Immunobiochemical profile of clear cell renal cell carcinoma (ccRCC): A preliminary study.

Clear cell renal cell carcinoma (ccRCC) is the most common variant of RCC. It is an aggressive disease with an unfavorable prognosis. The rich immune infiltrates present in the tumor microenvironment (TME) of ccRCC produce various signaling molecules, especially cytokines, which primarily activate the Jak/STAT pathway and significantly influence tumor pathogenesis. STAT3 has a well-defined oncogenic character. Using multiplex assays and ELISA, we have measured the concentrations of 27 cytokines and STAT3 in tumor and healthy renal tissue from 16 patients with histologically verified ccRCC. We have detected significantly higher levels of G-CSF, IL-6, CXCL10, CCL3, and CCL4 in tumor tissue than in their healthy counterparts. There were significant differences in the levels of IL-1ß and PDGF-BB between tumors of different nuclear grades (NG). Intratumoral IL-12p70 and IL-15 showed a significant positive correlation with intratumoral STAT3. The concentration of STAT3 in tumors was significantly lower than in the kidney. An increase in tumor STAT3 levels was associated with an increase in the pathological stage of the disease (TNM), but not with NG. The results of our study confirm the significant role of various cytokines and STAT3 in the pathogenesis of ccRCC and indicate their clinical relevance.

Cytokines in Renal Cell Carcinoma: A Step Towards Earlier Detection and Targeted Therapy.

Symptoms of renal cell carcinoma (RCC) have typically late onset and correlate with its advanced stage. No biomarkers of RCC are currently available. The present study analyzed the immuno-biochemical profile of RCC by measuring the levels of cytokines engaged in RCC pathophysiology. Cytokines were examined by capture sandwich immunoassays in tumor tissue and urine. Specimens of cancer and nearby healthy kidney tissues were obtained during nephrectomy from 60 RCC patients. The urine was obtained from both patients and healthy subjects. The findings in RCC tumor tissue compared to healthy renal tissues were following: (i) increases in interleukin-15 (IL-15), vascular endothelial growth factor (VEGF), interferon gamma-induced protein-10 (IP-10), macrophage inflammatory protein-1ß (MIP-1ß), monocyte chemoattractant protein-1 (MCP-1), and eotaxin, with VEGF, IP-10, and MIP-1ß significantly associated with the histologic tumor nuclear grading (NG); (ii) increases in platelet-derived growth factor (PDGF), IL-15, MIP-1ß, eotaxin, and MCP-1 in urine, with significant associations noticed between cytokines and disease stages for eotaxin and MCP-1; and (iii) decreases in PDGF, IL-15, MCP-1, VEGF, MIP-1ß, and eotaxin in urine from six patients on the third day after nephrectomy. We conclude that cytokines may play a critical role in the local pathogenesis of RCC, which opens the way for potential targeting of these molecules in novel therapies and their use as biomarkers for early noninvasive detection of RCC.

Effects of Inhalation of STIM-Orai Antagonist SKF 96365 on Ovalbumin-Induced Airway Remodeling in Guinea Pigs.

Airway remodeling (AR) consists of wall thickening and hyperreactivity. STIM (stromal interaction molecule) and Orai protein pathways mediate extracellular Ca2+ signals involved in AR. This study aims to define the effects on AR of the STIM-Orai antagonist SKF 96365 given by inhalation in three increasing doses in ovalbumin-induced AR. In the control group, the antiasthmatic budesonide and salbutamol were given in the same model. The airway structure was evaluated by histological and immunohistochemistry and reactivity by specific airway resistance, contraction strength of isolated airway smooth muscles, and mucociliary clearance expressed by ciliary beating frequency. The immuno-biochemical markers of chronic inflammation were evaluated by BioPlex and ELISA assays. The AR was mediated by inflammatory cytokines and growth factors. The findings show significant anti-remodeling effects of SKF 96365, which were associated with a decrease in airway hyperreactivity. The anti-remodeling effect of SKF 96365 was mediated via the suppression of IL-4, IL-5, and IL-13 synthesis, and IL-12-INF-γ-TGF-ß pathway. The budesonide-related AR suppression had to do with a decrease in proinflammatory cytokines and an increase in the anti-inflammatory IL-10, with negligible influence on growth factors synthesis and mucous glands activity.

Negative impact of anesthesia with midazolam, sufentanil, and propofol used in pediatric flexible bronchoscopy on the tracheal ciliary beat frequency in guinea pigs.

There is no direct evidence for the exact cilia-inhibitory effects of opioids, which are generally used to achieve general anesthesia in combination with other anesthetic drugs. These are the reasons, why we analysed direct concentration-dependent or systemic effects of anesthetics (propofol, sufentanil, and midazolam) at a recommended doses administered individually or simultaneously on the tracheal ciliary beat frequency (CBF) in in vitro experimental conditions. Brush biopsy technique was used to remove the tracheal epithelia of guinea pigs for microscopy evaluation of ciliary beating monitored by high-speed video camera and analysed by Ciliary Analysis software. The tracheal CBF was significantly lower in the presence of sufentanil (10-8 mol/L) than in the control group; similarly for midazolam-sufentanil (10-8 - 10-5 mol/L), as well as for midazolam-propofol (10-5 and 10-3 mol/L) combinations. The fact that concurrent administration of benzodiazepine significantly increased the risk of sufentanil-induced cilia-inhibition was pharmacologically confirmed using GABAA receptor antagonist, bicuculline methiodide. The benefit of propofol on the potent cilia-inhibitory effect achieved by benzodiazepine-opioid combination was non-significant. We highlight the pharmacodynamics interaction between anesthetic drugs mediated via GABAA receptor with negative impact on the CBF in a respiratory epithelium under experimental condition rather than the effect of individual anesthetic.

Different adaptive NO-dependent Mechanisms in Normal and Hypertensive Conditions.

Myocardial infarction (MI) remains the leading cause of death worldwide. We aimed to investigate the effect of NO deficiency on selective biochemical parameters within discreet myocardial zones after experimentally induced MI. To induce MI, the left descending coronary artery was ligated in two groups of 16-week-old WKY rats. In one group, NO production was inhibited by L-NAME (20 mg/kg/day) administration four weeks prior to ligation. Sham operations were performed on both groups as a control. Seven days after MI, we evaluated levels of nitric oxide synthase (NOS) activity, eNOS, iNOS, NFÒ¡B/p65 and Nrf2 in ischemic, injured and non-ischemic zones of the heart. Levels of circulating TNF-α and IL-6 were evaluated in the plasma. MI led to increased NOS activity in all investigated zones of myocardium as well as circulating levels of TNF-α and IL-6. L-NAME treatment decreased NOS activity in the heart of sham operated animals. eNOS expression was increased in the injured zone and this could be a compensatory mechanism that improves the perfusion of the myocardium and cardiac dysfunction. Conversely, iNOS expression increased in the infarcted zone and may contribute to the inflammatory process and irreversible necrotic changes.

Involvement of calcium regulating ion channels in contractility of human isolated urinary bladder.

This study specified the role of several key calcium-operating ion channels in contraction/relaxation of human detrusor muscle as possible target for overactive bladder (OAB) treatment. Detrusor samples, obtained from 18 males (average age 61.5 ± 5.9 years), were investigated by organ tissue bath method with following agents: diltiazem for L-type voltage-gated calcium channels; 3-fluropyridine-4-carboxylic acid (FPCA) for Orai-STIM channels; SKF 96365-hydrochloride for transient receptor potential (TRP) channels, T-type channels and Orai-STIM channels; 2- aminoethoxydiphenyl borate (2-APB) for inositol-triphosphate receptors (IP3Rs) and Orai-STIM channels. Oxybutynin and mirabegron were tested under the same conditions as controls. Mirabegron, 2-APB and FPCA exhibited the best suppressive effect on carbachol-induced detrusor contractility. As expressed by area under the contractile curve (AUCC), 2-APB, FPCA and mirabegron have similar AUCC: 1.79, 1.73, 1.73. The highest AUCC was 3.64 for diltiazem+SKF, followed by 3.21 for diltiazem, 3.16 for SKF and 2.94 for oxybutynin. The lowest median amplitude and contraction variability is for 2-APB followed by mirabegron and FPCA. There were significant differences between: 2-APB/FPCA vs.: ditiazem, diltiazem+SKF and SKF. Summary of results suggested the principal role of IP3Rs, Orai-STIM coupling and large-conductance calcium-activated potassium channels in detrusor contraction and pointed on Orai-STIM channels as possible targets for OAB pharmacotherapy.

Antitussive activity of Withania somnifera and opioid receptors.

Arabinogalactan is a polysaccharide isolated from the roots of the medicinal plant Withania somnifera L. It contains 65% arabinose and 18% galactose. The aim of the present study was to evaluate the antitussive activity of arabinogalactan in conscious, healthy adult guinea pigs and the role of the opioid pathway in the antitussive action. A polysaccharide extract was given orally in a dose of 50 mg/kg. Cough was induced by an aerosol of citric acid in a concentration 0.3 mol/L, generated by a jet nebulizer into a plethysmographic chamber. The intensity of cough response was defined as the number of cough efforts counted during a 3-min exposure to the aerosol. The major finding was that arabinogalactan clearly suppressed the cough reflex; the suppression was comparable with that of codeine that was taken as a reference drug. The involvement of the opioid system was tested with the use of a blood-brain barrier penetrable, naloxone hydrochloride, and non-penetrable, naloxone methiodide, to distinguish between the central and peripheral mu-opioid receptor pathways. Both opioid antagonists acted to reverse the arabinogalactan-induced cough suppression; the reversion was total over time with the latter antagonist. We failed to confirm the presence of a bronchodilating effect of the polysaccharide, which could be involved in its antitussive action. We conclude that the polysaccharide arabinogalactan from Withania somnifera has a distinct antitussive activity consisting of cough suppression and that this action involves the mu-opioid receptor pathways.

Orai1 protein expression and the role of calcium release-activated calcium channels in the contraction of human term-pregnant and non-pregnant myometrium.

AIM: This experimental in vitro study examined differences in the expression and activity of calcium release-activated calcium (CRAC) channels of human term-pregnant and non-pregnant myometrium. MATERIAL AND METHODS: The tissue samples were obtained from term-pregnant myometrium in labor of women undergoing cesarean section and from non-pregnant myometrium of women undergoing total hysterectomy due to uterine myoma. The expression of Orai1 protein, a pore-forming subunit of CRAC channels, in human myometrium was examined using immunohistochemistry. CRAC channel involvement in the amplitude and frequency of myometrial contractions was evaluated in vitro using a tissue bath method with a CRAC ion channel blocker 3-fluropyridine-4-carboxylic acid (FPCA). RESULTS: Decreased Orai1 expression was observed in human term-pregnant laboring myometrium compared with non-pregnant myometrium. However, the initial oxytocin-induced contraction of myometrium was significantly suppressed at different doses of FPCA in both non-pregnant human isolated myometrium and non-pregnant myometrium. The frequency of contractions was the most significantly reduced at the lowest dose of FPCA in non-pregnant myometrium and remained suppressed at all doses of FPCA in term-pregnant myometrium. Salbutamol was shown as more effective in suppression of amplitude in term-pregnant isolated myometrium. CONCLUSION: Our results provide the first information about the changes in the Orai1 protein expression and activity of human myometrial CRAC channels in term-pregnant laboring myometrium.

The effect of long-term administered CRAC channels blocker on the functions of respiratory epithelium in guinea pig allergic asthma model.

Previously, therapeutic potency of CRAC channels blocker was evidenced as a significant decrease in airway smooth muscle hyperreactivity, antitussive and anti-inflammatory effects. The major role of the respiratory epithelium in asthma pathogenesis was highlighted only recently and CRAC channels were proposed as the most significant route of Ca2+ entry into epithelial cells. The aim of the study was to analyse the impact of long-term administered CRAC channels blocker on airway epithelium, e.g. cytokine production and ciliary beat frequency (CBF) using an animal model of allergic asthma. Ovalbumin-induced allergic airway inflammation of guinea pigs was followed by long-term (14 days lasted) therapy by CRAC blocker (3-fluoropyridine-4-carboxylic acid, FPCA). The influence of long-term therapy on cytokines (IL-4, IL-5 and IL-13) in BALF and in plasma, immunohistochemical staining of pulmonary tissue (c-Fos positivity) and CBF in vitro were used for analysis. Decrease in cytokine levels and in c-Fos positivity confirmed an anti-inflammatory effect of long-term administered FPCA. Cytokine levels in BALF and distribution of c-Fos positivity suggested that FPCA was a more potent inhibitor of respiratory epithelium secretory functions than budesonide. FPCA and budesonide reduced CBF only insignificantly. All findings supported CRAC channels as promising target in the new strategy of antiasthmatic treatment.

Immune analysis of urine and plasma samples from patients with clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the third most common type of urological malignancy worldwide, and it is associated with a silent progression and late manifestation. Patients with a metastatic form of ccRCC have a poor prognosis; however, when the disease is diagnosed early, it is largely curable. Currently, there are no biomarkers available in clinical practice for ccRCC. Thus, the aim of the present study was to measure 27 biologically relevant cytokines in preoperative and postoperative urine samples, and in preoperative plasma samples from 34 patients with ccRCC, and to evaluate their diagnostic significance. The concentrations of cytokines were assessed by multiplex immune assay. The results showed significantly higher levels of IL-1 receptor antagonist, IL-6, IL-15, chemokine (C-C motif) ligand (CCL)2, CCL3, CCL4, C-X-C motif ligand (CXCL)10, granulocyte-macrophage colony stimulating factor (GM-CSF) and platelet-derived growth factor-BB (PDGF-BB), and lower levels of granulocyte colony stimulating factor (G-CSF) in urine samples from patients prior to surgery compared with those in the controls. Notably, the urine levels of G-CSF, IL-5 and vascular endothelial growth factor differed following tumor removal compared with the preoperative urine levels. In addition, urinary G-CSF, GM-CSF, IL-6, CXCL10, CCL5 and PDGF-BB appeared to be potential markers of tumor grade. Plasma from patients with ccRCC contained significantly higher levels of IL-6 and lower levels of CCL2 than control plasma. In conclusion, the present findings indicated that urinary and circulating cytokines may represent a promising novel tool for the early diagnosis of ccRCC and/or prediction of tumor grade.

The long-term administration of Orai 1 antagonist possesses antitussive, bronchodilatory and anti-inflammatory effects in experimental asthma model.

The best-studied store-operated Ca2+ channels (SOCs), Ca2+ release activated Ca2+ (CRAC) channels, are activated by depleting endoplasmic reticulum Ca2+ pool and mediate Ca2+ influx vitally important for Ca2+ restoration and many cellular function. CRAC channels were identified on immune and airway smooth muscle (ASM) cells. Emerging evidence points to its involvement in allergic airways diseases. This article evaluated therapeutic potency of CRAC antagonist in experimental animal model of allergic asthma. Allergic asthma, induced by repetitive exposure of guinea pigs to ovalbumine, was followed by 14 days therapy by CRAC antagonist (3-fluoropyridine-4-carboxylic acid, FPCA). In vivo changes of specific airways resistance (sRaw) evaluated bronchodilatory effect of FPCA and salbutamol. The method of citric acid-induced cough reflex assessed antitussive activity of FPCA and codeine. The measurement of exhaled NO (ENO), expression of inducible NO-synthase (iNOS) by RT-PCR and immunohistochemical staining of airways tissue verified anti-inflammatory effect of FPCA. Long-term administration of FPCA resulted in significant cough suppression and bronchodilation, both comparable to the effect of control drugs. FPCA significantly decreased ENO and iNOS expression, which together with immunohistochemical analysis validated its anti-inflammatory effect. Presented data confirmed CRAC channels as a promising target for treatment of respiratory diseases associated with allergic inflammation.

Effects of TRPV4 channel blocker on airway inflammation and airway defense reflexes in experimentally induced model of allergic asthma.

The transient receptor potential (TRP) channels regulate physiological and pathological processes. Changes in their activity and sensitivity may be involved in the pathophysiology of asthma. The present study investigates the effect of an inhaled TRPV4 channel blocker HC-067047 in an experimental guinea pig model of ovalbumin-induced allergic asthma. We monitored the effect of 50 nM, 100 nM, and 150 nM HC-067047 concentrations on airway defense reflexes in vivo and tracheal smooth muscle contractility in vitro. The anti-inflammatory action of HC-067047 was investigated by analysis of chronic inflammation markers from lung homogenates. The results suggest that HC-067047 can suppress airway defense reflexes in vivo and acetylcholine-induced contractility in vitro. Immunological analysis revealed that TRPV4 channel blockade leads to a decrease in the levels of inflammatory cytokines. An effect on airway defence reflexes and airway inflammation was observed using tested concentrations (50 mM, 100 mM, 150 mM) of HC-067047. The effects of HC-067047 on both airway defense reflexes and inflammation underline the role of TRPV4 channels in asthma and uncover therapeutic targets for developing innovative drugs in asthma therapy.

The anti-asthmatic potential of Rho-kinase inhibitor hydroxyfasudil in the model of experimentally induced allergic airway inflammation.

OBJECTIVE: This experimental study evaluated the anti-asthmatic potential of the Rho-kinase inhibitor hydroxyfasudil in the settings of allergen-induced allergen-induced experimental asthma. METHODS: Chronic allergic airway inflammation was caused by 28 days-sensitisation of guinea pigs with ovalbumin (OVA). Hydroxyfasudil was administered intraperitoneally in two doses for the last two weeks (1 mg/kg b.w.; 10 mg/kg b.w.). The degree of allergic inflammation was determined based on concentrations of inflammatory Th2 cytokines (IL-4, IL-13), Th1 cytokines (TNF-α and IFN-γ) in the lung homogenate and leukocyte count in the bronchoalveolar lavage fluid (BALF). The markers of remodelling and fibrosis, the growth factors (TGF-ß1, EGF), EGF receptor, collagen type III and V were estimated in lung homogenate. The changes in specific airway resistance (sRaw) were used as an in vivo bronchial hyperreactivity parameter. RESULTS: Hydroxyfasudil administration at both doses significantly reduced sRaw after a week of therapy. We observed a decline of IL-13, TNF-α and IFN-γ in lung homogenate and a lower presence of lymphocytes in BALF after 14 days of hydroxyfasudil administration at both tested doses. Hydroxyfasudil 14 days-treatment at both doses effectively reduced the concentrations of TGF-ß1, EGF receptors, collagen type III and V in BALF and modulated EGF levels. CONCLUSIONS: These findings indicate that RhoA/Rho-kinase is involved in the pathophysiology of allergic airway inflammation and suggest that Rho-kinase inhibitor hydroxyfasudil has therapeutic potential for asthma management.

Dysfunctional mucociliary clearance in asthma and airway remodeling - New insights into an old topic.

Bronchial asthma is a heterogeneous respiratory condition characterized by chronic airway inflammation, airway hyperresponsiveness and airway structural changes (known as remodeling). The clinical symptoms can be evoked by (non)specific triggers, and their intensity varies over time. In the past, treatment was mainly focusing on symptoms' alleviation; in contrast modern treatment strategies target the underlying inflammation, even during asymptomatic periods. Components of airway remodeling include epithelial cell shedding and dysfunction, goblet cell hyperplasia, subepithelial matrix protein deposition, fibrosis, neoangiogenesis, airway smooth muscle cell hypertrophy and hyperplasia. Among the other important, and frequently forgotten aspects of airway remodeling, also loss of epithelial barrier integrity, immune defects in anti-infectious defence and mucociliary clearance (MCC) dysfunction should be pointed out. Mucociliary clearance represents one of the most important defence airway mechanisms. Several studies in asthmatics demonstrated various dysfunctions in MCC - e.g., ciliated cells displaying intracellular disorientation, abnormal cilia and cytoplasmic blebs. Moreover, excessive mucus production and persistent cough are one of the well-recognized features of severe asthma and are also associated with defects in MCC. Damaged airway epithelium and impaired function of the ciliary cells leads to MCC dysfunction resulting in higher susceptibility to infection and inflammation. Therefore, new strategies aimed on restoring the remodeling changes and MCC dysfunction could present a new therapeutic approach for the management of asthma and other chronic respiratory diseases.

Analysis of HIF-1α expression and genetic polymorphisms in human clear cell renal cell carcinoma.

Introduction: Clear cell renal cell carcinoma (ccRCC) is mostly diagnosed incidentally and has relatively high recurrence rates. Alterations in VHL/HIF and mTOR pathways are commonly present in ccRCC. The present study attempted to identify potential diagnostic markers at the biochemical and molecular level. Methods: In total, 54 subjects (36 patients with ccRCC and 18 cancer-free controls) were enrolled. ELISA was used to measure the levels of HIF-1α in the tumor and healthy kidney tissue. The association between five selected SNPs (rs779805, rs11549465, rs2057482, rs2295080 and rs701848) located in genes of pathologically relevant pathways (VHL/HIF and mTOR) and the risk of ccRCC in the Slovak cohort was studied using real-time PCR. Results: Significant differences in HIF-1α tissue levels were observed between the tumor and healthy kidney tissue (p < 0.001). In the majority (69%) of cases, the levels of HIF-1α were higher in the kidney than in the tumor. Furthermore, the concentration of HIF-1α in the tumor showed a significant positive correlation with CCL3 and IL-1ß (p (R2) 0.007 (0.47); p (R2) 0.011 (0.38). No relationship between intratumoral levels of HIF-1α and clinical tumor characteristics was observed. Rs11549465, rs2057482 in the HIF1A gene did not correlate with the expression of HIF-1α either in the tumor or in the normal kidney. None of the selected SNPs has influenced the susceptibility to ccRCC. Conclusion: More research is neccesary to elucidate the role of HIF-1α in the pathogenesis of ccRCC and the association between selected SNPs and susceptibility to this cancer.

Participation of BKCa2+ and KATP potassium ion channels in the contractility of human term pregnant myometrium in in vitro conditions.

AIM: The aim of this study was to assess the participation of ligand-sensitive potassium large conductance calcium-activated ion channels (BK(Ca2+) ) and adenosine triphosphate (ATP)-sensitive potassium ion channels (K(ATP) ) using its openers (NS1619 and pinacidil) in the contractility of human term pregnant myometrium in in vitro conditions. METHODS: Human myometrium tissue samples were collected from term pregnant laboring women who had to undergo cesarean section. The contractility of myometrium was induced by the application of oxytocin into the organ bath. Myometrial strips were incubated with the opener of BK(Ca2+) potassium ion channels NS1619 and its antagonist tetraethylammonium or with the opener of K(ATP) potassium ion channels pinacidil and its antagonist glibenclamide. RESULTS: K(ATP) potassium ion channel's opener pinacidil significantly decreased amplitude of myometrial contractions (P < 0.05) as well as frequency of myometrial contractions (P < 0.05) provoked by oxytocin in human term pregnant myometrium in in vitro conditions. The inhibition of the human myometrial contractions of pinacidil was significantly antagonized by its specific antagonist glibenclamide (P < 0.05). BK(Ca2+) potassium ion channel's opener NS1619 did not significantly affect the contractile activity of human term pregnant myometrium induced by the application of oxytocin in in vitro conditions. CONCLUSION: In our experimental study we found that the participation of BK(Ca2+) and K(ATP) potassium ion channels in the contractility of human term pregnant myometrium in labor is probably different.

The Changes in Expression of NaV1.7 and NaV1.8 and the Effects of the Inhalation of Their Blockers in Healthy and Ovalbumin-Sensitized Guinea Pig Airways.

BACKGROUND: The presented study evaluated the suppositional changes in the airway expression of Nav1.8 and Nav1.7 and their role in the airway defense mechanisms in healthy animals and in an experimental asthma model. METHODS: The effects of the blockers inhalation on the reactivity of guinea pig airways, number of citric-acid-induced coughs and ciliary beating frequency (CBF) were tested in vivo. Chronic inflammation simulating asthma was induced by repetitive exposure to ovalbumin. The expression of Nav1.7 and Nav1.8 was examined by ELISA. RESULTS: The Nav 1.8 blocker showed complex antitussive and bronchodilatory effects and significantly regulated the CBF in healthy and sensitized animals. The Nav1.7 blockers significantly inhibited coughing and participated in CBF control in the ovalbumin-sensitized animals. The increased expression of the respective ion channels in the sensitized animals corresponded to changes in CBF regulation. The therapeutic potency of the Nav1.8 blocker was evidenced in combinations with classic bronchodilators. CONCLUSION: The allergic-inflammation-upregulated expression of Nav1.7 and Nav1.8 and corresponding effects of blocker inhalation on airway defense mechanisms, along with the Nav1.8 blocker's compatibility with classic antiasthmatic drugs, bring novel possibilities for the treatment of various respiratory diseases. However, the influence of the Nav1.8 blocker on CBF requires further investigation.

The anti-asthmatic potential of flavonol kaempferol in an experimental model of allergic airway inflammation.

Flavonol kaempferol possesses a broad spectrum of potent pharmacological activities that seem to be effective in the modulation of allergic respiratory diseases. In our study, an experimental animal model of ovalbumin (OVA)-induced allergic airway inflammation in guinea pigs was used to determine the anti-asthmatic potential of kaempferol. The parameters of specific airway resistance (sRaw) and cough reflex response were evaluated in vivo. In vitro, an assessment of tracheal smooth muscle (TSM) contractility and analyses of inflammatory cytokines (IL-4, IL-5, IL-13, GM-CSF, IFN-γ), transforming growth factor (TGF-ß1), immune cells count and ciliary beating frequency (CBF) were performed. Both single (6, 20 mg/kg b. w. p. o.) and long-term administered doses of kaempferol (20 mg/kg b. w. p. o., 21 days) suppressed sRaw provoked by histamine in conscious animals. The administration of kaempferol for 21 days attenuated histamine-induced TSM contractility in vitro and ameliorated the progression of chronic airway inflammation by decreasing the levels of IL-5, IL-13, GM-CSF, eosinophil count in bronchoalveolar lavage (BAL) fluid and TGF-ß1 protein level in lung tissue. Kaempferol also eliminated the alterations in cough reflex sensitivity invoked by OVA-sensitization, but it did not affect CBF. The results demonstrate that flavonol kaempferol can modulate allergic airway inflammation and associated asthma features (AHR, aberrant stimulation of cough reflex).

Pharmacodynamic evaluation of dihydroxyflavone derivate chrysin in a guinea pig model of allergic asthma.

OBJECTIVE: This experimental study evaluated the anti-asthmatic capacity of the dihydroxyflavone chrysin in the settings of ovalbumin (OVA)-induced allergic inflammation. METHODS: The parameters that were used to assess the anti-asthmatic activity of chrysin included the specific airway resistance to histamine, the sensitivity to a chemically induced cough and the activity of chrysin on the ciliary beat frequency (CBF) of the respiratory epithelium. The anti-inflammatory potential was confirmed by the measurement of cytokine concentrations Th2 (IL-4, IL-5 and IL-13), Th1 (Granulocyte-macrophage colony-stimulating factor [GM-CSF], INF-γ and IL-12), leucocyte count in the bronchoalveolar lavage fluid (BALF) and growth factor TBF-ß1 in lung homogenate. KEY FINDINGS: Chronic administration of chrysin (30 mg/kg/day for 21 days) to OVA-sensitised guinea pigs showed bronchodilatory activity comparable to that of long-acting ß 2 receptors agonist (LABA) salmeterol. Chrysin revealed antitussive efficiency but was not able to abolish the negative effect of OVA on CBF. Chrysin managed to ameliorate the progression of chronic airway inflammation by decreasing the count of eosinophils, lymphocytes and basophils, IL-5, L-13, GM-CSF, INF-γ in BALF, and TGF-ß1 in lung homogenate. CONCLUSIONS: The acquired results support the complex anti-asthmatic profile of chrysin. The flavone may represent an attractive compound for further studies concerning the prevention or treatment of asthma.

Respiratory Cilia as a Therapeutic Target of Phosphodiesterase Inhibitors.

Mucociliary clearance is an essential airway defense mechanism dependent predominantly on the proper ciliary function and mucus rheology. The crucial role of cilia is evident in `a variety of respiratory diseases, as the ciliary dysfunction is associated with a progressive decline in lung function over time. The activity of cilia is under supervision of multiple physiological regulators, including second messengers. Their role is to enable a movement in coordinated metachronal waves at certain beat frequency. Ciliary function can be modulated by various stimuli, including agents from the group of beta2 agonists, cholinergic drugs, and adenosine triphosphate (ATP). They trigger cilia to move faster in response to elevated cytoplasmic Ca2+ originated from intracellular sources or replenished from extracellular space. Well-known cilia-stimulatory effect of Ca2+ ions can be abolished or even reversed by modulating the phosphodiesterase (PDE)-mediated breakdown of cyclic adenosine monophosphate (cAMP) since the overall change in ciliary beating has been dependent on the balance between Ca2+ ions and cAMP. Moreover, in chronic respiratory diseases, high ATP levels may contribute to cAMP hydrolysis and thus to a decrease in the ciliary beat frequency (CBF). The role of PDE inhibitors in airway cilia-driven transport may help in prevention of progressive loss of pulmonary function often observed despite current therapy. Furthermore, administration of selective PDE inhibitors by inhalation lowers the risk of their systemic effects. Based on this review we may conclude that selective (PDE1, PDE4) or dual PDE inhibitors (PDE3/4) increase the intracellular level of cyclic nucleotides in airway epithelial cells and thus may be an important target in the development of new inhaled mucokinetic agents. Further research is required to provide evidence of their effectiveness and feasibility regarding their cilia-modulating properties.