Impaired activation of Notch-1 signaling hinders repair processes of bronchial epithelial cells exposed to cigarette smoke.

Notch-1 intervenes in the reparative processes of mucosa by controlling cell proliferation, differentiation and stem cell maintenance. Cigarette smoke alters airway epithelial homeostasis. The present study explored whether: Smokers showed altered Notch-1 expression; and whether in bronchial epithelial cells (16HBE): a) cigarette smoke extracts (CSE) altered the expression of Notch-1, of its ligand Jagged-1 (Jag-1) and the nuclear translocation of Notch-1; b) Notch-1 signaling activation as well as CSE modified Ki67, PCNA, p21, IL-33 expression, cell proliferation and repair processes. Notch-1 expression was assessed in the epithelium from large airway surgical samples from non-smoker and smoker subjects by immunohistochemistry.16HBE were cultured with/without CSE and Jag-1. A Notch-1 inhibitor (DAPT) was used as control. The expression of Notch-1, Jag-1, Ki67, PCNA, p21, IL-33 and cell proliferation (by CFSE) were all assessed by flow cytometry. Notch-1 nuclear expression was evaluated by immunofluorescence and western blot analysis. Repair processes were assessed by wound assay. Smokers had cytoplasmic but not nuclear Notch-1 expression. Although CSE increased Notch-1 expression, it counteracted Notch-1 signaling activation since it reduced Jag-1 expression and Notch-1 nuclear translocation. Notch-1 signaling activation by Jag-1 increased Ki67, PCNA and repair processes but reduced intracellular IL-33 and p21 expression without affecting cell proliferation. DAPT counteracted the effects of Notch-1 activation on PCNA and IL-33. CSE increased Ki67, PCNA, p21 and IL-33 expression but reduced cell proliferation and repair processes. In conclusion, cigarette smoke exposure, limiting Notch-1 signaling activation and hindering repair processes, amplifies injury processes in bronchial epithelial cells.

Cigarette smoke extract modulates E-Cadherin, Claudin-1 and miR-21 and promotes cancer invasiveness in human colorectal adenocarcinoma cells.

BACKGROUND: Cigarette smoke is considered a risk factor for lung and colorectal cancer. A convincing link between epithelial-to-mesenchymal transition (EMT) with colorectal cancer progression and therapeutic resistance has emerged. Deregulated expression of E-Cadherin and Claudin-1 and increased miR-21 expression and invasiveness represent hallmarks of EMT. The effects of cigarette smoke exposure on EMT in colorectal adenocarcinoma cells are largely unknown. AIM: The aim of the study is to evaluate the effect of cigarette smoke extract (CSE) on miR-21, Claudin-1 and E-Cadherin, molecules associated to EMT in colorectal cancer cells. METHODS: A human colorectal adenocarcinoma cell line (Caco-2) was treated with CSE at different concentration (5% and 10%) and for different time points (3 h and 24 h). Metabolic activity (by MTS assay), cell necrosis/cell apoptosis (evaluating Propidium Iodide/Annexin V expression by flow cytometry), miR-21, Claudin-1 and E-Cadherin gene expression were evaluated by Real time PCR. Cell permeability, actin polymerization and cancer cell migration was assessed by Trans-Epitelial Electrical Resistance (TEER), Phalloidin expression and matrigel system, respectively. RESULTS: CSE at all the tested concentrations and at all time points reduced cell necrosis. CSE at 10% increased miR-21 and reduced the metabolic activity, cell necrosis, Claudin-1 and E-cadherin mRNA at 3 h. Cell permeability, actin polymerization and cancer cell migration were all increased upon CSE exposure. CONCLUSION: These results showed that CSE increasing miR-21, Claudin-1 and E-Cadherin and enhancing the aggressiveness of cancer cells, may concur to colorectal cancer progression.

Budesonide, Aclidinium and Formoterol in combination limit inflammaging processes in bronchial epithelial cells exposed to cigarette smoke.

Inflammation and cellular senescence (also called inflammaging) are involved in the pathogenesis of premature lung aging, a key driver of chronic obstructive pulmonary disease (COPD). Downregulation of histone deacetylases and FoxO3 expression, activation of the ERK 1/2 pathway and IL-8 increase are hallmarks of lung inflammaging. The effects of Budesonide (BUD), Aclidinium (ACL) and Formoterol (FO) on lung inflammaging are unknown. This study was aimed to assess the effects of BUD, ACL and FO in bronchial epithelial cells exposed to cigarette smoke extract (CSE) by evaluating: a) Expression of TLR4 and survivin and LPS binding by flow cytometry; b) expression of HDAC2, HDAC3, SIRT1 and FoxO3 and activation of the ERK 1/2 pathway by western blot; c) IL-8 mRNA levels and release by Real Time-PCR and ELISA, respectively. Reported results show that CSE increased TLR4 and survivin, LPS binding, ERK 1/2 activation, IL-8 release and mRNA levels but decreased SIRT1, HDAC2, HDAC3 and FoxO3 nuclear expression. Combined therapy with BUD, ACL and FO counteracted the effects of CSE on LPS binding, FoxO3 nuclear expression, ERK 1/2 activation, survivin and IL-8 release and mRNA levels. These findings suggest a new role of combination therapy with BUD, ACL and FO in counteracting inflammaging processes induced by cigarette smoke exposure.

Notch-1 signaling activation sustains overexpression of interleukin 33 in the epithelium of nasal polyps.

BACKGROUND: Alterations in the nasal epithelial barrier homeostasis and increased interleukin 33 (IL-33) expression contribute to the pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP). AIMS: As Notch-1 signaling is crucial in repair processes of mucosa, the current study assessed Notch-1/Jagged-1 signaling and IL-33 in the epithelium of nasal polyps biopsies from allergic (A-CRSwNP; n = 9) and not allergic (NA-CRSwNP; n = 9) subjects by immunohistochemistry. We also assessed, in a model of nasal epithelial cells, the effects of stimulation of Notch-1 with Jagged-1 on the expression of IL-33 (by flow cytometry, immunofluorescence, and immunocytochemistry), Jagged-1 (by flow cytometry), and p-CREB transcription factor (by western blot analysis). RESULTS: Ex vivo (a) in normal epithelium, the expression of Notch-1 and IL-33 were higher in NA-CRSwNP than in A-CRSwNP; (b) in metaplastic epithelium, the expression of Notch-1, Jagged-1, and IL-33 were higher in NA-CRSwNP than in A-CRSwNP; (c) in hyperplastic epithelium, the expression of Notch-1, Jagged-1, and IL-33 were higher in A-CRSwNP than in NA-CRSwNP; and (d) in basal epithelial cells, no differences were observed in the expression of Jagged-1, IL-33, and Notch-1. The expression of Notch-1 significantly correlated with the expression of IL-33. In vitro, stimulation of Notch-1 with Jagged-1 induced the expression of (a) Jagged-1; (b) IL-33; and (c) p-CREB transcription factor. The inhibitor of Notch-1, DAPT, reduced all the effects of Jagged-1 on nasal epithelial cells. CONCLUSIONS: The data herein provided support, for the first time, a putative role of Notch-1/Jagged-1 signaling in the overexpression of IL-33 in the epithelium of nasal polyps from patients with CRSwNP.

Ceftaroline modulates the innate immune and host defense responses of immunocompetent cells exposed to cigarette smoke.

BACKGROUND: Cigarette smoke, the principal risk factor for chronic obstructive pulmonary disease (COPD), negatively influences the effectiveness of the immune system's response to a pathogen. The antibiotic ceftaroline exerts immune-modulatory effects in bronchial epithelial cells exposed to cigarette smoke. AIMS AND METHODS: The present study aims to assess the effects of ceftaroline on TLR2 and TLR4 expression, LPS binding and TNF-α and human beta defensin (HBD2) release in an undifferentiated and PMA-differentiated human monocyte cell line (THP-1) exposed or not to cigarette smoke extracts (CSE). TLR2, TLR4, and LPS binding were assessed by flow cytometry, TNF-α and HBD2 release were evaluated by ELISA. RESULTS: The constitutive expression of TLR2 and TLR4 and LPS binding were higher in differentiated compared to undifferentiated THP-1 cells. In undifferentiated THP-1 cells, CSE increased TLR2 and TLR4 protein levels, LPS binding and TNF-α release and reduced HBD2 release and ceftaroline counteracted all these effects. In differentiated THP-1, CSE did not significantly affect TLR2 and TLR4 expression and LPS binding but reduced HBD2 release and increased TNF-α release. Ceftaroline counteracted the effects of CSE on HBD2 release in differentiated THP-1. CONCLUSION: Ceftaroline counteracts the effect of CSE in immune cells by increasing the effectiveness of the innate immune system. This effect may also assist in reducing pathogen activity and recurrent exacerbations in COPD patients.

Formoterol and fluticasone propionate combination improves histone deacetylation and anti-inflammatory activities in bronchial epithelial cells exposed to cigarette smoke.

BACKGROUND: The addition of long-acting beta2-agonists (LABAs) to corticosteroids improves asthma control. Cigarette smoke exposure, increasing oxidative stress, may negatively affect corticosteroid responses. The anti-inflammatory effects of formoterol (FO) and fluticasone propionate (FP) in human bronchial epithelial cells exposed to cigarette smoke extracts (CSE) are unknown. AIMS: This study explored whether FP, alone and in combination with FO, in human bronchial epithelial cellline (16-HBE) and primary bronchial epithelial cells (NHBE), counteracted some CSE-mediated effects and in particular some of the molecular mechanisms of corticosteroid resistance. METHODS: 16-HBE and NHBE were stimulated with CSE, FP and FO alone or combined. HDAC3 and HDAC2 activity, nuclear translocation of GR and NF-κB, pERK1/2/tERK1/2 ratio, IL-8, TNF-α, IL-1ß mRNA expression, and mitochondrial ROS were evaluated. Actin reorganization in neutrophils was assessed by fluorescence microscopy using the phalloidin method. RESULTS: In 16-HBE, CSE decreased expression/activity of HDAC3, activity of HDAC2, nuclear translocation of GR and increased nuclear NF-κB expression, pERK 1/2/tERK1/2 ratio, and mRNA expression of inflammatory cytokines. In NHBE, CSE increased mRNA expression of inflammatory cytokines and supernatants from CSE exposed NHBE increased actin reorganization in neutrophils. FP combined with FO reverted all these phenomena in CSE stimulated 16-HBE cells as well as in NHBE cells. CONCLUSIONS: The present study provides compelling evidences that FP combined with FO may contribute to revert some processes related to steroid resistance induced by oxidative stress due to cigarette smoke exposure increasing the anti-inflammatory effects of FP.

Effects of ceftaroline on the innate immune and on the inflammatory responses of bronchial epithelial cells exposed to cigarette smoke.

The tobacco smoking habit interferes with the innate host defence system against infections. Recurrent infections accelerated the functional respiratory decline. The present study assessed the effects of ceftaroline on TLR2 and TLR4 and on pro-inflammatory responses in airway epithelial cells (16HBE cell line and primary bronchial epithelial cells) with or without cigarette smoke extracts (CSE 10%). TLR2, TLR4, LPS binding and human beta defensin 2 (HBD2) were assessed by flow cytometry, NFkB nuclear translocation by western blot analysis, IL-8 and HBD2 mRNA by Real Time PCR; the localization of NFkB on the HBD2 and IL-8 promoters by ChiP Assay. CSE increased TLR4, TLR2 expression, LPS binding and IL-8 mRNA; CSE decreased HBD2 (protein and mRNA), activated NFkB and promoted the localization of NFkB on IL-8 promoter and not on HBD2 promoter. Ceftaroline counteracted the CSE effect on TLR2 expression, on LPS binding, on IL-8 mRNA, HBD2 and NFkB in 16HBE. The effects of ceftaroline on HBD2 protein and on IL-8 mRNA were confirmed in primary bronchial epithelial cells. In conclusion, ceftaroline is able to counteract the effects of CSE on the innate immunity and pro-inflammatory responses modulating TLR2, LPS binding, NFkB activation and activity, HBD2 and IL-8 expression in bronchial epithelial cells.

Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells.

BACKGROUND: Cigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. AIMS: Aim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. METHODS: 16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. RESULTS: CSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. CONCLUSIONS: The study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure.

Surgical buccinator muscle myotomy in dentoskeletal Class II alterations.

Recent studies on autoptic findings have underlined the complexity of perioral muscles; the discovery of a fourth band of buccinator muscle and the effects it can have on teeth, alveolar bone, and oral mucosa during growth has stressed the importance of muscular influence on the genesis of and therapy for basal class II dentofacial alterations. Frederick proposed a surgical marginal myotomy to elongate the buccinator bundle together with inferior vestibuloplasty to unload the jaw from the overcontraction of the muscle and to reduce the inferior lip hypotone. In our investigation, we have applied this technique on a group of 50 patients with basal class II defect selected according to our protocol before functional or fixed orthodontics, and we have recorded and compared cephalometric measurements and clinical facial profile changes 12 (T1) and 24 (T2) months after the end of therapy to a control group with the same defect treated only with orthodontic functional devices. We obtained improvement of cephalometric parameters in 80% of patients who underwent surgery and orthodontics, and in only 50% of patients who underwent only orthodontics. Results suggest that neutralizing buccinator and mental muscles pressure on the jaw during growth can help the clinician dealing with II basal malocclusion therapy and relapse.

Transforming growth factor-beta1 down-regulation of major histocompatibility complex class I in thyrocytes: coordinate regulation of two separate elements by thyroid-specific as well as ubiquitous transcription factors.

Transforming growth factor (TGF)-beta1-decreased major histocompatibility complex (MHC) class I gene expression in thyrocytes is transcriptional; it involves trans factors and cis elements important for hormone- as well as iodide-regulated thyroid growth and function. Thus, in rat FRTL-5 thyrocytes, TGF-beta1 regulates two elements within -203 bp of the transcription start site of the MHC class I 5'-flanking region: Enhancer A, -180 to -170 bp, and a downstream regulatory element (DRE), -127 to -90 bp, that contains a cAMP response element (CRE)-like sequence. TGF-beta1 reduces the interaction of a NF-kappaB p50/fra-2 heterodimer (MOD-1) with Enhancer A while increasing its interaction with a NF-kappaB p50/p65 heterodimer. Both reduced MOD-1 and increased p50/p65 suppresses class I expression. Decreased MOD-1 and increased p50/p65 have been separately associated with the ability of autoregulatory (high) concentrations of iodide to suppress thyrocyte growth and function, as well as MHC class I expression. TGF-beta1 has two effects on the downstream regulatory element (DRE). It increases DRE binding of a ubiquitously expressed Y-box protein, termed TSEP-1 (TSHR suppressor element binding protein-1) in rat thyroid cells; TSEP-1 has been shown separately to be an important suppressor of the TSH receptor (TSHR) in addition to MHC class I and class II expression. It also decreases the binding of a thyroid-specific trans factor, thyroid transcription factor-1 (TTF-1), to the DRE, reflecting the ability of TGF-beta1 to decrease TTF-1 RNA levels. TGF-beta1-decreased TTF-1 expression accounts in part for TGF-beta1-decreased thyroid growth and function, since decreased TTF-1 has been shown to decrease thyroglobulin, thyroperoxidase, sodium iodide symporter, and TSHR gene expression, coincident with decreased MHC class I. Finally, we show that TGF-beta1 increases c-jun RNA levels and induces the formation of new complexes involving c-jun, fra-2, ATF-1, and c-fos, which react with Enhancer A and the DRE. TGF-beta1 effects on c-jun may be a pivotal fulcrum in the hitherto unrecognized coordinate regulation of Enhancer A and the DRE.

Thymosin-alpha1 regulates MHC class I expression in FRTL-5 cells at transcriptional level.

In this study we examined the effect of the synthetic peptide thymosin-alpha1 (T(alpha)1) on MHC class I expression in FRTL-5 cells. Treatment with T(alpha)1 increased expression of MHC class I surface molecules and mRNA, which reached its peak (153 +/- 8 % of the control value) after 12 h. Chloramphenicol acetyltransferase (CAT) analysis, following transfection with a plasmid containing the regulatory sequence of MHC class I (or its deletion derivatives) with the CAT reporter gene, and electrophoretic mobility shift assay experiments demonstrated that the action of T(alpha)1 was at the transcriptional level, and its mechanism of action is likely due to increased binding between the complex p50/fra-2 and the enhancer A sequence of the 5' flanking region of a swine class I gene (PD1). An increase in the expression of MHC class I surface molecules was also observed by flow cytometry in murine and human tumor cell lines and in primary cultures of human macrophages. This study shows for the first time an effect of Talpha1 on the regulation of gene expression at the molecular level, and may further contribute to explaining the results obtained using Talpha1 in the control of infectious diseases and tumor growth.

[The clinical use of human recombinant TSH]. / Impiego clinico del TSH umano ricombinante.

The recent cloning of human TSH-beta gene has allowed the production of recombinant human TSH (rhTSH) by recombinant DNA technology in mammalian cells (Chinese hamster ovary cells). Studies aimed at biochemical and biological characterization have shown that rhTSH, unlike pituitary TSH, is highly sialylated and is biological active in stimulating c-AMP accumulation in FRTL-5 cells. Phase I/II and phase III clinical studies have been performed to evaluate the safety and efficacy of rhTSH in stimulating radioactive iodine uptake in patients after total thyroidectomy for differentiated thyroid cancer. In these patients therapy with thyroid hormones is performed to replace hormone production and to suppress TSH-stimulated tumor growth. To detect residual or recurrent cancer, the therapy has to be withdrawn in order to obtain rise in endogenous TSH to perform a total body scan. rhTSH, as a source of exogenous human TSH, has been shown as an additional diagnostic tool in the follow-up of patients with thyroid cancer. Used in patients maintained on thyroid hormone suppressive therapy, rhTSH enhances the sensitivity of serum Tg testing. Although the sensitivity of scans obtained after rhTSH administration is slightly lower than that after thyroid hormone withdrawal, the use of rhTSH avoids the clinical signs and symptom of hypothyroidism and can be used in selected patients.

Effect of clonidine on glucose, insulin and glucagon responses to a protein meal in type 2 diabetics.

The authors investigated the effects of clonidine (alpha-2 stimulating agent) on blood glucose, insulin and glucagon levels in order to assess the alpha-adrenergic regulation of endocrine pancreatic secretion. Ten hypertensive female subjects affected with type 2 diabetes were studied; each subject was given a protein meal (boiled beef 200 g); blood samples were taken at -30, 0, 30, 60, 90 and 120 min; after this test each subject was treated for 4 days with clonidine (0.150 mg, 3 times/day per os); at the 5th day the protein meal was repeated under the same conditions except for the added administration of clonidine. Plasma glucose, insulin and glucagon were estimated. The administration of a protein meal caused a significant increase of blood glucose (peak at 60 min), insulin (peak at 90 min) and glucagon (peak at 90 min) levels; the association of clonidine caused an increase of blood glucose (single values and total areas) without changes of insulin and glucagon levels, when compared to those obtained before clonidine treatment. In conclusion, the association of clonidine to a protein meal caused impaired glucose tolerance presumably due to a direct glycogenolytic effect, occurring in the liver on account of an alpha-2 receptor stimulation, insulin and glucagon not being involved in this phenomenon.

Effect of alpha-1-blockade by prazosin on blood sugar, insulin and glucagon levels in normals and non-insulin dependent diabetics.

The AA. studied the glycometabolic activity of prazosin, a hypotensive drug with an adrenergic blocking activity of alpha-1-selective type. Twenty-two moderately hypertensive subjects were studied. Their ages ranged from 37 to 57 years; 10 of the patients had non-insulin dependent diabetes mellitus. After overnight fasting every patient underwent an oral glucose test (g 100) at 09:00. Blood samples were withdrawn at 0, 30, 60, 90 and 120 minutes. Each patient was then given in randomized and double-blind fashion placebo or prazosin (4 mg, 2 pills of Minipress Pfizer per day) for the following 7 days; then another glucose load was administered. Glucose (enzymatic method), insulin and glucagon (RIA method) were measured in each blood sample. In non diabetic subjects glucose levels (60, 90, 120 min and total area) after oral glucose and prazosin were statistically higher (p less than 0.05, p less than 0.01, p less than 0.05) than after glucose only. No significant difference between the two curves was observed in the diabetic group. IRI levels in normal subjects were statistically higher after 120 min and in the total area, while no evident changes were noted in the diabetic group. The glucagon curve seen after oral glucose was not modified by prazosin in either group.

Effect of metformin on blood glucose, insulin and C-peptide responses to glucagon in non-insulin dependent diabetics.

Since the C-peptide Radioimmuno active/Immuno Reactive Insulin (CPR/IRI) molar ratio is considered as an index of insulin hepatic extraction and tissue receptor binding, the AA. investigated the effects of metformin on this index after glucagon infusion in non-insulin dependent diabetics. Fourteen lean subjects (aged 48 to 67 years, mean 54 +/- 7) with non-insulin dependent diabetes were studied. At 9.00 a.m. each subject after overnight fasting, underwent glucagon infusion (1 mg i.v. diluted in 250 ml of saline, infused at a rate of 8.3 gamma/min for 2 hours); blood specimen were obtained at --15, 0, 30, 60, 90, 120 min. This test was repeated after a five-day treatment with metformin (1.5 g per os). For each sample plasma glucose by glucose oxidase method, plasma insulin and C-peptide by Radio Immuno Assay (RIA) method were determined. After treatment with metformin the hyperglycemia induced by glucagon was not influenced; nevertheless insulin and C-peptide plasma levels showed an evident reduction while CPR/IRI molar ratio was unchanged. The AA. suppose an indirect effect of metformin upon beta cells, namely a less pancreatic insulin requirement, mediated by an improvement of glucose utilization.