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.

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.

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.

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.

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.

Chemical characteristics and significant antitussive effect of the Erigeron canadensis polyphenolic polysaccharide-protein complex.

ETHNOPHARMACOLOGICAL RELEVANCE: Erigeron canadensis has been used in traditional medicine to treat a variety of respiratory diseases, including acute upper and lower respiratory tract infections and cough-related asthma. There is as yet no relevant experimental or clinical study in the scientific literature evaluating the efficacy of plants in these disorders. AIM OF THE STUDY: To investigate the active ingredients in Erigeron canadensis, a complex isolated from flowering parts of a plant was tested for airway defense reflexes, in particular for cough reflexes and airway reactivity. Both were experimentally induced by a chemical irritant that simulated the inflammatory conditions of their formation. MATERIAL AND METHODS: The polyphenolic polysaccharide-protein (PPP) complex was isolated from the flowering parts of Erigeron canadensis by hot alkaline extraction and a multi-stage purification process. The antitussive activity was confirmed as a decrease in the number of citric acid-induced coughs and the bronchodilator effect was verified as a decrease in specific airway resistance (sRaw) in conscious guinea pigs. RESULTS: The dark brown Erigeron complex with a molecular weight of 38,000 g/mol contained phenolics (13.2% wt%), proteins (16.3% wt%), and uronic acids (6.3% wt%). The neutral carbohydrate part of Erigeron consisted mainly of xylose (12.1 wt%), glucose (13.3 wt%), arabinose (24.1 wt%), and galactose (41.0 wt%) residues. Arabinogalactan and 4-OMe-glucuronoxylan have been found to be the major polysaccharides in the Erigeron complex. Using a method of chemically-induced cough reflex and guinea pigs test system the Erigeron complex exhibited statistically significant, the dose-dependent antitussive activity, which was similar to that of the centrally-acting opioid agonist codeine. CONCLUSION: Pharmacological tests have revealed a new pharmacodynamic effect of the Erigeron complex, namely an antitussive effect. Its activity was most pronounced in comparison with all previously tested compounds from other medicinal plants and approached the effect of codeine, the most potent antitussive used in clinical practice. The results provide the scientific basis for the application of this herb in traditional medicine.

Structural characteristics and biological effects of exopolysaccharide produced by cyanobacterium Nostoc sp.

Complex structure of cyanobacterium Nostoc sp. exopolysaccharide (EPS), with apparent molecular weight 214 × 103 g/mol, can be deduced from its composition. Chemical and NMR analyses found four dominant sugar monomers, namely (1 → 4)-linked α-l-arabinopyranose, ß-d-glucopyranose, ß-d-xylopyranose and (1 → 3)-linked ß-d-mannopyranose, two different uronic acids and a lactyl group, with (1 → 4,6)-linked ß-d-glucopyranose as the only branch point suggest a complex structure of this polymer. The dominant uronic acid is α-linked, but it remained unidentified. ß-d-Glucuronic acid was present in lower amount. Their position as well as that of lactyl remained undetermined too. Different doses of orally administered EPS in guinea pigs evoked a significant decrease in cough effort and a decrease in airway reactivity. The antitussive efficacy and bronchodilator effect of higher doses of EPS were found to be similar to that of the antitussive drug codeine and the antiasthmatic salbutamol. Without significant cytotoxicity on the RAW 264.7 cells, EPS stimulated the macrophage cells to produce pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), and prostaglandins (PGs) and nitric oxide (NO) via induction of COX-2 and iNOS expression, respectively, suggesting that this biopolymer potentiates an early innate immune response and can therefore be used as a new immune modulator.

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.

The Possibility of Therapeutic Drug Monitoring of the Most Important Interactions in Nursing Homes.

BACKGROUND: Therapeutic drug monitoring is a relevant tool in drug treatment of elderly patients. The aim of this study was to assess the possibility of therapeutic drug monitoring of the most important potential interactions in nursing homes. METHODS: A material of prescribed drugs to 446 patients in three nursing homes in Bergen, Norway from a single day in March 2016 was analysed. Clinically relevant drug interactions (pharmacodynamic or pharmacokinetic) were identified and classified with Stockley`s Interaction Alerts. The most important interaction among several in each patient were ranked by recommended action > severity > evidence according to Stockley`s. The possibility of therapeutic drug monitoring of drug combinations involved in the most important interactions was retrieved from a database of all laboratories performing clinical pharmacology in Norway (the Pharmacology Portal). RESULTS: Two or more drugs were used by 443 (99.3%) of 446 patients. Three-hundred and eightyfour patients (86.1%) had > 1 interaction. About 95% of the most important interactions were pharmacodynamic. In 280 (72.9%) of these interactions, Stockley`s recommended adjust dose or monitoring. Among the 384 most important interactions, 93% involved one drug and 41% involved two drugs available for therapeutic drug monitoring. CONCLUSION: In this pilot study, therapeutic drug monitoring was possible in the majority of the most important interactions in Norwegian nursing homes. This option is of importance since adjust dose or monitoring were frequently recommended actions associated with these interactions.

Degenerative Lumbar Spondylolisthesis: Biochemical Aspects and Evaluation of Stabilization Surgery Extent in Terms of Adjacent Segment Disease Theory.

OBJECTIVE: In lumbar degenerative spondylolisthesis (DSL), the criteria and extent of surgical treatment have not been strictly defined owing to the adjacent segment disease theory and unclear molecular pathogenesis. The present study analyzed the clinical and radiographic findings of patients after lower lumbar fusion surgery with single and 2-level DSL and explored the inflammatory mediator's role in DSL evolution and symptoms. METHODS: The prospective follow-up of patients with DSL, stratified by the stabilization extent (L4-L5, L5-S1, and L4-S1), included the Back Illness Pain and Disability 9-item questionnaire and native and dynamic radiographs to evaluate the intervertebral disc height and adjacent segments' angular motion. Follow-up examinations were performed at 3, 12, and 24 months. The pathological cytokine concentrations in the intervertebral disc and facet joints of the subchondral bone were assessed using the BioPlex assay in perioperatively collected patient samples and compared with those of control subjects obtained during multiorgan procurement. These findings were correlated with pain localization and severity. RESULTS: Statistical analysis of the questionnaire data revealed significant postoperative improvement in all patients, in particular, the L4-L5 group. Also, we found radiographic evidence of angular motion reduction in both adjacent segments near the limits of statistical significance and a meaningful correlation with subjective status improvement at 24 months. BioPlex analysis revealed platelet-derived growth factor 2 B subunits, interleukin-6, interleukin-8, and tumor necrosis factor-α were elevated in spinal unit segments and the interleukin-1ß levels correlated significantly with the intensity of low backache. CONCLUSIONS: Our findings did not support the adjacent segment disease theory. However, later development of these changes could not be excluded. The cytokines, chemokines, and growth factors play a significant role in DSL pathogenesis and symptoms.

Cytokine and chemokine profile changes in patients with lower segment lumbar degenerative spondylolisthesis.

BACKGROUND: Lumbar degenerative spondylolisthesis (DS) develops as a result of inflammatory and remodeling processes in facet joints (FJs). Several inflammatory cytokines are involved in the osteoarthritic and remodeling changes that occur and in low-back and/or radicular pain, the most prevalent clinical symptom of disease. This study improves knowledge related to the roles that 27 cytokines, chemokines and growth factors play in the pathophysiology of lumbar DS. MATERIAL AND METHODS: Cytokine levels were examined using capture sandwich immunoassay using the Bio-Plex® 200 System and the Bio-PlexTM Human Cytokine Standard 27-Plex, Group I (Bio-Rad, Hercules, California, USA) separately in intervertebral discs (IVDs) and FJ bone tissue. The samples were obtained during primary spinal surgery from 9 patients suffering from lower segment lumbar DS. The pain intensity was assessed using a visual analog scale. The controls were tissue samples collected from both lower lumbar segment levels of 6 male subjects during a multiorgan procurement procedure. RESULTS: The Bio-Plex® assay revealed significant differences between the patients and controls in cytokines, chemokines and growth factor profiles: i, The elevated interleukin-6 (IL-6), IL-7, IL-13, tumor necrosis factor α (TNF-α), interferon γ and platelet-derived growth factor levels in lumbar DS samples of subchondral FJ bone. These indicated ongoing inflammation, bone formation and increased fibroblasts activity in the FJ bone. ii, The elevated levels of IL-6, IL-8, TNF-α, granulocyte-macrophage colony-stimulating factor and monocyte chemoattractant protein-1 in anulus fibrosus together with increased IL-6, IL-8, TNF-α and eotaxin and decreased IL-1-receptor antagonist in nucleus pulposus confirmed advanced IVD degeneration in the patient samples. CONCLUSION: This study identified, for the first time, protective levels of cytokines, chemokines and growth factors in healthy subjects and supported their significant involvement in the pathogenesis of lumbar DS. The control samples and analytical methods used avoided any false changes in the cytokine levels due to secondary factors (e.g., death of donor and limited cytokine stability).

Bronchodilatory, antitussive and anti-inflammatory effect of morin in the setting of experimentally induced allergic asthma.

OBJECTIVE: Using an experimental model of allergic asthma, we evaluated the anti-asthmatic potential of polyphenol flavonol derivate morin after either acute or long-term treatment of male OVA-sensitised guinea pigs. METHODS: The following methods were used in experiments: the in-vitro tracheal smooth muscle contraction induced by histamine; the changes in specific airway resistance (sRaw) to histamine and the sensitivity of a chemically induced cough reflex both via an in-vivo method; the serum and BALF concentrations' analysis of the inflammatory cytokines interleukin IL-4, IL-5, IL-13; and lung tissue infiltration by eosinophils and mastocytes. KEY FINDINGS: Our data show that acute morin (30 mg/kg) and chronic 21-day morin (30 mg/kg/day) administration had a comparable antitussive efficiency with opioid antitussive codeine. Acute morin bronchodilatory activity defined by in-vivo sRaw decline did not reach SABA salbutamol effect. However, bronchodilatory efficiency of morin after long-term administration was by 34% higher as effect of LABA salmeterol. The 21-day morin treatment of OVA-sensitised guinea pigs reduced the serum, BALF levels of IL-4 and IL-13, lung tissue eosinophil and mastocyte infiltration comparable with corticosteroid budesonide. CONCLUSIONS: In summary, morin represents very rational target for additional studies as potential substance for control as well as prevention of asthma inflammation and symptoms.

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.

Pharmacologic modulation of experimentally induced allergic asthma.

Allergic asthma is the most frequent disease of the respiratory tract. The aim of the current experimental and clinical studies was to find new sources of drugs able to control asthmatic inflammation and airway hyperresponsiveness. Our experimental studies were focused on efficiency evaluation of substances able to influence activities of ion channels, phosphodiesterase (PDE) isoforms, substances from the group of polyphenols and NO metabolism modulators during experimentally induced allergic asthma.

Red wine polyphenolic compounds inhibit tracheal smooth muscle contraction during allergen-induced hyperreactivity of the airways.

The aims of the study were to investigate the short and long-term effects of Provinol (red wine polyphenolic compounds) on tracheal smooth muscle reactivity using an in-vitro model of ovalbumin-induced airway inflammation in guinea-pig trachea, and to evaluate the role of nitric oxide (NO) in the bronchodilatory effect of Provinol. The amplitude of tracheal smooth muscle contraction in response to mediators of bronchoconstriction - histamine (10 nM-1 mM), acetylcholine (10 nM-1 mM) and to allergen (ovalbumin 10(-5)-10(-3) g mL(-1)) was used as a parameter of tracheal smooth muscle reactivity. To test the short-term effects of Provinol, isolated tracheal strips were pre-treated for 30 min with Provinol (10(-4) mg mL(-1)) alone or in combination with Nomega-nitro-L-arginine methyl ester (L-NAME; 10(-6) mol L(-1)). To test the long-term effects of Provinol, isolated tracheal strips were prepared from guinea pigs that had been treated for 14 days with Provinol (20 mg kg(-1) per day) alone or in combination with L-NAME (40 mg kg(-1) per day). Incubation of tracheal smooth muscle with Provinol decreased the amplitude of contraction in response to ovalbumin, histamine and acetylcholine. The non-selective NO synthase inhibitor L-NAME partially abolished the effect of Provinol on acetylcholine and ovalbumin-induced but not histamine-induced bronchoconstriction. A similar profile was observed after 14 days' oral administration of Provinol. In conclusion, Provinol inhibited the allergen- and spasmogen-induced contraction of tracheal smooth muscle in ovalbumin-sensitized guinea pigs via a mechanism that was mediated at least partially through the metabolism of NO.