Protective Effects of Pinus halepensis Bark Extract and Nicotine on Cigarette Smoke-induced Oxidative Stress in Keratinocytes.

BACKGROUND/AIM: Cigarette smoke (CS) is a major environmental health threat. The oxidative stress induced by CS on keratinocytes and the possible protective effect of nicotine, its receptor inhibitors, and Pinus halepensis bark extract in relation to known antioxidants were investigated. MATERIALS AND METHODS: Primary mouse keratinocytes were exposed to cigarette smoke in the presence and absence of Pinus halepensis bark extract (1 µg/ml), rutin (50 µM) and ascorbic acid (250 µM), nicotine (1 µM) with or without mecamylamine (5 µM) and α-bungarotoxin (0.1 µM). Keratinocyte viability and oxidative stress were evaluated by MTT and fluorescence assays. RESULTS: Pinus halepensis bark extract decreased the oxidative stress and increased the viability of keratinocytes, and moreover, these effects were more pronounced compared to the mixture of rutin and L-ascorbic acid. Nicotine significantly enhanced the viability potentiation of the beneficial effect induced by Pinus halepensis bark extract. Mecamylamine and α-bungarotoxin showed no specific effect. CONCLUSION: Pinus halepensis bark extract in combination with nicotine may successfully reverse skin damage induced by cigarette smoke.

A historic perspective on the current progress in elucidation of the biologic significance of non-neuronal acetylcholine.

The "5th International Symposium on Non-neuronal Acetylcholine: from bench to bedside" was held on September 27-29, 2019 in Hyatt Regency, Long Beach, CA, USA. Approximately 50 scientists from 11 countries over 6 continents participated in this meeting. The major topics included an overall biologic significance of non-neuronal acetylcholine (ACh) and the roles of the non-neuronal cholinergic systems in mucocutaneous, respiratory, digestive, immunologic, endocrine, cardiovascular, musculoskeletal and kidney diseases, and cancer. This meeting facilitated continued work to advance the fundamental science and translational aspects of the interdisciplinary studies on non-neuronal ACh. The progress made has opened a new chapter in the field of cholinergic pharmacology, and advanced our knowledge beyond regulation of individual cell- and tissue-types, defining a new paradigm of selective pharmacological regulation of vital function of practically all types of non-neuronal cells. It is now clear that the autocrine and paracrine control of non-neuronal cells by non-neuronal ACh is implemented through synergistic, additive, and reciprocal effects triggered by two different cholinergic receptor classes. Each biologic effect of ACh is determined by a unique combination of cholinergic receptors subtype expressed at each stage of cell development and differentiation. The plasticity of the non-neuronal cholinergic system helps adjust homeostasis to new environmental conditions.

Mechanisms of synergy of autoantibodies to M3 muscarinic acetylcholine receptor and secretory pathway Ca2+/Mn2+-ATPase isoform 1 in patients with non-desmoglein pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing a devastating blistering disease affecting oral and/or esophageal surfaces and, sometimes, also the skin. Anti-keratinocyte AuAbs developed by the desmoglein (Dsg) 1/3 AuAb-negative acute PV patients are pathogenic, as they induced acantholysis and epidermal split in the experimental models of PV in vitro and in vivo. These PV patients have various combinations of AuAbs to keratinocyte muscarinic acetylcholine receptor subtype M3 (M3AR), the secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1), and desmocollin 3 whose relative concentrations correlate with the disease activity. In this study, we identified new molecular mechanisms of the synergistic cooperation of AuAbs to M3AR and SPCA1 in inducing acantholysis in the anti-Dsg 1/3 AuAb-negative PV patients. Anti-M3AR AuAb was found to play an important role in determining the level of intraepidermal split just above the basal cells, caspase to mediate early pro-apoptotic events triggered by anti-SPCA1 AuAb, and the neonatal Fc receptor (FcRn) to contribute to the pathobiological actions of both anti-M3AR and anti-SPCA1 AuAbs. Altogether, these novel results support our original hypothesis that pemphigus acantholysis is a complex disease process (also known as apoptolysis) initiated by AuAbs directed against different keratinocyte proteins that play important roles in supporting cell viability and regulating vital cell functions.

Real-world experience of dupilumab treatment for atopic dermatitis in adults: a retrospective analysis of patients' records.

BACKGROUND: Clinical trial data for dupilumab, a monoclonal antibody against the interleukin-4 receptor (IL-4Rα), have shown that it is safe and effective for the treatment of moderate to severe atopic dermatitis in patients whose disease is resistant to other therapies. However, little real-world experience with dupilumab use has been reported thus far. The aim of this retrospective study was to assess overall outcomes in adult patients with atopic dermatitis (AD) treated with dupilumab. METHODS: A retrospective review of electronic medical records was conducted for patients treated with dupilumab in the Department of Dermatology at the University of California, Irvine. RESULTS: We analyzed the medical records of 77 AD patients who received dupilumab according to standard dosing and had at least one documented follow-up visit. In 66 patients (86%), dupilumab improved clinical disease severity, with 23 patients (30%) experiencing complete clearance on dupilumab. Dupilumab was generally well-tolerated and caused no serious adverse events. The most common side effects included dry eyes, conjunctivitis, and keratitis. The most common reason for discontinuation of treatment was lack of substantial clinical improvement or progression of disease severity, followed by ophthalmologic side effects. CONCLUSIONS: Overall, dupilumab was well-tolerated and resulted in clinical improvement in our patient population. These results provide additional important information on the safety and utility of dupilumab treatment for moderate to severe atopic dermatitis in the real-world clinical setting.

Synergy among non-desmoglein antibodies contributes to the immunopathology of desmoglein antibody-negative pemphigus vulgaris.

Pemphigus vulgaris (PV) is a potentially lethal mucocutaneous blistering disease characterized by IgG autoantibodies (AuAbs) binding to epidermal keratinocytes and inducing this devastating disease. Here, we observed that non-desmoglein (Dsg) AuAbs in the sera of patients with Dsg1/3 AuAb-negative acute PV are pathogenic, because IgGs from these individuals induced skin blistering in neonatal mice caused by suprabasal acantholysis. Serum levels of AuAbs to desmocollin 3 (Dsc3), M3 muscarinic acetylcholine receptor (M3AR), and secretory pathway Ca2+/Mn2+-ATPase isoform 1 (SPCA1) correlated with the disease stage of PV. Moreover, AuAb absorption on recombinant Dsc3, M3AR, or SPCA1 both prevented skin blistering in the passive transfer of AuAbs model of PV in BALB/c mice and significantly decreased the extent of acantholysis in a neonatal mouse skin explant model. Although acantholytic activities of each of these immunoaffinity-purified AuAbs could not induce a PV-like phenotype, their mixture produced a synergistic effect manifested by a positive Nikolskiy sign in the skin of neonatal mice. The downstream signaling of all pathogenic non-Dsg AuAbs involved p38 mitogen-activated protein kinase (MAPK)-mediated phosphorylation and elevation of cytochrome c release and caspase 9 activity. Anti-Dsc3 and anti-SPCA1 AuAbs also activated SRC proto-oncogene, nonreceptor tyrosine kinase (SRC). Of note, although a constellation of non-Dsg AuAbs apparently disrupted epidermal integrity, elimination of a single pathogenic AuAb could prevent keratinocyte detachment and blistering. Therefore, anti-Dsg1/3 AuAb-free PV can be a model for elucidating the roles of non-Dsg antigen-specific AuAbs in the physiological regulation of keratinocyte cell-cell adhesion and blister development.

Paraneoplastic autoimmune multiorgan syndrome (PAMS): Beyond the single phenotype of paraneoplastic pemphigus.

Paraneoplastic autoimmune multiorgan syndrome (PAMS) is characterized by a heterogenous group of signs and symptoms including severe desquamative stomatitis, a polymorphous cutaneous eruption, humoral immunity against plakin proteins, contribution of cell-mediated autoimmunity and commonly a progressive respiratory failure. Autoantibodies in PAMS target a wide array of antigens including plakins, cadherins, alpha-2-macroglobulin like 1 (A2ML1), BP180, plakophilin-3, and several neuromuscular antigens. Originally described as paraneoplastic pemphigus in 1990 due to some of its clinical and immunologic similarities to classic pemphigus (pemphigus vulgaris and pemphigus foliaceus), PAMS is a multiorganopathy with several distinct features from these classic forms of pemphigus. Epidemiologically, PAMS is associated with underlying neoplasia and has a differing HLA-II allele predisposition compared to classic forms of pemphigus. Clinically, lesion morphology is polymorphous, and lesion distribution fundamentally differs from that seen in classic pemphigus. PAMS has a significantly higher mortality rate and a poorer response to treatments typically effective in pemphigus. Histologically, PAMS is characterized by the presence of interface dermatitis, vacuolar changes, and dyskeratotic keratinocytes which are not seen in classic pemphigus. PAMS demonstrates not only intercellular IgG as seen in classic pemphigus, but the presence of linear basement membrane zone deposition. Antibodies against desmoglein 3 (Dsg3) map to a broader array of epitopes than in pemphigus vulgaris and there is a higher prevalence of complement binding anti-Dsg3 IgG autoantibodies in PAMS. Autoantibodies can in rare cases be absent in the more cell-mediated form of PAMS. Considering these numerable differences, we review the entity of PAMS, and provide similarities and differences to classic forms of pemphigus.

A case report of bullous pemphigoid associated with a melanoma and review of the literature.

The association of bullous pemphigoid with melanoma remains controversial and poorly understood. Recent studies report the presence of the bullous pemphigoid antigen, BP180, in melanoma cells, yet not normal melanocytes, suggesting an underlying mechanism for cases of melanoma-associated bullous pemphigoid. We report on an 88-year-old woman who showed a temporal relationship between the development of bullous pemphigoid and melanoma. The patient did not receive programmed death ligand 1 inhibitor therapy and improved rapidly following complete excision of her melanoma, with clobetasol, doxycycline, and niacinamide. We review the literature on the relationship between bullous pemphigoid and melanoma, and propose a mechanism underlying a melanoma-associated bullous pemphigoid.

Recent progress in revealing the biological and medical significance of the non-neuronal cholinergic system.

This special issue of International Immunopharmacology is the proceedings of the Fourth International Symposium on Non-neuronal Acetylcholine that was held on August 28-30, 2014 at the Justus Liebig University of Giessen in Germany. It contains original contributions of meeting participants covering the significant progress in understanding of the biological and medical significance of the non-neuronal cholinergic system extending from exciting insights into molecular mechanisms regulating this system via miRNAs over the discovery of novel cholinergic cellular signaling circuitries to clinical implications in cancer, wound healing, immunity and inflammation, cardiovascular, respiratory and other diseases.

Mechanisms of growth-promoting and tumor-protecting effects of epithelial nicotinic acetylcholine receptors.

Although the role of nicotine as a carcinogen is debatable, it is widely accepted that it contributes to cancer by promoting growth and survival of mutated cell clones and protecting them from the chemo- and radiotherapy-induced apoptosis. On the cell membrane (cm), the nicotinic acetylcholine (ACh) receptors (nAChRs) implement upregulation of proliferative and survival genes. Nicotine also can permeate cells and activate mitochondrial (mt)-nAChRs coupled to inhibition of the mitochondrial permeability transition pore (mPTP) opening, thus preventing apoptosis. In this study, we sought to pin down principal mechanisms mediating the tumor-promoting activities of nicotine resulting from activation of cm- and mt-nAChRs in oral and lung cancer cells, SCC25 and SW900, respectively. Activated cm-nAChRs were found to form complexes with receptors for EGF and VEGEF via the α7 and ß2 nAChR subunits, respectively, whereas activated mt-nAChRs physically associated with the intramitochondrial protein kinases PI3K and Src via the α7 and ß4 subunits. This was associated with upregulated expression of cyclin D1/activation of ERK1/2 and inhibition of mPTP opening, respectively, as well as upregulated proliferation and resistance to H(2)O(2)-induced apoptosis. The molecular synergy between cm-nAChRs and growth factor receptors helps explain how one biological mediator, such as ACh, can modulate activity of the other, such as a growth factor, and vice versa. Establishment of functional coupling of mt-nAChRs to regulation of mPTP opening provides a novel mechanism of nicotine-dependent protection from cell death. Further elucidation of this novel mechanism of tumor-promoting activities of nicotine should have a strong translational impact, because extraneuronal nAChRs may provide a novel molecular target to prevent, reverse, or retard progression of both nicotine-related and unrelated cancers.

Molecular mechanisms of synergy of corneal muscarinic and nicotinic acetylcholine receptors in upregulation of E-cadherin expression.

Corneal epithelial erosion is one of the most common problems in clinical ophthalmology. Despite significant progress in understanding how the cornea heals, clinically available pharmacological therapies that can promote repair and prevent visual complications remain limited. We have recently demonstrated that the acetylcholine (ACh) axis of corneal epithelium plays an important role in regulation and coordination of distinct activities of corneal epithelial cells (CEC) mediating re-epithelialization, but mechanisms remained unclear. We hypothesized that the grounds for synergistic effects of corneal ACh receptors lie within the signaling pathways linking different receptors to specific elements of the CEC pro-epithelialization activities. In this study, we sought to elucidate the molecular mechanisms of cooperation of corneal muscarinic and nicotinic ACh receptors (mAChRs and nAChRs) in upregulation of E-cadherin expression. The roles of individual corneal mAChRs and nAChRs subtypes were investigated by in-cell western assay of the ACh-treated CEC, in which different ACh receptor genes were silenced by receptor-specific shRNAs. Functional inactivation of M3, but not M4, mAChR subtype, or α3 or α7, but not α9, nAChR subunit significantly inhibited E-cadherin expression. To gain a mechanistic insight, we blocked the key steps of the downstream signaling pathways. Results demonstrated that cholinergic agonists can upregulate E-cadherin expression by activating M3 mAChR, and α3ß2 and α7 nAChRs via the common signaling cascade Ca(2+)-CaMKII-PKC-Ras-Raf-MEK-ERK. Activation of α7 nAChR can launch the Ras-Raf-MEK-ERK cascade both indirectly, through the Ca(2+)-CaMKII-PKC step, and directly, perhaps, due to its direct interaction with Ras. Although the biological significance of such redundancy remained to be elucidated, results of the present study point to a new direction to pharmacologically accelerate corneal re-epithelialization, and should have salient clinical implication.

The nicotinic acetylcholine receptor-mediated reciprocal effects of the tobacco nitrosamine NNK and SLURP-1 on human mammary epithelial cells.

Recent research has demonstrated that the nicotinergic signaling network of mammary epithelium can both mediate the physiological control of normal breast epithelial cells (BECs) and exhibit tumor-promoting effects on malignant BECs. Therefore, mammary nicotinic acetylcholine (ACh) receptors (nAChRs) may become a specific target for novel anti-breast cancer therapies. Toward this goal, we investigated the difference in the ACh receptor repertoires between normal and malignant BECs, determined effects of nicotinic ligands on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-dependent activation of ERK1/2 and tumorigenic transformation of MCF10A cells, and characterized reciprocal effects of NNK and SLURP (secreted mammalian Ly-6/urokinase plasminogen activator receptor related protein-1)-1 on the expression of nAChR subunits and several oncogenes and tumor-suppressing genes in BECs. Both the non-malignant MCF10A and malignant MCF7 breast cells expressed α3, α5, α7, α9, α10, ß1, ß2, γ, δ and ε nAChR subunits and M(1), M(3), M(4) and M(5) muscarinic receptor subtypes. The malignancy was associated with expression of α1, α4 and ß4 nAChR subunits and M(2) subtype. Malignant transformation of BECs was also associated with overexpression of α7-, and α9-made nAChRs. NNK upregulated ERK1/2 phosphorylation, stimulated expression of the gene encoding the tumor-promoter HGF, downregulated expression of the tumor suppressor gene CDKN2A, and induced tumorigenic transformation of MCF10A cells. Compared to the canonical nAChR antagonists, SLURP-1 showed the highest ability to abolish the nAChR-mediated effects of NNK in both cell-signaling and cell-transformation assays and reversed many effects of NNK on gene expression. SLURP-1 also markedly upregulated the tumor suppressor genes CDKN2B, RUNX3 and TP73. Altogether, the obtained results provided new insight into the molecular mechanisms of nAChR-mediated oncogenic effects of NNK on BECs and demonstrated the ability to abolish or reverse these effects by SLURP-1.

Pemphigus vulgaris antibodies target the mitochondrial nicotinic acetylcholine receptors that protect keratinocytes from apoptolysis.

The mechanism of detachment and death of keratinocytes in pemphigus vulgaris (PV) involves pro-apoptotic action of constellations of autoantibodies determining disease severity and response to treatment. The presence of antibodies to nicotinic acetylcholine receptors (nAChRs) and the therapeutic efficacy of cholinomimetics in PV is well-established. Recently, adsorption of anti-mitochondrial antibodies abolished the ability of PVIgGs to cause acantholysis, demonstrating their pathophysiological significance. Since, in addition to cell membrane, nAChRs are also present on the mitochondrial outer membrane, wherein they act to prevent activation of intrinsic (mitochondrial apoptosis), we hypothesized that mitochondrial (mt)-nAChRs might be targeted by PVIgGs. To test this hypothesis, we employed the immunoprecipitation-western blot assay of keratinocyte mitochondrial proteins that visualized the α3, α5, α7, α9, α10, ß2 and ß4 mt-nAChR subunits precipitated by PV IgGs, suggesting that functions of mt-nAChRs are compromised in PV. To pharmacologically counteract the pro-apoptotic action of anti-mitochondrial antibodies in PV, we exposed naked keratinocyte mitochondria to PVIgGs in the presence of the nicotinic agonist nicotine ± antagonists, and measured cytochrome c (CytC) release. Nicotine abolished PVIgG-dependent CytC release, showing a dose-dependent effect, suggesting that protection of mitochondria can be a novel mechanism of therapeutic action of nicotinic agonists in PV. The obtained results indicated that the mt-nAChRs targeted by anti-mitochondrial antibodies produced by PV patients are coupled to inhibition of CytC release, and that nicotinergic stimulation can abolish PVIgG-dependent activation of intrinsic apoptosis in KCs. Future studies should determine if and how the distinct anti-mt-nAChR antibodies penetrate KCs and correlate with disease severity.

Persistence of Autoreactive IgA-Secreting B Cells Despite Multiple Immunosuppressive Medications Including Rituximab.

IMPORTANCE: Immunobullous diseases mediated by IgA are often difficult to manage, but to date no mechanism has been proposed. Rituximab is an anti-CD20 monoclonal antibody that has demonstrated good efficacy in the treatment of refractory mucous membrane pemphigoid. However, not all cases of mucous membrane pemphigoid respond to rituximab. Herein we present a case of treatment-refractory mucous membrane pemphigoid and propose a mechanism to explain the lack of response to therapy. OBSERVATIONS: Before treatment, direct immunofluorescent examination of a biopsy sample from the patient's perilesional skin demonstrated linear deposition of IgG and IgA along the dermoepidermal junction. After a multidrug immunosuppressive regimen that included rituximab, results of a second biopsy demonstrated only IgA along the dermoepidermal junction. This finding correlated well with flow cytometry data from the same patient that demonstrated a persistent population of IgA-secreting plasmablasts/plasma cells, despite depletion of CD20⁺ cells. In addition, results of immunohistochemical analysis of the perilesional skin remained positive for CD19 and CD138 immune cells (plasmablast/plasma cell markers). CONCLUSIONS AND RELEVANCE: These findings suggest that current available immunosuppressive medications, including rituximab, cannot eliminate IgA-secreting plasmablasts/plasma cells, which are likely central to the pathophysiology of IgA-mediated immunobullous diseases. Future studies are needed to develop alternative therapeutic strategies that target autoreactive IgA-secreting plasmablasts/plasma cells.

Mechanisms of tumor-promoting activities of nicotine in lung cancer: synergistic effects of cell membrane and mitochondrial nicotinic acetylcholine receptors.

BACKGROUND: One of the major controversies of contemporary medicine is created by an increased consumption of nicotine and growing evidence of its connection to cancer, which urges elucidation of the molecular mechanisms of oncogenic effects of inhaled nicotine. Current research indicates that nicotinergic regulation of cell survival and death is more complex than originally thought, because it involves signals emanating from both cell membrane (cm)- and mitochondrial (mt)-nicotinic acetylcholine receptors (nAChRs). In this study, we elaborated on the novel concept linking cm-nAChRs to growth promotion of lung cancer cells through cooperation with the growth factor signaling, and mt-nAChRs - to inhibition of intrinsic apoptosis through prevention of opening of mitochondrial permeability transition pore (mPTP). METHODS: Experiments were performed with normal human lobar bronchial epithelial cells, the lung squamous cell carcinoma line SW900, and intact and NNK-transformed immortalized human bronchial cell line BEP2D. RESULTS: We demonstrated that the growth-promoting effect of nicotine mediated by activation of α7 cm-nAChR synergizes mainly with that of epidermal growth factor (EGF), α3 - vascular endothelial growth factor (VEGF), α4 - insulin-like growth factor I (IGF-I) and VEGF, whereas α9 with EGF, IGF-I and VEGF. We also established the ligand-binding abilities of mt-nAChRs and demonstrated that quantity of the mt-nAChRs coupled to inhibition of mPTP opening increases upon malignant transformation. CONCLUSIONS: These results indicated that the biological sum of simultaneous activation of cm- and mt-nAChRs produces a combination of growth-promoting and anti-apoptotic signals that implement the tumor-promoting action of nicotine on lung cells. Therefore, nAChRs may be a promising molecular target to arrest lung cancer progression and re-open mitochondrial apoptotic pathways.

Targeting histone deacetylase in lung cancer for early diagnosis: (18)F-FAHA PET/CT imaging of NNK-treated A/J mice model.

Elevated levels of histone deacetylases (HDACs) have been indicated in the development of some cancers. HDAC has been imaged using (18)F-FAHA and may serve as a marker to study epigenetics. We report evaluation of (18)F-FAHA as a probe in the early diagnosis of lung cancer using (18)F-FAHA PET/CT studies of A/J mice treated with NNK. (18)F-FAHA radiosynthesis was carried out in specific activity of ~2 Ci/µmol. A/J mice were divided into 2 groups: 1. Controls; 2. NNK treatment group with NNK (100 mg/kg, ip, weekly for 4 wks). Mice were injected 100-200 µCi i.v. (18)F-FAHA and then scanned in Inveon PET/CT under anesthesia using 2.0% isoflurane. Midbrain, cerebellum and brainstem uptake of (18)F-FAHA was displaced by the known HDAC inhibitor, suberanilohydroxamic acid (SAHA) with less than 10% activity remaining. CT revealed presence of lung nodules in 8 to 10-month old NNK mice while control mice were free of tumors. Little uptake of (18)F-FAHA was observed in the control mice lungs while significant (18)F-FAHA uptake occurred in the lungs of NNK-treated mice with tumor/nontumor >2.0. Ex vivo scans of the excised NNK and control mice lungs confirmed presence of extensive amounts of lung nodules seen by CT and confirmed by (18)F-FAHA in the NNK mice with tumor/nontumor >6.0. Our preliminary imaging studies with A/J mice lung cancer model suggest (18)F-FAHA PET may allow the study of epigenetic mechanisms involved in NNK-induced tumorigenesis in the lungs.

Anti-inflammatory effects of the nicotinergic peptides SLURP-1 and SLURP-2 on human intestinal epithelial cells and immunocytes.

A search for novel and more efficient therapeutic modalities of inflammatory bowel disease (IBD) is one of the most important tasks of contemporary medicine. The anti-inflammatory action of nicotine in IBD might be therapeutic, but its toxicity due to off-target and nonreceptor effects limited its use and prompted a search for nontoxic nicotinergic drugs. We tested the hypothesis that SLURP-1 and -2--the physiological nicotinergic substances produced by the human intestinal epithelial cells (IEC) and immunocytes--can mimic the anti-inflammatory effects of nicotine. We used human CCL-241 enterocytes, CCL-248 colonocytes, CCRF-CEM T-cells, and U937 macrophages. SLURP-1 diminished the TLR9-dependent secretion of IL-8 by CCL-241, and IFN γ-induced upregulation of ICAM-1 in both IEC types. rSLURP-2 inhibited IL-1 ß-induced secretion of IL-6 and TLR4- and TLR9-dependent induction of CXCL10 and IL-8, respectively, in CCL-241. rSLURP-1 decreased production of TNFα by T-cells, downregulated IL-1 ß and IL-6 secretion by macrophages, and moderately upregulated IL-10 production by both types of immunocytes. SLURP-2 downregulated TNFα and IFNγ R in T-cells and reduced IL-6 production by macrophages. Combining both SLURPs amplified their anti-inflammatory effects. Learning the pharmacology of SLURP-1 and -2 actions on enterocytes, colonocytes, T cells, and macrophages may help develop novel effective treatments of IBD.

Connections of nicotine to cancer.

This Opinion article discusses emerging evidence of direct contributions of nicotine to cancer onset and growth. The list of cancers reportedly connected to nicotine is expanding and presently includes small-cell and non-small-cell lung carcinomas, as well as head and neck, gastric, pancreatic, gallbladder, liver, colon, breast, cervical, urinary bladder and kidney cancers. The mutagenic and tumour-promoting activities of nicotine may result from its ability to damage the genome, disrupt cellular metabolic processes, and facilitate growth and spreading of transformed cells. The nicotinic acetylcholine receptors (nAChRs), which are activated by nicotine, can activate several signalling pathways that can have tumorigenic effects, and these receptors might be able to be targeted for cancer therapy or prevention. There is also growing evidence that the unique genetic makeup of an individual, such as polymorphisms in genes encoding nAChR subunits, might influence the susceptibility of that individual to the pathobiological effects of nicotine. The emerging knowledge about the carcinogenic mechanisms of nicotine action should be considered during the evaluation of regulations on nicotine product manufacturing, distribution and marketing.

Tropisetron suppresses collagen synthesis in skin fibroblasts via α7 nicotinic acetylcholine receptor and attenuates fibrosis in a scleroderma mouse model.

OBJECTIVE: There is increasing evidence that serotonin (5-hydroxytryptamine [5-HT]) and distinct 5-HT receptors are involved in the pathogenesis of systemic sclerosis. The aim of this study was to test the hypothesis that tropisetron, a routinely used antiemetic agent previously characterized as a 5-HT(3/4) receptor-modulating agent, can directly affect collagen synthesis in vitro and attenuate experimentally induced fibrosis in vivo. METHODS: Functional in vitro studies were performed using human dermal fibroblasts (HDFs). Signal transduction studies included immunofluorescence analysis, Western immunoblotting, promoter reporter assays, cAMP/Ca(2+) measurements, and use of pharmacologic activators and inhibitors. Gene silencing was performed using small interfering RNA. Putative receptors of tropisetron were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. The murine model of bleomycin-induced scleroderma was used to assess the antifibrogenic and antifibrotic effects of tropisetron in vivo. Collagen expression in vitro, ex vivo, and in situ was determined by real-time RT-PCR analysis, Western immunoblotting, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunohistochemical analysis. RESULTS: Tropisetron suppressed collagen synthesis induced by transforming growth factor ß1 (TGFß1). This effect was independent of 5-HT(3/4) receptor but was mediated via α7 nicotinic acetylcholine receptor (α7nAChR). Suppression of TGFß1-induced collagen synthesis occurred via an unknown molecular mechanism not involving modulation of the Smad, cAMP, Akt, c-Jun, or MAPK pathway. In vivo, tropisetron not only prevented skin fibrosis but also reduced the collagen content in established dermal fibrosis induced by bleomycin. CONCLUSION: Tropisetron directly reduces collagen synthesis in HDFs via an α7nAChR-dependent mechanism. The antifibrogenic and antifibrotic effects of this agent observed in a mouse model of bleomycin- induced scleroderma indicate the future potential of tropisetron in the treatment of fibrotic diseases such as scleroderma.

The tobacco carcinogen nitrosamine induces a differential gene expression response in tumour susceptible A/J and resistant C3H mouse lungs.

The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), an important carcinogen found in tobacco products, causes lung cancer in genetically susceptible animals. In addition to mutations of the K-Ras gene, NNK has non-mutagenic effects that include alterations in gene expression and immunomodulation in the lung. Here we report the identification of two gene sets associated with NNK-induced pulmonary tumourigenesis. First, to identify genes involved in the susceptibility to NNK, we compared the lung transcriptomes of NNK-resistant C3H mice with that of the NNK-susceptible A/J mice, identifying differential expression of genes related to innate immunity and inflammation. Second, to identify gene expression induced by NNK, we compared the lung transcriptomes of C3H and A/J mice post-treatment. The Resistin-like alpha (Retnla) gene was highly upregulated in response to NNK only in susceptible mice. This gene product is known to recruit immune cells to the lung, and accumulation of CD45 positive cells in A/J lungs correlated with increased Retnla expression. Genetic susceptibility to NNK-induced lung tumourigenesis may relate in part to gene expression changes and alterations in the immune response to create a pro-tumourigenic environment, acting in concert with NNK's mutagenic effects.