diminished antibacterial and potent anti-inflammatory activity. In addition, the anti-inflammatory activity observed in vitro was confirmed in an in vivo model of lipopolysaccharide-induced inflammation.

Macrolide antibiotics broadly and distinctively inhibit cytokine and chemokine production by COPD sputum cells in vitro.

Macrolide antibiotics are known to exert anti-inflammatory actions in vivo, including certain effects in COPD patients. In order to investigate the immunomodulatory profile of activity of macrolide antibiotics, we have studied the effects of azithromycin, clarithromycin, erythromycin and roxithromycin on the in vitro production of a panel of inflammatory mediators from cells isolated from human, steroid-naïve, COPD sputum samples. Macrolide effects were compared to three other commonly used anti-inflammatory compounds, the corticosteroid dexamethasone, the PDE4 inhibitor, roflumilast and the p38 kinase inhibitor, SB203580. Three of the four tested macrolides, azithromycin, clarithromycin and roxithromycin, exhibited pronounced, concentration-related reduction of IL-1ß, IL-6, IL-10, TNF-α, CCL3, CCL5, CCL20, CCL22, CXCL1, CXCL5, and G-CSF release. Further slight inhibitory effects on IL-1α, CXCL8, GM-CSF, and PAI-1 production were also observed. Erythromycin was very weakly active. Qualitatively and quantitatively, macrolides exerted distinctive and, compared to other tested classes of compounds, more pronounced immunomodulatory effects, particularly in terms of chemokine (CCL3, CCL5, CCL20, CCL22, and CXCL5), IL-1ß, G-CSF and PAI-1 release. The described modulation of inflammatory mediators could potentially contribute to further definition of biomarkers of macrolide anti-inflammatory activity in COPD.

Azithromycin and clarithromycin inhibit lipopolysaccharide-induced murine pulmonary neutrophilia mainly through effects on macrophage-derived granulocyte-macrophage colony-stimulating factor and interleukin-1beta.

Macrolide antibiotics possess immunomodulatory/anti-inflammatory properties. These properties are considered fundamental for the efficacy of macrolide antibiotics in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, the molecular mechanisms and cellular targets of anti-inflammatory/immunomodulatory macrolide activity are still not fully understood. To describe anti-inflammatory effects of macrolides in more detail and to identify potential biomarkers of their activity, we have investigated the influence of azithromycin and clarithromycin on the inflammatory cascade leading to neutrophil infiltration into lungs after intranasal lipopolysaccharide challenge in mice. Azithromycin and clarithromycin pretreatment reduced total cell and neutrophil numbers in bronchoalveolar lavage fluid and myeloperoxidase concentration in lung tissue. In addition, concentrations of several inflammatory mediators, including CCL2, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and sE-selectin in lung homogenates were decreased after macrolide treatment. Inhibition of cytokine production observed in vivo was also corroborated in vitro in lipopolysaccharide-stimulated monocytes/macrophages, but not in an epithelial cell line. In summary, results presented in this article confirm that macrolides can suppress neutrophil-dominated pulmonary inflammation and suggest that the effect is mediated through inhibition of GM-CSF and IL-1beta production by alveolar macrophages. Besides GM-CSF and IL-1beta, CCL2 and sE-selectin are also identified as potential biomarkers of macrolide anti-inflammatory activity in the lungs.

Homology modeling of human Fyn kinase structure: discovery of rosmarinic acid as a new Fyn kinase inhibitor and in silico study of its possible binding modes.

Tyrosine phosphorylation represents a unique signaling process that controls metabolic pathways, cell activation, growth and differentiation, membrane transport, apoptosis, neural, and other functions. We present here the three-dimensional structure of Fyn tyrosine kinase, a Src-family enzyme involved in T-cell receptor signal transduction. The structure of Fyn was modeled for homology using the Sybyl-Composer suite of programs for modeling. Procheck and Prosa II programs showed the high quality of the obtained three-dimensional model. Rosmarinic acid, a secondary metabolite of herbal plants, was discovered as a new Fyn kinase inhibitor using immunochemical and in silico methods. Two possible binding modes of rosmarinic acid were evaluated here, i.e., near to or in the ATP-binding site of kinase domain of Fyn. Enzyme kinetic experiments revealed that Fyn is inhibited by a linear-mixed noncompetitive mechanism of inhibition by rosmarinic acid. This indicates that rosmarinic acid binds to the second "non-ATP" binding site of the Fyn tyrosine kinase.

Anti-inflammatory activity of azithromycin attenuates the effects of lipopolysaccharide administration in mice.

Macrolide antibacterials inhibit the production of various cytokines and the migration of inflammatory cells. These anti-inflammatory actions of macrolides may be beneficial in attenuating inflammatory processes involved in bacterial sepsis. Therefore, we investigated the ability of azithromycin to attenuate the deleterious effects of lipopolysaccharide (LPS), in three different LPS-induced inflammatory models. Our results show that azithromycin (10 and 100 mg/kg) significantly attenuated the intraperitoneal LPS-induced increase in plasma TNF-alpha concentration. It also increased survival rate in a septic shock model in mice challenged with intravenous LPS. Oral treatment with azithromycin (up to 300 mg/kg) was less effective in suppressing neutrophil infiltration into the lungs 24 h after intranasal LPS challenge, possibly because of a slower onset of action or inadequate dosing. In the same model, azithromycin given intraperitoneally significantly improved inflammatory markers (total cell number, neutrophil percentage and MIP-2 concentration) in bronchoalveolar lavage fluid. In conclusion, azithromycin exhibits significant anti-inflammatory properties but the potency of such effects varies depending on the experimental model and route of administration.

Serum activities of adenosine deaminase, dipeptidyl peptidase IV and prolyl endopeptidase in patients with fibromyalgia: diagnostic implications.

Fibromyalgia (FM) is a chronic pain syndrome with number of symptoms that present challenge in terms of diagnosis and treatment. Patients with FM show abnormal profile of purines in plasma. In this work, we measured serum activities of enzymes involved in purine metabolism, namely total adenosine deaminase (ADE) and its isoforms (ADE1 and ADE2), ecto-ATPase, and 5'-nucleotidase (5'-NT). We also measured activity of dipeptidyl peptidase IV (DPPIV) and prolyl endopeptidase (PEP). Spectrophotometric and fluorometric methods were used for enzyme activity determinations. Enzyme activities were measured in sera of 24 patients with FM that were not undergoing pharmacological treatment during the study. Control group comprised 32 healthy control subjects. Significantly higher activities of total ADE (P = 0.025) and ADE2 (P = 0.011) were observed in FM patients, while no significant differences in ADE1, ecto-ATPase, and 5'-NT activities (P > 0.05) were found when compared to healthy controls. Moreover, increase in the activity of DPPIV (P = 0.015) and lower activity of PEP (P = 0.011) were also found in the FM group. ROC analysis pointed to different diagnostic sensitivities/specificities for individual enzyme activities measured as follows: ADE (50.0/87.5), ADE2 (41.7/90.6), DPPIV (62.5/71.9), and PEP (83.3/62.5). ADE2 and PEP were shown to be independent predictors of FM, while combination of the two gives AUC of 0.786 (95 % confidence interval of 0.656-0.885, P < 0.05). Our results are showing that serum activities of ADE2 and PEP could be useful as biomarkers for FM diagnosis. However, relatively low diagnostic sensitivity of ADE2 and specificity of PEP must be taken into account.

AMPK Phosphorylation Modulates Pain by Activation of NLRP3 Inflammasome.

AIMS: Impairment in adenosine monophosphate-activated protein kinase (AMPK) activity and NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation are associated with several metabolic and inflammatory diseases. In this study, we investigated the role of AMPK/NLRP3 inflammasome axis in the molecular mechanism underlying pain perception. RESULTS: Impairment in AMPK activation induced by compound C or sunitinib, two AMPK inhibitors, provoked hyperalgesia in mice (p<0.001) associated with marked NLRP3 inflammasome protein activation and increased serum levels of interleukin-1ß (IL-1ß) (24.56±0.82 pg/ml) and IL-18 (23.83±1.882 pg/ml) compared with vehicle groups (IL-1ß: 8.15±0.44; IL-18: 4.92±0.4). This effect was rescued by increasing AMPK phosphorylation via metformin treatment (p<0.001), caloric restriction diet (p<0.001), or NLRP3 inflammasome genetic inactivation using NLRP3 knockout (nlrp3(-/-)) mice (p<0.001). Deficient AMPK activation and overactivation of NLRP3 inflammasome axis were also observed in blood cells from patients with fibromyalgia (FM), a prevalent human chronic pain disease. In addition, metformin treatment (200 mg/daily), which increased AMPK activation, restored all biochemical alterations examined by us in blood cells and significantly improved clinical symptoms, such as, pain, fatigue, depression, disturbed sleep, and tender points, in patients with FM. INNOVATION AND CONCLUSIONS: These data suggest that AMPK/NLRP3 inflammasome axis participates in chronic pain and that NLRP3 inflammasome inhibition by AMPK modulation may be a novel therapeutic target to fight against chronic pain and inflammatory diseases as FM.

Modulation of neutrophil and inflammation markers in chronic obstructive pulmonary disease by short-term azithromycin treatment.

The anti-inflammatory potential of azithromycin in chronic obstructive pulmonary disease (COPD) patients was explored following a standard oral dosing regimen. Patients with moderate and severe COPD were treated with azithromycin (500 mg, n=16) or placebo (n=8) once daily for 3 days in a randomized, double blind design, to compare effects on inflammation markers with those seen in a previous study in healthy volunteers. A battery of tests was made on serum, blood neutrophils and sputum on days 1 (baseline), 3, 4, 11, 18 and 32. In comparison to placebo, azithromycin resulted in an early transient increase in serum nitrites plus nitrates (day 3), associated with a tendency towards an increase in the blood neutrophil oxidative burst to phorbol myristic acetate. Subsequently, prolonged decreases in blood leukocyte and platelet counts, serum acute phase protein (including C reactive protein) and soluble E-selectin and blood neutrophil lactoferrin concentrations and a transient decrease in serum interleukin-8 were observed. Blood neutrophil glutathione peroxidase activity showed a prolonged increase after azithromycin treatment. The biphasic facilitatory-then-inhibitory response to azithromycin seen in healthy volunteers is not so clearly detectable in COPD patients, only potential anti-inflammatory effects. Treatment for longer periods may give therapeutic anti-inflammatory benefit in these patients.

Azithromycin modulates neutrophil function and circulating inflammatory mediators in healthy human subjects.

Effects on human neutrophils and circulating inflammatory mediators were studied in 12 volunteers who received azithromycin (500 mg/day, p.o.) for 3 days. Blood was taken 1 h before treatment, 2.5, 24 h and 28 days after the last dose. An initial neutrophil degranulating effect of azithromycin was reflected in rapid decreases in azurophilic granule enzyme activities in cells and corresponding increases in serum. The oxidative response to a particulate stimulus was also acutely enhanced. These actions were associated with high plasma and neutrophil drug concentrations. A continuous fall in chemokine and interleukin-6 serum concentrations, within the non-pathological range, accompanied a delayed down-regulation of the oxidative burst and an increase in apoptosis of neutrophils up to 28 days after the last azithromycin dose. Neutrophils isolated from blood at this time point still contained detectable drug concentrations. Acute neutrophil stimulation could facilitate antibacterial effects of azithromycin, while delayed, potentially anti-inflammatory activity may curtail deleterious inflammation.

NLRP3 inflammasome is activated in fibromyalgia: the effect of coenzyme Q10.

AIMS: Fibromyalgia (FM) is a prevalent chronic pain syndrome characterized by generalized hyperalgesia associated with a wide spectrum of symptoms such as fatigue and joint stiffness. Diagnosis of FM is difficult due to the lack of reliable diagnostic biomarkers, while treatment is largely inadequate. We have investigated the role of coenzyme Q10 (CoQ10) deficiency and mitochondrial dysfunction in inflammasome activation in blood cells from FM patients, and in vitro and in vivo CoQ10 deficiency models. RESULTS: Mitochondrial dysfunction was accompanied by increased protein expression of interleukin (IL)-1ß, NLRP3 (NOD-like receptor family, pyrin domain containing 3) and caspase-1 activation, and an increase of serum levels of proinflammatory cytokines (IL-1ß and IL-18). CoQ10 deficiency induced by p-aminobenzoate treatment in blood mononuclear cells and mice showed NLRP3 inflammasome activation with marked algesia. A placebo-controlled trial of CoQ10 in FM patients has shown a reduced NLRP3 inflammasome activation and IL-1ß and IL-18 serum levels. INNOVATION: These results show an important role for the NLRP3 inflammasome in the pathogenesis of FM, and the capacity of CoQ10 in the control of inflammasome. CONCLUSION: These findings provide new insights into the pathogenesis of FM and suggest that NLRP3 inflammasome inhibition represents a new therapeutic intervention for the disease.

Can coenzyme q10 improve clinical and molecular parameters in fibromyalgia?

Fibromyalgia (FM) is a complex disorder that affects up to 5% of the general population worldwide. Its pathophysiological mechanisms are difficult to identify and current drug therapies demonstrate limited effectiveness. Both mitochondrial dysfunction and coenzyme Q10 (CoQ10) deficiency have been implicated in FM pathophysiology. We have investigated the effect of CoQ10 supplementation. We carried out a randomized, double-blind, placebo-controlled trial to evaluate clinical and gene expression effects of forty days of CoQ10 supplementation (300 mg/day) on 20 FM patients. This study was registered with controlled-trials.com (ISRCTN 21164124). An important clinical improvement was evident after CoQ10 versus placebo treatment showing a reduction of FIQ (p<0.001), and a most prominent reduction in pain (p<0.001), fatigue, and morning tiredness (p<0.01) subscales from FIQ. Furthermore, we observed an important reduction in the pain visual scale (p<0.01) and a reduction in tender points (p<0.01), including recovery of inflammation, antioxidant enzymes, mitochondrial biogenesis, and AMPK gene expression levels, associated with phosphorylation of the AMPK activity. These results lead to the hypothesis that CoQ10 have a potential therapeutic effect in FM, and indicate new potential molecular targets for the therapy of this disease. AMPK could be implicated in the pathophysiology of FM.

Association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-alpha (+489G/A) polymorphisms with COPD in Croatian population.

OBJECTIVE: To test for possible association of hsp70-2 (+1267A/G), hsp70-hom (+2437T/C), HMOX-1 (number of GT repeats) and TNF-α (+489G/A) polymorphisms with chronic obstructive pulmonary disease (COPD) in Croatian population. METHODS: Genotyping of DNA isolated from whole blood of 130 COPD patients (as defined by spirometry) and 95 healthy controls was performed. Fragment size analysis upon restriction enzyme digestion and/or sequencing was used for genotype/allele definition. Significance of findings was tested using χ(2) test. RESULTS: hsp70-2 (+1267A/G) polymorphism was significantly associated with COPD. Results of genotyping analysis indicated that a genotype carrying G allele was preferentially associated with COPD; odds ratio (OR)=1.50, 95% confidence interval (CI)=1.00-2.24 and P=0.061. OR for the GG genotype was 3.47 with CI=1.26-9.56 and P=0.04. No association for hsp70-hom (+2437T/C), TNF-α (+489G/A) and HMOX-1 (number of GT repeats) polymorphisms were found. In addition, comparison of genotype frequencies among different stages of disease severity (GOLD II-IV) revealed no discrimination for any of the tested polymorphisms. CONCLUSION: This study is supporting the association of hsp70-2 (+1267A/G) polymorphism and COPD. Higher frequency of G allele and GG genotype in Croatian COPD patients was observed. There was no evidence for the association of hsp70-hom (+2437T/C), TNF-α (+489G/A) SNPs and HMOX-1 (number of GT repeats) polymorphism with COPD. Allele and genotype frequencies for all of the tested polymorphisms show no association with disease severity (GOLD II-IV).

Porphyrins as new endogenous anti-inflammatory agents.

A series of porphyrins, tetrapyrrole natural organic compounds, are evaluated here as endogenous anti-inflammatory agents. They directly inhibit the activity of Fyn, a non-receptor Src-family tyrosine kinase, triggering anti-inflammatory events associated with down-regulation of T-cell receptor signal transduction, leading to inhibition of tumor necrosis factor alpha (TNF-α) production. This is one of the major pro-inflammatory cytokines, associated with diseases such as diabetes, tumorigenesis, rheumatoid arthritis, and inflammatory bowel disease. Porphyrins, as a chemical class, inhibited Fyn kinase activity in a non-competitive, linear-mixed fashion. In cell-based in vitro experiments on polymorphonuclear cells, porphyrins inhibited TNF-α cytokine production, T-cell proliferation, and the generation of free radicals in the oxidative burst, in a concentration-related manner. In vivo, lipopolysaccharide-induced TNF-α production in mice was inhibited by several of the porphyrins. These findings may be very important for the overall understanding of the role(s) of porphyrins in inflammation and their possible application as new anti-inflammatory agents.

Decreased soluble dipeptidyl peptidase IV activity as a potential serum biomarker for COPD.

OBJECTIVES: The objective of this study was to measure soluble dipeptidyl peptidase IV (sDPPIV) activity in sera of patients with stable chronic obstructive pulmonary disease (COPD) in comparison to healthy controls. The main goal was to assess changes in the enzyme activity in relation to severity of the disease, age and smoking history and to evaluate diagnostic accuracy for prediction of COPD by level of serum sDPPIV activity. DESIGN AND METHODS: The study included 106 patients with stable COPD (GOLD II-GOLD IV stages) and 38 healthy controls. Serum sDPPIV activity as well as some inflammatory markers (CRP, total and differential leukocyte counts) was measured. Multivariate logistic regression models were applied to analyze association of sDPPIV activity and inflammatory markers in risk estimation for COPD development. RESULTS: sDPPIV activity in COPD patients was significantly reduced when compared to healthy controls. Decrease was observed already in GOLD II stage. Age and smoking history did not influence sDPPIV activity. Very good diagnostic accuracy (AUC=0.833; sensitivity and specificity of 85.7% and 78.9%, respectively) for GOLD II and good diagnostic accuracy (AUC=0.801; sensitivity and specificity of 65.1% and 86.8%, respectively) for total cohort of COPD patients were found. The multivariate logistic regression model showed that the use of sDPPIV in combination with CRP and lymphocyte proportion improved diagnostic strength and gave an AUC of 0.933. CONCLUSIONS: sDPPIV activity is decreased in COPD patients as early as in GOLD II stage. Very good diagnostic accuracy of sDPPIV activity suggests it as a candidate biomarker for early diagnosis of COPD.

N'-substituted-2'-O,3'-N-carbonimidoyl bridged macrolides: novel anti-inflammatory macrolides without antimicrobial activity.

Macrolide antibiotics, like erythromycin, clarithromycin, and azithromycin, possess anti-inflammatory properties. These properties are considered fundamental to the efficacy of these three macrolides in the treatment of chronic inflammatory diseases like diffuse panbronchiolitis and cystic fibrosis. However, long-term treatment with macrolide antibiotics presents a considerable risk for promotion of bacterial resistance. We have examined antibacterial and anti-inflammatory effects of a novel macrolide class: N'-substituted 2'-O,3'-N-carbonimidoyl bridged erythromycin-derived 14- and 15-membered macrolides. A small focused library was prepared, and compounds without antimicrobial activity, which inhibited IL-6 production, were selected. Data analysis led to a statistical model that could be used for the design of novel anti-inflammatory macrolides. The most promising compound from this library retained the anti-inflammatory activity observed with azithromycin in lipopolysaccharide-induced pulmonary neutrophilia in vivo. Importantly, this study strongly suggests that antimicrobial and anti-inflammatory activities of macrolides are independent and can be separated, which raises development plausibility of novel anti-inflammatory therapeutics.