The effect of combining an inhaled corticosteroid and a long-acting muscarinic antagonist on human airway epithelial cells in vitro.

BACKGROUND: Airway epithelial cells (AECs) are a major component of local airway immune responses. Direct effects of type 2 cytokines on AECs are implicated in type 2 asthma, which is driven by epithelial-derived cytokines and leads to airway obstruction. However, evidence suggests that restoring epithelial health may attenuate asthmatic features. METHODS: We investigated the effects of passive sensitisation on IL-5, NF-κB, HDAC-2, ACh, and ChAT in human bronchial epithelial cells (HBEpCs) and the effects of fluticasone furoate (FF) and umeclidinium (UME) alone and in combination on these responses. RESULTS: IL-5 and NF-κB levels were increased, and that of HDAC-2 reduced in sensitised HEBpCs. Pretreatment with FF reversed the effects of passive sensitisation by concentration-dependent reduction of IL-5, resulting in decreased NF-κB levels and restored HDAC-2 activity. Addition of UME enhanced these effects. Sensitized HEBpCs also exhibited higher ACh and ChAT levels. Pretreatment with UME significantly reduced ACh levels, and addition of FF caused a further small reduction. CONCLUSION: This study confirmed that passive sensitisation of AECs results in an inflammatory response with increased levels of IL-5 and NF-κB, reduced levels of HDAC-2, and higher levels of ACh and ChAT compared to normal cells. Combining FF and UME was found to be more effective in reducing IL-5, NF-κB, and ACh and restoring HDAC-2 compared to the individual components. This finding supports adding a LAMA to established ICS/LABA treatment in asthma and suggests the possibility of using an ICS/LAMA combination when needed.

Early pathological changes of peri-coronal tissue in the distal area of erupted or partially impacted lower third molars.

AIM: This study was performed to histologically evaluate peri-coronal tissues of partially impacted and erupted third molars that did not exhibit pathologic peri-coronal radiolucency. MATERIALS AND METHODS: Healthy patients with erupted or partially erupted (with part or all of the dental crown present in the oral cavity) mandibular third molars (classified as IA and IIA according to the Pell and Gregory classification) and vertically positioned (according to the Winter classification or erupted third molars) associated with peri coronal radiolucency of equal to or less than 2.5 mm. Associated with third molar surgery, tissue sampling from the distal area was performed, which was subjected to an anatomopathological examination to determine the histological nature. RESULTS: One hundred teeth (100 patients) were selected, and 100 specimens were analyzed. 53% of the sample were included in the non-pathological group and 47% showed pathological changes (fibrotic tissue (n 15), periodontal cyst-like (n 9), squamous epithelial metaplasia (4 cases), islands of odontogenic epithelial residues organized micro-cyst with keratocystic/ameloblastic appearance (4 cases), granulation tissue (n 8), giant cell tumour (n 4) and lobular capillary hemangioma (n 4)). Pathological changes did not have differences in incidence between the gender (p value = 0.85) and did not show any correlation with age, (p value = 0,96). CONCLUSIONS: These findings suggest that radiographic appearance may not be a reliable indicator of the absence of disease within a dental follicle. Therefore, clinicians should pay attention to or follow up on even peri-coronal radiolucency of less than 2.5 mm.

Comparative study of the anaesthetic efficacy of 4% articaine versus 2% mepivacaine in mandibular third molar germectomy using different anaesthetic techniques: a split-mouth clinical trial.

BACKGROUND: Currently, one of the most discouraging aspects for many patients undergoing dental procedures is the administration of local anaesthesia. Therefore, there is a constant search for new techniques to avoid the invasive and painful nature of the injection. This study aimed to compare the clinical efficacy of local anaesthetics with articaine 4% or mepivacaine 2% (both with epinephrine 1:100.000), using different anaesthetic techniques to perform germectomy of lower third molars and to assess patients' feelings and pain during surgery. METHODS: Totally 50 patients (ranged 11-16 years) who required germectomy of mandibular third molars were recruited. Each patient received local anaesthesia on one side with articaine inoculated with plexus technique while on the other side with mepivacaine using inferior alveolar nerve block technique. The patients' evaluation was performed on pre and intraoperative tactile-pressure feelings and intraoperative pain with four levels on the Visual Analogue Scale (VAS). RESULTS: Surgical operations lasted less with more efficient analgesia when articaine was used. The additional intraosseous injection was required mainly in the mepivacaine group intraoperatively. A few patients had tactile-pressure feelings while intraoperative pain sensation was absent in 90% of cases with articaine. Significant differences were found in the cases who reported "absent" and "moderate" VAS values, favoring the use of articaine. CONCLUSIONS: Articaine injected with a plexus anaesthetic technique seems to be more clinically manageable than mepivacaine for the mandibular third molar germectomy. The discomfort of tactile-pressure feelings and pain experienced was lower using articaine anaesthetic technique used.

Current and emerging treatment modalities for bacterial rhinosinusitis in adults: a comprehensive review.

INTRODUCTION: Rhinosinusitis (RS) is defined as acute when it lasts up to 4 weeks and chronic when it lasts at least 12 weeks. Most acute forms begin with a viral upper respiratory infection that spreads into the paranasal sinuses and is followed by bacterial infection. It is uncertain how bacteria affect chronic rhinosinusitis (CRS). AREA COVERED: We review the current treatment of bacterial rhinosinusitis in adults, referring mainly to the two key documents published by the European Position Paper on Rhinosinusitis and Nasal Polyps 2020 and the International Consensus Statement 2021 on Allergy and Rhinology: Rhinosinusitis. EXPERT OPINION: The routine use of antibiotics should be avoided because most acute RS (ARS) have a viral origin. In patients with persistent/worsening symptoms, the most appropriate empirical therapy is a course of amoxicillin (with or without clavulanate). Macrolides are considered therapy options for CRS mainly because of their anti-inflammatory activity. The best agent, dose, and treatment duration still need to be identified due to a lack of solid evidence. Inflammation and symptoms must also be reduced, mainly by using nasal corticosteroids. Since antibiotic use in bacterial rhinosinusitis is questionable, the research focuses on non-antibiotic antimicrobial treatments.

HCC and Molecular Targeting Therapies: Back to the Future.

Hepatocellular carcinoma (HCC) is one of the leading causes of death from cancer in the world. Recently, the effectiveness of new antiviral therapies and the HBV vaccine have reduced HCC's incidence, while non-alcoholic steato-hepatitis is an emerging risk factor. This review focuses on antiangiogenic molecules and immune checkpoint inhibitors approved for HCC treatment and possible future approaches. Sorafenib was the first drug approved for the treatment of advanced HCC (aHCC) and it has been shown to increase survival by a few months. Lenvatinib, a multikinase inhibitor, has shown non-inferiority in survival compared with sorafenib and an improvement in progression-free survival (PFS). The combination of atezolizumab (an anti-PDL1 antibody) and bevacizumab (an anti-VEGF antibody) was the first drug combination approved for HCC, demonstrating improved survival compared with sorafenib (19.2 vs. 13.4 months). As a second line of therapy, three regimens (regorafenib, cabozantinib, and ramucirumab) have been approved for the treatment of aHCC after progression on sorafenib according to guidelines. Furthermore, nivolumab, pembrolizumab, and nivolumab plus ipilimumab have been approved by the FDA (2017, 2018, and 2020, respectively). Finally, immune target therapy, cancer vaccines, and epigenetic drugs represent three new possible weapons for the treatment of HCC.

An in vitro study to assess the effect of hyaluronan-based gels on muscle-derived cells: Highlighting a new perspective in regenerative medicine.

Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.

Pharmacological characterization of the interaction between tiotropium bromide and olodaterol on human bronchi and small airways.

Combining a long-acting ß2-agonist (LABA) with a long-acting muscarinic antagonist (LAMA) is the cornerstone to treat patients with chronic obstructive pulmonary disease (COPD). In this study we have characterized the interaction between the LAMA tiotropium bromide, and the LABA olodaterol, on the contractile tone of human medium bronchi and small airways. The response to a combination of tiotropium bromide and olodaterol was assessed at sub-maximal contractile tone induced by carbachol. The duration of action was studied in tissue contracted by transmural stimulation. Relaxation of bronchial tone was expressed as % of maximal response to papaverine. Drug interactions were analyzed by the Bliss Independence method and Unified Theory. Tiotropium bromide/olodaterol combination induced a significant synergistic relaxant response (P < 0.05 vs. expected additive effect) in medium bronchi and small airways pre-contracted by carbachol, by enhancing relaxation +22.13 ± 4.42% and +26.31 ± 12.39%, respectively. The combination of tiotropium bromide and olodaterol also reduced the airway smooth muscle contractility elicited by transmural stimulation by 73.60 ± 3.10%. The extent of synergy was strong to very strong, and was supported by the release of neuronal acetylcholine, cyclic adenosine monophosphate levels, and activation of iberiotoxin-sensitive KCa++ channels. Conversely, the interaction between tiotropium bromide and olodaterl was independent of the activity at M2 muscarinic receptors. These results indicate that tiotropium bromide/olodaterol combination leads to a potent and durable synergistic relaxation of human medium bronchi and small airways. Further pharmacological studies are needed to confirm these results in clinical settings.

AT1-receptor blockade: Protective effects of irbesartan in cardiomyocytes under hypoxic stress.

Hypoxia induces myocardial injury through the activation of inflammatory and oxidative processes. The pivotal role of the renin angiotensin system (RAS) in the pathogenesis of cardiovascular diseases has been firmly established in clinical trials and practice; in fact many experimental and clinical data have highlighted that its inhibition has a cardioprotective role. Activated RAS also stimulates inflammation directly inducing proinflammatory and oxidative gene expression. This study aimed to investigate the protective role of a pre-treatment (10 and 100 µM) with irbesartan on injury induced by 24 h of hypoxia in HL-1 cardiomyocytes; in particular, we have analyzed the natriuretic peptide (BNP) expression, a biomarker able to modulate inflammatory reaction to cardiac injury and some markers involved in oxidative stress and inflammation. Our results demonstrated that a pre-treatment with 100 µM irbesartan significantly increased SOD activity and catalase expression of 15 and 25%, respectively, compared to hypoxic cells (P<0.05). On the other hand, it was able to reduce the release of peroxynitrite and iNOS protein expression of 20 and 50% respectively (P<0.05). In addition irbesartan exerts an anti-inflammatory activity reducing Toll-like receptors (TLRs)-2 and -4 mRNA expression, TNF-alpha expression and activity (20%) and increasing the expression of the cytokine IL-17 (40%) (P<0.05 vs hypoxia). Our findings also showed that BNP induced by ischemia was significantly and in a concentration-dependent manner reduced by irbesartan. The findings of our study demonstrated that the AT1 receptor antagonist irbesartan exerts a protective role in an in vitro hypoxic condition reducing oxidative stress and inflammation.

Role of statins and mevalonate pathway on impaired HDAC2 activity induced by oxidative stress in human airway epithelial cells.

In patients with chronic obstructive pulmonary disease (COPD) the inflammatory response is often steroid-resistant, likely since oxidative stress and cigarette smoking impair histone deacetylase 2 (HDAC2) activity. Since it has been demonstrated that statins may restore the HDAC2 activity in cultured human endothelial cells, the aim of this study was to investigate the effects of statins in reversing the steroid-resistance induced by oxidative stress. We evaluated the effects of simvastatin and dexamethasone on HDAC2 expression and activity, and the role of mevalonate and Rho/ROCK pathways in A549 cells, a human lung type II epithelial cell line stressed with H2O2. Our results documented that H2O2 significantly reduced the HDAC2 expression and activity. In H2O2 treated cells dexamethasone was unable to restore the activity of HDAC2, whereas simvastatin restored both the expression and the activity of this enzyme. Our data also showed that mevalonate reduced the activity of HDAC2 whereas Y27632, a Rho/ROCK inhibitor, had no effect on HDAC2 activity when co-administered with simvastatin. Our data suggest that statins could have the potential to restore corticosteroid sensitivity in A549 cells. The evidences of this study suggest that, although both mevalonate and Rho/ROCK pathways are involved in the detrimental effect elicited by oxidative stress, statins may restore the function and expression of depleted HDAC2 via modulating the mevalonate cascade, at least in A549 cells. In conclusion, the modulation of histone acetyltransferase/deacetylase activity may lead to the development of novel anti-inflammatory approaches to inflammatory lung diseases that are currently difficult to treat.

N-Acetylcysteine protects human bronchi by modulating the release of neurokinin A in an ex vivo model of COPD exacerbation.

AIMS: N-Acetylcysteine (NAC) reduces the risk of exacerbation of chronic obstructive pulmonary disease (COPD). Although NAC also has anti-inflammatory activity, the detailed mechanism leading to its protective role remains to be elucidated. We tested the impact of NAC against the effects of lipopolysaccharide (LPS) in an ex vivo model of COPD exacerbation, and investigated the role of neurokinin A (NKA) in this context. MAIN METHODS: Isolated airways from COPD patients were incubated overnight with LPS (100 ng/ml). NAC was tested at concentrations resembling the plasma levels elicited by oral administration of NAC at 200 mg/day (very low dose), 600 mg/day (low dose) and 1.200 mg/day (high dose). KEY FINDINGS: NAC at high concentrations normalized the peroxidase activity, H2O2, malondialdehyde (MDA), nitric oxide, glutathione (GSH), total antioxidant capacity (TAC), and interleukin 6 (IL-6) (overall change 34.32% ±â€¯4.22%, P < 0.05 vs. LPS-treated). NAC at low concentrations modulated peroxidase activity, H2O2, MDA, GSH, TAC, and IL-6 (overall change 34.88% ±â€¯7.39%, P < 0.05 vs. LPS-treated). NAC at very-low concentrations was effective on peroxidase activity, H2O2, GSH, and IL-6 (overall change 35.05 ±â€¯7.71%, P < 0.05 vs. LPS-treated). Binary logistic regression analysis indicated that the modulatory effect of NAC on NKA levels was associated with a reduction of pro-oxidant factors and IL-6, and selectively blocking the NK2 receptor abolished such an association. SIGNIFICANCE: This study demonstrates that, along with its well-known antioxidant activity, the protective effect of NAC against the detrimental effect of LPS is due to the modulation of NKA and IL-6 levels.

Pharmacogenetic and pharmacogenomic considerations of asthma treatment.

INTRODUCTION: Pharmacogenetic and pharmacogenomic approaches are already utilized in some areas, such as oncology and cardiovascular disease, for selecting appropriate patients and/or establishing treatment and dosing guidelines. This is not true in asthma although many patients have different responses to drug treatment due to genetic factors. Areas covered: Several genetic factors that affect the pharmacotherapeutic responses to asthma medications, such as ß2-AR agonists, corticosteroids, and leukotriene modifiers and could contribute to significant between-person variability in response are described. Expert opinion: An expanding number of genetic loci have been associated with therapeutic responses to asthma drugs but the individual effect of one single-nucleotide polymorphism is partial. In fact, epigenetic changes can modify genetic effects in time-, environment-, and tissue-specific manners, genes interact together in networks, and nongenetic components such as environmental exposures, gender, nutrients, and lifestyle can significantly interact with genetics to determine the response to therapy. Therefore, well-designed randomized controlled trials or observational studies are now mandatory to define if response to asthma medications in individual patients can be improved by using pharmacogenetic predictors of treatment response. Meanwhile, routine implementation of pharmacogenetics and pharmacogenomics into clinical practice remains a futuristic, far-off challenge for many clinical practices.

Inhibition of p110δ PI3K prevents inflammatory response and restenosis after artery injury.

Inflammatory cells play key roles in restenosis upon vascular surgical procedures such as bypass grafts, angioplasty and stent deployment but the molecular mechanisms by which these cells affect restenosis remain unclear. The p110δ isoform of phosphoinositide 3-kinase (PI3K) is mainly expressed in white blood cells. Here, we have investigated whether p110δ PI3K is involved in the pathogenesis of restenosis in a mouse model of carotid injury, which mimics the damage following arterial grafts. We used mice in which p110δ kinase activity has been disabled by a knockin (KI) point mutation in its ATP-binding site (p110δD910A/D910A PI3K mice). Wild-type (WT) and p110δD910A/D910A mice were subjected to longitudinal carotid injury. At 14 and 30 days after carotid injury, mice with inactive p110δ showed strongly decreased infiltration of inflammatory cells (including T lymphocytes and macrophages) and vascular smooth muscle cells (VSMCs), compared with WT mice. Likewise, PI-3065, a p110δ-selective PI3K inhibitor, almost completely prevented restenosis after artery injury. Our data showed that p110δ PI3K plays a main role in promoting neointimal thickening and inflammatory processes during vascular stenosis, with its inhibition providing significant reduction in restenosis following carotid injury. p110δ-selective inhibitors, recently approved for the treatment of human B-cell malignancies, therefore, present a new therapeutic opportunity to prevent the restenosis upon artery injury.

Management of Chronic Obstructive Pulmonary Disease in Patients with Cardiovascular Diseases.

Chronic obstructive pulmonary disease (COPD) and cardiovascular diseases often coexist. The mechanistic links between these two diseases are complex, multifactorial and not entirely understood, but they can influence the therapeutic approach. Therapy can be primarily directed towards treating the respiratory symptoms and reducing lung inflammation. Smoking cessation, bronchodilators and inhaled corticosteroids are central to this therapeutic approach. The underlying pathophysiological mechanisms that are responsible for the increased cardiovascular risk in COPD remain unclear, but might include arterial stiffness, inflammation and endothelial dysfunction as a consequence of systemic exposure to chemicals in cigarette smoke or airborne pollution. Therefore, it is plausible that treatment of cardiovascular co-morbidities might reduce morbidity and mortality in patients with COPD and, consequently, therapy of COPD should be shifted to the treatment of cardiovascular diseases and systemic inflammation. In support of this approach, early data suggest that patients with COPD treated with angiotensin-converting enzyme inhibitors, angiotensin II type 1 receptor blockers, statins, anti-platelet drugs or ß-adrenoceptor blockers may have improved survival and reduced hospitalisation from acute exacerbations of COPD. In this review, the potential impact of traditional therapies for COPD that are centred on treating the lungs and newer strategies potentially able to affect and mitigate cardiovascular risks in patients with COPD are discussed.

Long-term administration of ranolazine attenuates diastolic dysfunction and adverse myocardial remodeling in a model of heart failure with preserved ejection fraction.

BACKGROUND: To investigate the effects of chronic administration of ranolazine (RAN) on experimental model of heart failure with preserved ejection fraction. METHODS: Seven-weeks old Dahl salt-sensitive rats were fed a high salt diet for 5weeks to induce hypertension. Afterwards, rats continued with a high salt diet and were administered either with vehicle or RAN (20mg/kg/die, ip) for the following 8weeks. Control rats were maintained on a low salt diet. RESULTS: While systolic parameters were not altered, diastolic parameters were changed in high salt animals. Hemodynamic analysis showed a decreased dP/dt min, increased LVEDP, longer time constant and steeper slope of the end-diastolic pressure-volume relationship. Treatment with RAN attenuated these alterations and determined a reduction in mortality. Additionally, the magnitude of myocardial hypertrophy and activation of PI3K/Akt pathway were reduced. Alteration in diastolic compliance as a consequence of elevated myocardial stiffness was confirmed by an increase of collagen deposition and activation of pro-fibrotic TGF-ß/SMAD3/CTGF signaling. These effects were counteracted by RAN. High salt rats had a decrease in SERCA2 and an increase in Na(+)/Ca(2+) exchanger (NCX). Treatment with RAN reduced NCX expression and determined an increment of SERCA2. Moreover, the levels of nitrotyrosine and oxidized dyhydroethidium were higher in high salt rats. RAN induced a decrement of oxidative stress, supporting the concept that reduction in ROS may mediate beneficial effects. CONCLUSIONS: Our findings support the possibility that diastolic dysfunction can be attenuated by RAN, indicating its ability to affect active relaxation and passive diastolic compliance.

G-CSF contributes at the healing of tunica media of arteriotomy-injured rat carotids by promoting differentiation of vascular smooth muscle cells.

Restenosis is a complex pathophysiological disease whose causative mechanisms are not fully understood. Previous studies allowed us to demonstrate the efficacy of bone marrow mesenchymal stromal cells (MSCs) transplantation in limiting the pathophysiological remodeling in a model of arteriotomy-induced (re) stenosis. In the current research we studied the effectiveness of G-CSF treatment on male rate rats that were subjected carotid arteriotomy in order to evaluate a potentially effective non-invasive strategy that recapitulates the MSC-mediated recovery of injured vessels. WKY male rats were subjected carotid arteriotomy and given a nine day treatment (3 days pre- to 6 days post-arteriotomy) with G-CSF or saline. Carotids were harvested 7 and 30 days following arteriotomy (early- and late-phase, respectively). Although morphometrical analysis did not reveal differences in lumen narrowing between G-CSF- and PBS-carotids 30 days following arteriotomy, we detected a noticeable conservative effect of G-CSF treatment on vascular wall morphology. Histological and molecular analysis revealed an increase in cellularity within the tunica media with a concomitant increase of the VSMCs differentiation markers both at early- and late-phases of (re) stenotic response in G-CSF-treated carotids (Sm22-alpha, Myocd, and Smtn). These findings were accompanied by the downregulation of oxidative stress-related genes in G-CSF-injured rats. The effect exerted by G-CSF in our model of arteriotomy-induced (re) stenosis seemed support the recovery of the architecture of the tunica media of injured vessels by: (i) inducing VSMCs differentiation; and (ii) limiting the oxidative-stress response induced by arteriotomy.

AXL is an oncotarget in human colorectal cancer.

AXL is a tyrosine kinase receptor activated by GAS6 and regulates cancer cell proliferation migration and angiogenesis. We studied AXL as new therapeutic target in colorectal cancer (CRC). Expression and activation of AXL and GAS6 were evaluated in a panel of human CRC cell lines. AXL gene silencing or pharmacologic inhibition with foretinib suppressed proliferation, migration and survival in CRC cells. In an orthotopic colon model of human HCT116 CRC cells overexpressing AXL, foretinib treatment caused significant inhibition of tumour growth and peritoneal metastatic spreading. AXL and GAS6 overexpression by immunohistochemistry (IHC) were found in 76,7% and 73.5%, respectively, of 223 human CRC specimens, correlating with less differentiated histological grading. GAS6 overexpression was associated with nodes involvement and tumour stage. AXL gene was found amplified by Fluorescence in situ hybridization (FISH) in 8/146 cases (5,4%) of CRC samples. Taken together, AXL inhibition could represent a novel therapeutic approach in CRC.

Contribution of sensory nerves to LPS-induced hyperresponsiveness of human isolated bronchi.

AIMS: Bacterial lipopolysaccharide (LPS) can induce bronchial hyperresponsiveness (BHR), but the underlying mechanisms remain to be determined. Here, the possible contribution of sensory nerves to LPS-induced BHR was examined in human isolated bronchi to pharmacologically identify the mechanisms underlying this phenomenon. MAIN METHODS: Human isolated bronchial tone was induced by electrical field stimulation (EFS). The responses of airways to LPS, with or without capsaicin desensitization or thiorphan treatment were studied and the transient receptor potential vanilloid type 1 (TRPV1) expression was assessed. We performed similar experiments in the presence of a TRPV1 or a neurokinin (NK) 2 receptor antagonist using SB366791 and GR159897, respectively. KEY FINDINGS: LPS increased (≃2.3-fold, P<0.001) the contraction induced by EFS, compared to control tissues. Acute administration of capsaicin enhanced (≃2.3-fold, P<0.001) the EFS-mediated contraction, but did not potentiate the effect of LPS. Thiorphan increased (≃1.3-fold, P<0.05) the contractile response of LPS treated tissues and, at lower frequencies, it enhanced (≃1.7-fold, P<0.001) the capsaicin-induced contraction. In capsaicin-desensitized bronchi, LPS did not modify (P>0.05) the EFS contractile response, nor after treatment with thiorphan. Capsaicin desensitization reduced (≃0.4-fold, P<0.001) the LPS-induced BHR. SB366791 and GR159897 prevented the LPS-induced BHR and the release of NKA. LPS increased (+85.3±9.5%, P<0.01) the surface membrane expression of TRPV1 in parasympathetic ganglia. SIGNIFICANCE: Our results demonstrate the involvement of capsaicin-sensitive sensory nerves and neutral endopeptidases in LPS-induced BHR of the human bronchi, associated with an upregulation of TRPV1 and release of NKA.

Primary and Acquired Resistance of Colorectal Cancer to Anti-EGFR Monoclonal Antibody Can Be Overcome by Combined Treatment of Regorafenib with Cetuximab.

PURPOSE: In colorectal cancer, the activation of the intracellular RAS-RAF and PIK3CA-AKT pathways has been implicated in the resistance to anti-EGFR mAbs. We have investigated the role of regorafenib, an oral multikinase inhibitor, in combination with cetuximab, an anti-EGFR mAb, to overcome anti-EGFR resistance. EXPERIMENTAL DESIGN: We have tested, in vitro and in vivo, the effects of regorafenib in a panel of human colorectal cancer cell lines with a KRAS mutation (SW480, SW620, HCT116, LOVO, and HCT15) or with a BRAF mutation (HT29), as models of intrinsic resistance to cetuximab treatment, and in two human colorectal cancer cell lines (GEO and SW48) that are cetuximab-sensitive, as well as in their derived cells with acquired resistance to cetuximab (GEO-CR and SW48-CR). RESULTS: Treatment with regorafenib determined a dose-dependent growth inhibition in all colorectal cancer cell lines. The combined treatment with cetuximab and regorafenib induced synergistic antiproliferative and apoptotic effects in cetuximab-resistant cell lines by blocking MAPK and AKT pathways. Nude mice were injected s.c. with HCT116, HCT15, GEO-CR, and SW48-CR cells. The combined treatment caused significant tumor growth inhibition. Synergistic antitumor activity of regorafenib plus cetuximab was also observed in an orthotopic colorectal cancer model of HCT116 cells. In particular, the combined treatment induced a significant tumor growth inhibition in the primary tumor site (cecum) and completely prevented metastasis formation. CONCLUSIONS: The combined treatment with cetuximab and regorafenib could be a strategy to overcome resistance to anti-EGFR therapies in metastatic colorectal cancer patients.

Effects of sildenafil on the gastrocnemius and cardiac muscles of rats in a model of prolonged moderate exercise training.

Moderate exercise training improves energetic metabolism, tissue perfusion and induces cardiac and skeletal muscle remodeling. Sildenafil, a potent phosphodiesterase-5 inhibitor used to treat erectile dysfunction, reduces infarct size and increases tissue oxygenation in experimental models of cardiovascular disease. We have evaluated the effects of prolonged moderate exercise training and a repeat administration of sildenafil on the rat gastrocnemius and cardiac muscles. Animals were divided into two groups: sedentary and trained. Each group was subdivided into animals treated with vehicle or with two doses of sildenafil (10 or 15 mg/kg/day) during the last week of training. Physical exercise did not induce cardiac hypertrophy, whereas it increased mRNA levels of the PGC-1α, HIF-1α and VEGF genes, which are involved in mitochondrial biogenesis and angiogenesis, and reduced mRNA levels of FoxO3a, MuRF-1 and Atrogin-1. Sildenafil dose-dependently promoted both angiogenesis, as shown by increased capillary density, and muscle atrophy, as shown by muscle fibre size. These effects were more pronounced in trained animals. Our data confirm the beneficial effects of a moderate and prolonged training on cardiovascular and skeletal systems and document the positive and negative effects of sildenafil on these tissues at doses higher than those used in clinical practice. This report may impact on the use of sildenafil as a substance able to influence sports performance.

Stem cell therapy for arterial restenosis: potential parameters contributing to the success of bone marrow-derived mesenchymal stromal cells.

PURPOSE: Restenosis is a complex and heterogeneous pathophysiological phenomenon occurring in patients submitted to revascularization procedures. Previous studies proved the antirestenotic properties of injected allogenic mesenchymal stromal cells (MSCs) in an experimental model of rat carotid (re)stenosis induced through arteriotomy. In this study we describe some of the effects subsequent to MSC treatment of rats submitted to carotid arteriotomy and possibly responsible for their antirestenotic effect. METHODS: Rat MSCs were isolated from bone marrow, expanded in vitro and characterized. Subsequently, we evaluated the effects of MSC administration via tail vein at 3 and 7 days after carotid arteriotomy both in rat serum and in injured carotids, focusing on DNA oxidative damage (8-oxo-dG detection), cell proliferation index (BrdU incorporation assay), apoptotic index (TUNEL assay), the expression of inflammation- and proliferation-related genes (RT-PCR), the release of growth factors and of inflammation-related cytokines (antibody arrays and ELISA). RESULTS: MSC administration induced a greater cell proliferation in carotids after arteriotomy, together with an increased level of VEGF in the serum and with the higher expression of VEGF mRNA in injured carotids. Serum analysis also revealed a decreased level of the pro-inflammatory cytokines CXCL1, CXCL5, L-Selectin, ICAM-1 and LIX, and of TIMP1 and SDF-1alpha in MSC-treated rats. The MSC immunomodulatory activity was confirmed by the decreased expression of TLR2 and TLR4 in injured carotids. CONCLUSIONS: MSCs play an immunomodulatory paracrine role when injected in rats submitted to carotid arteriotomy, accompanied by the release of VEGF, possibly contributing to the accelerated repair of the injured vascular wall.