Expression of protease-activated receptor-2 (PAR-2) in patients with nasopharyngeal carcinoma: Correlation with clinicopathological features and prognosis

We aimed at determining whether the expression of protease-activated receptor 2 (PAR-2) is involved in the progression of nasopharyngeal carcinoma (NPC) and correlated with latent membrane protein 1 (LMP-1), matrix metalloproteinases-9 (MMP9), and angiogenesis of tumor.     

Protease and protease-activated receptor-2 signaling in the pathogenesis of atopic dermatitis

Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/ PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD.     

Activation of protease-activated receptor-2 reduces airways inflammation in experimental allergic asthma

BACKGROUND: Proteinase-activated receptors (PAR)-2 are members of the family of G-protein-coupled receptors activated by proteases. These receptors are widely expressed in several tissues and in virtually all cells involved in rhinitis and asthma. In particular, proteinases activating PAR-2 may affect airway functions and play a role in human diseases. OBJECTIVE: Assessment of the role of PAR-2 in bronchoconstriction, airway responsiveness and immune response after allergic challenge, in rabbits sensitized to Par j 1, the major allergen of Parietaria judaica pollen. METHODS: Evaluation of antigen challenge in rabbits treated with PAR-2-activating peptide (PAR-2AP) (SLIGRL) or the scrambled peptide LSIGRL or vehicle immediately before allergen exposure measuring airway responsiveness. Characterization of bronchoalveolar lavage (BAL) following histamine challenge and phenotype analysis of cells by flow cytometry and analysis of cytokine production by quantitative PCR. RESULTS: PAR-2AP pre-treatment, but not the scrambled peptide, was able to significantly inhibit bronchoconstriction, airway hyper-responsiveness and to modulate the immune response induced by allergic challenge in sensitized rabbits. The phenotype analysis of the cells recovered from BAL showed an increase in RLA-DR-positive cells while RTLA-positive cells were unchanged. IFN-gamma and IL-2 production were inhibited, with a concomitant increase in IL-10 of about 10-fold over the control values. CONCLUSIONS: In this experimental model, PAR-2 modulates bronchoconstriction interfering with antigen challenge-induced immune response in rabbits sensitized and challenged to Par j 1.     

TREM-2对小鼠肺成纤维细胞凋亡的影响

目的:初步探讨髓样细胞触发受体-2(triggering receptors expressed on myeloid cells-2,TREM-2)过表达对小鼠肺成纤维细胞凋亡的影响.方法:将小鼠肺成纤维细胞分成脂多糖( lipopolysaccharide,LPS)组、LPS+转染试剂组、LPS+空质粒组和LPS+...     

Uterine fibroblast growth factor-2 and embryonic fibroblast growth factor receptor-1 at the beginning of gastrulation in the rabbit

Fibroblast growth factor-2 (FGF-2) induces gastrulation of rabbit blastocysts in vitro and is present in the uterine secretion at day 6 after mating. The following study was made in order to show if changes in the uterine FGF-2 concentration or in the FGF receptor concentration of the embryonic tissues point to a regulation of this event. By the use of the ELISA technique and immunohistochemistry, FGF-2 concentration was determined in the endometrical tissue, uterine secretion and blastocyst between day 4 and day 8 of pregnancy, in the uterine secretion after induction of pseudopregnancy, in day 6 blastocysts after in vitro culture, and FGF immunoreactivity was localized in the endometrial tissue. FGF receptor-1 (FGFR-1) concentration was examined correspondingly in the blastocyst. Cross-linking experiments using ¹²⁵I-FGF-2 were done to identify binding proteins in the blastocyst. In the uterine secretion, FGF-2 was constantly high up to day 6.5 but showed an increase thereafter. Similar values in pseudopregnant uterine secretions indicated that the growth factor was of uterine origin. It was probably synthesized by the uterine epithelium as shown by immunohistochemistry. Under culturing conditions, the blastocyst produced small amounts of FGF-2. In the blastocyst, FGFR-1 as well as binding of ¹²⁵I-FGF-2 showed a dramatic increase from day 6.0 to day 6.5, coinciding with the onset of gastrulation. Receptor antigenicity was located in the embryonic disc at day 6.5 and day 7.0. Two binding proteins of about 200 and 130 kDa were found by cross-linking. The results indicate that a regulation of growth factor influence on embryonic differentiation is more probable via expression of the embryonic receptor than via differential release of the uterine growth factor.     

Intracellular Ca2+ concentrations are not elevated in resting cultured muscle from Duchenne (DMD) patients and in MDX mouse muscle fibres

The free intracellular calcium concentration, [Ca²⁺]_i, was studied in single myotubes using the fluorescent Ca²⁺ indicator fura-2. Myotubes cultured from satellite cells of small muscle specimens from Duchenne muscular dystrophy (DMD) patients were compared with human control myotubes and with myotubes cultured from MDX and control mouse muscle satellite cells. The resting [Ca²⁺]_i levels in DMD and control myotubes were not significantly different, i. e. 104 ±26 nM (mean ± SD, n=190 cells from eight DMD patients) compared with 97±25 nM (175/seven controls) and were not significantly lower than the corresponding murine values (154±33 nM, n=135 MDX myotubes; 159±34 nM, n=135 controls). All myotubes reacted to 10 M acetylcholine or 40 mM KCl with fast transient increases of [Ca²⁺]_i. After application of a hyposmotic (130 mOsm) solution, [Ca²⁺]_i was increased 1.5- to 3-fold within 2–3 min, the DMD myotubes tending to stronger reactions (significantly higher [Ca²⁺]_i in 2 out of 6 cases). The response was usually transient, [Ca²⁺]_i decreasing to the initial level within 10 min. Gadolinium (50 M) reduced the response by 50%–70%, indicating that the osmotic shock increased Ca²⁺ influx. During exposure to high (15 mM) [Ca²⁺]e, [Ca²⁺]_i of DMD and control cells was 1.5- to 2-fold higher. Adult muscle fibres from MDX mice and controls showed identical Ca²⁺ resting levels (n=45 fibres from three mice in each case), but did not respond to decreased external osmolarity with a change in [Ca²⁺]_i. The results indicate that lack of dystrophin in muscle fibres does not necessarily lead to increased [Ca²⁺]_i. It is suggested that increased [Ca²⁺]_i is probably a secondary consequence of fibre damage.     

Bile acids increase cellular free calcium in cultured kidney cells (LLC-PK1)

Suspensions of LLC-PK₁ cells were used to determine the effect of bile acids on the cellular homeostasis of morganic ions. It is determined that bile acids alter cellular free calcium (Ca_i) levels in LLC-PK₁ cells. A series of bile acids were compared and found to produce increases in Ca_i in the order: lithocholate sulfate (LCS) > deoxycholate > chenodeoxycholate > lithocholate glucuronide > cholate. LCS (300 M) produces changes in Ca_i (measured using Fura-2) qualitatively similar to those produced by 1 M monomycin, except that only ionomycin is able to release calcium from intracellular stores. The effect on Ca_i is roughly proportional to LCS concentration between 50 and 300 M. The presence of 40 mM Na in the extracellular medium reduces the LCS-induced rise in Ca_i to 20% of that observed in the absence of Na. This effect is specific for Na versus 150 mM extracellular K, Li, or TMA. The effect is not dependent on the Na gradient across the membrane. At concentrations of LCS which induce changes in Ca_i, no significant effect of LCS is observed on either cellular Na or K levels, or intracellular pH.Abbreviations used BCECF 2,7-bis-(2-carboxyethyl)-5,6-carboxy-fluorescein - HEPES 4-(-2-hydroxyethyl)-1-piperazine ethanesulfonate - TMA tetramethylammonium - PCA perchloric acid - LCS lithocholate sulfate - EGTA ethylene glycol-bis-(-aminoethyl ether)N,N-tetra-acetic acid - Ca_i intracellular free calcium - pH intracellular pH     

Nuclear factor erythroid-derived 2-like 2 (Nrf2) and DJ1 are prognostic factors in lung cancer

Nuclear factor erythroid-derived 2-like 2 (Nrf2) controls the expression of several enzymes that are protective against oxidative stress. We investigated the expression of nuclear factor erythroid-derived 2-like 2, DJ1 (Nrf2 stabilizer), and sulfiredoxin in a large set of lung carcinomas. The cases were analyzed immunohistochemically with antibodies to nuclear factor erythroid-derived 2-like 2, DJ1, and sulfiredoxin with the results being compared with histologic and clinical data. Significant differences were observed in the expression of DJ1 and sulfiredoxin between various types of lung tumors, while expression of nuclear factor erythroid-derived 2-like 2 was more constant. Patients with tumors with cytoplasmic (P = .033) or nuclear (P = .003) DJ1 positivity exhibited worse survival. Separately in squamous cell carcinomas, there was a tendency toward worse survival with both cytoplasmic (P = .013) and nuclear (P = .071) DJ1 positivity. Patients with a strong nuclear factor erythroid-derived 2-like 2 expression in their tumors had worse survival (P = .006). In the Cox regression analysis, nuclear factor erythroid-derived 2-like 2 was an independent prognostic factor (P = .012) along with the T status (P = .008) and DJ1 cytoplasmic positivity (P = .028). Interestingly, smokers and ex-smokers had significantly more sulfiredoxin expression in their tumors (P < .001); and in patients receiving cytostatic drugs or radiation therapy, sulfiredoxin expression predicted a poor prognosis (P = .038). Nuclear factor erythroid-derived 2-like 2 and its stabilizing protein DJ1 affect the prognosis of patients with lung cancer by inducing an elevated stress response to oxidative damage. There were differences in the expression of sulfiredoxin and DJ1 between different lung tumor types, suggesting that the pathways involved in combating oxidative stress vary in different lung cancer types.     

金纳多对内毒素诱导小鼠急性肺损伤保护作用机制的初步研究

目的：初步探讨银杏叶提取物金纳多（Ginaton）对内毒素（lipopolysaccharide, LPS）诱导小鼠的急性肺损伤(acute lung injury, ALI)保护作用的可能机制。方法：于小鼠腹腔注射LPS（10 mg/kg）复制ALI动物模型。将小鼠随机分为对照组、LPS组、Ginaton组和Ginaton＋LPS组。观察各组肺组织病理学改变,测量肺湿/干重比,支气管肺泡灌洗液蛋白含量及乳酸脱氢酶（lactate dehydrogenase, LDH）活性,测量丙二醛（malondialdehyde, MDA）、一氧化氮合酶（nitric oxide synthase, iNOS）和髓过氧化物（myeloperoxidase,MPO),免疫组织化学方法检测血红素加氧酶（heme oxygenase HO-1）及iNOS蛋白表达。结果： 金纳多可有效减轻LPS所致肺组织病理学变化,并降低肺湿/干重比和肺泡灌洗液中蛋白含量,降低肺泡灌洗液中LDH活性、肺组织MPO和iNOS活性,同时MDA和NO含量下降。免疫组织化学结果显示,LPS组iNOS表达上升（P＜0.01）,而血红素加氧酶（HO-1）蛋白表达未见明显变化;而预先给予Ginaton可显著提高HO-1的表达,降低iNOS的表达（P＜0.01）。结论：Ginaton可减轻LPS所致急性肺组织损伤,其机制可能与诱导HO-1的表达,下调iNOS的表达和活性有关。     

Role of STAT6 and SMAD2 in a model of chronic allergen exposure: a mouse strain comparison study

Background Asthma is a disease characterized by variable and reversible airway obstruction and is associated with airway inflammation, airway remodelling (including goblet cell hyperplasia, increased collagen deposition and increased smooth muscle mass) and increased airway responsiveness. It is believed that airway inflammation plays a critical role in the development of airway remodelling, with IL‐13 and TGF‐β1 pathways being strongly associated with the disease progression. Mouse models of asthma are capable of recapitulating some components of asthma and have been used to look at both IL‐13 and TGF‐β1 pathways, which use STAT6 and SMAD2 signalling molecules, respectively. Objectives Using brief and chronic models of allergen exposure, we utilized BALB/c and C57Bl/6 to explore the hypothesis that observed differences in responses to allergen between these mouse strains will involve fundamental differences in IL‐13 and TGF‐β1 responses. Methods The following outcome measurements were performed: airway physiology, bronchoalveolar lavage cell counts/cytokine analysis, histology, immunoblots and gene expression assays. Results We demonstrate in BALB/c mice an IL‐13‐dependent phosphorylation of STAT6, nuclear localized in inflammatory cells, which is associated with indices of airway remodelling and development of airway dysfunction. In BALB/c mice, phosphorylation of SMAD2 is delayed relative to STAT6 activation and also involves an IL‐13‐dependent mechanism. In contrast, despite an allergen‐induced increase in IL‐4, IL‐13 and eosinophils, C57Bl/6 demonstrates a reduced and distinct pattern of phosphorylated STAT6, no SMAD2 phosphorylation changes and fail to develop indices of remodelling or changes in airway function. Conclusion The activation of signalling pathways and nuclear translocation of signalling molecules downstream of IL‐13 and TGF‐β1 further support the central role of these molecules in the pathology and dysfunction in animal models of asthma. Activation of signalling pathways downstream from IL‐13 and TGF‐β1 may be more relevant in disease progression than elevations in airway inflammation alone.     

Salivary gland extract from engorged Ixodes ricinus (Acari: Ixodidae) stimulates in vitro growth of Borrelia burgdorferi sensu lato

In vitro effect of salivary gland extract from fed Ixodes ricinus, the competent vector of Lyme borreliosis in Europe, on the growth of Borrelia burgdorferi sensu lato (B. garinii, B. afzelii and B. burgdorferi sensu stricto) was examined in BSK‐H medium. Motility rate, concentration of motile spirochetes and their morphology were estimated at intervals of 0, 2, 4, 6 and 8 days using darkfield microscopy. Salivary gland extract derived from I. ricinus stimulated markedly the growth of three genomic species of borreliae. The results confirm a substantial role of salivary glands in the mechanism of pathogen transmission to vertebrate host. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Reduced susceptibility to dextran sulphate sodium-induced colitis in the interleukin-2 heterozygous (IL-2) mouse

Summary Mice homozygous for an inactivation of the interleukin-2 (IL-2) gene develop a T-cell dependent colitis. Heterozygous (IL-2+/-) mice are clinically healthy but have been shown to express reduced levels of IL-2 in the colon. Splenocytes from the IL-2+/- mice had a poorer proliferative response to polyclonal T-cell activation and these mice have reduced numbers of intestinal regulatory T cells (CD4+ CD25+ cells) when compared to wild type mice. When exposed to dextran sulphate sodium (DSS) IL-2+/- mice showed a markedly reduced susceptibility to DSS-induced colitis. While DSS treatment caused a marked increase in both CD4+ and CD8+ colonic T cells expressing increased levels of IL-2, IL-4, and IL-10 in wild type mice none of these changes were seen in IL-2+/- mice. On the contrary, cytokine expression in intestinal T cells of IL-2+/- mice was actually reduced after DSS treatment. These results suggest that reduced levels of IL-2 leads to attenuated activation and function of intestinal T cells in IL-2+/- mice and a failure to react adequately to DSS exposure.     

Small (SKCa) Ca2+-activated K+ channels in cultured rat hippocampal pyramidal neurones

 Small (SK_Ca) Ca²⁺-activated K⁺ channels were identified in membrane patches excised from cultured CA1-CA3 pyramidal neurones of the neonatal rat hippocampus. When recorded in low-K⁺ extracellular solution ([K⁺]_o=2.5 mM), SK_Ca channels had a low conductance (@3 pS at 0 mV), were activated by ≥175 nM Ca²⁺ (P _o=0.54 at 500 nM Ca²⁺) and there were two open-time components (2.1 and @70 ms) to their activity. These properties of single SK_Ca channels are similar to those of slow after-hyperpolarization channels (sAHP) previously inferred from fluctuation analysis of the sAHP current. It is concluded that the SK_Ca channel reported here may be the channel that generates the sAHP in hippocampal pyramidal neurones. Received: 9 July 1998 / Received after revision: 5 October 1998 / Accepted: 7 October 1998     

N-(2-mercaptopropionyl)-glycine but not allopurinol prevented cigarette smoke-induced alveolar enlargement in mouse

We investigated the possible protective effects of the Allopurinol (A), N-(2-mercaptopropionyl)-glycine (M) and N-acetylcysteine (N) against lung injury caused by long-term exposure to cigarette smoke (CS) in mouse. C57BL6 mice were exposed to 12 cigarettes a day for 60 days and concomitantly treated with either one of the antioxidant drugs diluted in saline (CS+A-50 mg/kg; CS+M-200 mg/kg/day; CS+N-200 mg/kg/day). Control groups were sham-smoked (AA). Long-term CS exposure results in extensive parenchyma destruction in CS group. Both CS+N and CS+M groups showed preserved alveolar structure and showed preserved lung function when compared to CS group. Macrophage and neutrophil counts were decreased in CS+M, and CS+N groups when compared to CS group (p<0.05). Antioxidant enzyme activities were reduced in all treated groups. CS+A showed the highest reduction in catalase activity (-25%, p<0.01). We conclude that M treatment reduced long-term CS-induced inflammatory lung parenchyma destruction and lung function, comparable to N treatment, however, antioxidant administration did not reverse CS-induced antioxidant enzyme activity reduction.     

Multi-step lung carcinogenesis model induced by oral administration of N-nitrosobis(2-hydroxypropyl)amine in rats

N-Nitrosobis(2-hydroxypropyl)amine (BHP) was first synthesized by Krüger et al. (1974), and has been shown to primarily induce pancreatic duct adenocarcinomas by a subcutaneous injection in Syrian hamsters. By contrast, the carcinogenic effect of BHP has been indicated at the different target organs in rats, namely the lung. When rats are received by an oral administration of BHP in drinking water for 25 weeks, a high incidence of lung carcinomas are induced, which include adenocarcinomas, squamous cell carcinomas and combined squamous cell and adenocarcinomas. So many similarities are observed in terms of not only histological appearances but also gene alterations between human and BHP-induced rat lung cancers. Moreover, the step by step development of lung lesions, from preneoplastic lesions to cancers in rat lung carcinogenesis by BHP offers a good model to investigate the mechanisms underlying the pathogenesis of lung cancers. Because data for genetic and epigenetic alterations have indeed been accumulated during the BHP-induced rat lung carcinogenesis, we will introduce them in this review and hence demonstrate that this lung carcinogenesis model provides a useful opportunity for the research on the pathogenesis of lung cancers of both humans and rats.     

Absence of significant Th2 response in diabetes-prone non-obese diabetic (NOD) mice.

Accumulating evidence suggests that Th1 T cells play a pivotal role in the development of autoimmune diabetes. Conversely, promoting a Th2 response inhibits disease progression. However, it has not been determined whether Th2 cells are regulatory T cells that fail at the time of diabetes development in naive non-diabetic NOD mice. Therefore, in order to evaluate cytokine secretion by spleen and islet infiltrating T cells in NOD mice at different stages of the autoimmune process, we developed an ELISPOT assay that detects IL-2, IL-4, and interferon-gamma (IFN-gamma) secretion in vitro at the single-cell level. We showed that, whatever the age considered, IFN-gamma is predominantly secreted, and that no IL-4-secreting cells are detected in the islets of male and female NOD mice. Spleen cells from 8-week-old female NOD mice, which include regulatory suppressor T cells, do not secrete IL-4, either upon presentation of islet cell antigens in vitro, or after transfer in vivo, but do secrete IFN-gamma. IFN-gamma secretion by T cells from diabetic mice results from CD4 but not CD8 T cells in transfer experiments into NOD/severe combined immunodeficient (SCID) recipients. These results suggest that (i) detection of regulatory CD4 T cells in NOD mice is not paralleled by a Th2 response; (ii) beta cell destruction does not depend on a switch from a Th2 to a Th1-type response; and (iii) CD8 T cells do not participate in induction of diabetes by secreting IFN-gamma.     

Anchored polymerase chain reaction based analysis of the Vβ repertoire in the non-obese diabetic (NOD) mouse

We have performed extensive analyses of T cell receptor Vβ usage in the thymus, the spleen and the infiltrated islets of preclinical non-obese diabetic (NOD) mice. A semiquantitative anchored polymerase chain reaction (An-PCR) protocol has been developed for this purpose. The validity of the method has been first assessed by antibody staining with a panel of anti-Vβ monoclonal antibodies (mAb). The results obtained by An-PCR are accurate, reproducible, and in good agreement with cell surface protein staining. A strict comparison between thymus and spleen repertoires reveals no major Vβ-specific deletion except the already reported Vβ3 deletion due to Mtv-3. Certain Vβ such as Vβ15, 18, 20 are found with a low frequency in the spleen, but the fact that they are also scarce in the thymus probably reflects a poor availability of these genetic elements during β chain rearrangement rather than negative selection. Other Vβ, such as Vβ2, Vβ12 and Vβ14 are significantly more abundant in the spleen than in the thymus. This finding was confirmed by mAb staining for Vβ2 and Vβ14. The expansion asymetrically affects the CD4⁺ subset and can be traced back to the mature, single-positive thymocyte subset, suggesting an intrathymic positive selection event. Vβ repertoires in infiltrated islets of 13- and 18-week-old, non-diabetic mice are polymorphic. Practically all the Vβ found in the peripheral lymphoid tissues are present in the islets, in similar proportions. The major exception is Vβ12, one of the Vβ which is subject to expansion during intrathymic differentiation and which is further augmented in the islets, both at 13 and 18 weeks. This increase probably reflects further peripheral amplification of the Vβ12-bearing subset due to encounter with the same ligand as in the thymus or with a cross-reactive motif. Finally, the nucleotide sequencing of all the Vβ segments in usage in the NOD strain confirms the absence of allelic polymorphism of Vβ-coding regions.