Combination of fluticasone propionate and salmeterol potentiates the suppression of cigarette smoke-induced IL-8 production by macrophages

Cigarette smoke is the major risk factor for the development of chronic obstructive pulmonary disease (COPD). Macrophages are suggested to orchestrate the chronic inflammatory response and tissue destruction associated with COPD by secreting interleukin (IL)-8, a major neutrophil chemoattractant. The combination of inhaled corticosteroids and long-acting beta(2)-adrenoceptor agonists are increasingly used as maintenance therapy in patients with COPD. The aim of this study was to determine whether combined fluticasone propionate, a corticosteroid, and salmeterol, a long-acting beta(2)-adrenoceptor agonist, can suppress IL-8 production by human macrophages. To mimic resident macrophages in the lung, human monocytes were cultured for 5 days in medium containing Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF). In human Monocyte-Derived Macrophages, we found that cigarette smoke medium strongly enhanced IL-8 release in a time- and concentration-dependent manner. IL-8 release by cigarette smoke was significantly suppressed in a concentration-dependent manner by fluticasone and salmeterol. Coincubation of the drugs potentiated the inhibitory effect on cigarette smoke medium-induced IL-8 production and longer preincubation times resulted in more IL-8 inhibition. Interestingly, preincubation of cells with suboptimal concentration of salmeterol for 4 h before fluticasone administration for 30 min potentiates the inhibitory effect of fluticasone on IL-8 release. In conclusion, combination therapy may provide benefits over monotherapy for the treatment of COPD patients.     

Expression of recombinant parvalbumin from wolf-herring fish and determination of its IgE-binding capability

In this study, we produced the recombinant form of parvalbumin from wolf-herring fish and determined its IgE reactivity. Parvalbumin cDNA was sub-cloned into pET28 and expressed in Escherichia coli BL-21. The immunoreactivities of the recombinant and native parvalbumins were compared, and the effect of calcium binding was determined by sera from 25 fish-allergic patients. ELISA and Western blotting confirmed similar IgE-reactivities of the recombinant and native proteins and confirmed that this phenomenon is highly dependent on calcium binding. The recombinant protein was 94.5% similar to carp parvalbumin (Cyp c1). Approximately 72% of patients reacted strongly with recombinant parvalbumin, 80% of them reacted with the native form and only 56% showed IgE reactivity with crude extract. Because the IgE-binding capacity of recombinant wolf-herring parvalbumin is retained and is highly similar to Cyp c1, the wild and hypoallergenic forms of this allergen could be used for diagnosis and immunotherapy of fish allergy, respectively.     

A randomized trial to promote health belief and to reduce environmental tobacco smoke exposure in pregnant women

Exposure to environmental tobacco smoke (ETS) is widespread among women in Iran. This study aimed to explore the impact of education on health belief and environmental tobacco smoke exposure in pregnant women. This randomized trial was administrated to 130 pregnant women exposed to ETS. The face-to-face education was provided for the intervention group after completing the questionnaire compiled on the constructs of the health belief model and self-reports of weekly ETS exposure. The theoretical constructs and weekly ETS exposure were compared in the study groups at the intake, third, fourth and fifth sections. In the intervention group, perceived susceptibility/severity and perceived benefits increased and the weekly ETS exposure decreased on the third as opposed to the first section (P < 0.05). Perceived susceptibility/severity and benefits significantly correlated with weekly ETS exposure in the intervention group (P < 0.05). The findings of this study point to the fact that education about the impacts of ETS exposure of pregnant women is an effective way to increase the theoretical constructs according to the health belief model and is associated with a reduction of ETS exposure. But this is not sufficient for making smoke-free homes.     

In vitro differences between venous and arterial-derived smooth muscle cells: potential modulatory role of decorin

OBJECTIVE: We analyzed the phenotypic and functional differences between venous and arterial smooth muscle cells (SMC) and the role of decorin in modulating these differences. METHODS AND RESULTS: SMC were isolated from the jugular veins and carotid arteries of male white New Zealand rabbits. Venous SMC demonstrated increased proliferation (2-fold, p<0.001), migration (1.7-fold, p<0.001), and collagen synthesis (4-fold, p<0.001), with decreased adhesion to collagen and fibronectin (1.2-fold, p<0.01) compared to arterial SMC. Higher levels of gelatinase activity (MMP-2 and MMP-9) and tissue inhibitor of metalloproteinase (TIMP) were also observed in venous SMC. Venous SMC demonstrated increased expression of SMemb and decreased expression of SM1--markers of a dedifferentiated and differentiated phenotype, respectively. Arterial SMC produced increased levels of the inhibitory proteoglycan, decorin, compared to venous SMC. Conditioned medium from arterial SMC (ASMC-CM) significantly decreased DNA synthesis, collagen synthesis, and gelatinase activity in venous SMC. Removal of decorin from ASMC-CM by immunoprecipitation significantly reversed the inhibitory effects of ASMC-CM on venous SMC proliferation and collagen synthesis but did not affect gelatinase activities. CONCLUSION: Venous SMC are more dedifferentiated and demonstrate increased proliferative and synthetic capacity than arterial SMC. Differential decorin expression between arterial and venous SMC contributes to these differences in biologic behavior. Venous SMC properties may contribute to accelerated atherosclerosis in venous bypass grafts.     

Arterial repair after stenting and the effects of GM6001, a matrix metalloproteinase inhibitor

OBJECTIVES: This study compared the extracellular matrix (ECM) and cellular responses after stenting to balloon angioplasty (BA) and to determine the late effects of matrix metalloproteinase (MMP) inhibition on arterial repair after stenting. BACKGROUND: Although stenting is the predominant form of coronary intervention, there is limited understanding of the early and late arterial response. METHODS: In a double-injury rabbit model, adjacent iliac arteries in 87 animals received BA (3.0 mm diameter) or stenting (3.0 mm NIR). Rabbits were treated for 1 week postprocedure with either GM6001 (100 mg/kg per day), an MMP inhibitor or placebo and sacrificed at 1 week or at 10 weeks' postprocedure. Arteries were analyzed for morphometry, collagen content, gelatinase activity, cell proliferation and DNA content.RESULTS: Stented arteries had significant increases in collagen content (2-fold) at 10 weeks compared to BA-treated arteries. At one week, overall gelatinase activity was increased >2-fold in stented arteries, with both 72 kD and 92 kD gelatinase activity. Stented arteries also had increases in both intimal DNA content (1.5-fold) and absolute cell proliferation (4-fold). Compared to placebo, GM6001 significantly inhibited intimal hyperplasia and intimal collagen content, and it increased lumen area in stented arteries without effects on proliferation rates. CONCLUSIONS: Stenting causes a more vigorous ECM and MMP response than BA, which involves all layers of the vessel wall. Inhibition by MMP blocks in-stent intimal hyperplasia and offers a novel approach to prevent in-stent restenosis.     

Leishmania major heat shock protein 70 (HSP70) is not protective in murine models of cutaneous leishmaniasis and stimulates strong humoral responses in cutaneous and visceral leishmaniasis patients

Heat shock proteins (HSP) are highly conserved molecules that play important roles in protein folding, assembly of protein complexes and translocation of proteins across cellular compartments, as well as in several immunological processes. In this study, we first immunized susceptible BALB/c and resistant C57BL/6 mice with the complete open-reading frame of Leishmania HSP-70 (pcDNA-HSP70) and boosted mice with rHSP-70 (amino acid 221-604 cloned in pQE-HSP70 and referred to as rHSP70) mixed with Montanide 720. When we evaluated the effects of HSP70 in both mouse strains, we found that the entire fragment (amino acids 221-604) and rCT-HSP70 (amino acids 491-604 cloned in pQE-CT), but not rNT-HSP70 (amino acids 221-291 cloned pQE-NT), contained the highest immunogenicity. However, after infectious challenge with Leishmania major, no efficient protective responses were observed in either mouse strain. The humoral immune responses against the different truncated forms of HSP70 suggested a mixed TH1/TH2 response in vivo. We then assessed infected susceptible and resistant mice for lymphoproliferative and cytokine responses against the truncated forms of HSP70. At 9-week post-infection, we observed no differences in those responses between vaccinated and control mice. Next, we initiated comparative studies in human patient samples, finding no significant proliferation against all three truncated forms of HSP70 in the cellular immune responses of 16 cured cutaneous leishmaniasis patients and 5 normal individuals. Sera from active cutaneous and visceral leishmaniasis patients, however, were reactive to all three forms of HSP70. This study demonstrates the potential of HSP70 in stimulating humoral responses in humans and mice and indicates there is a need to further explore and examine the value of this important molecule in the control of leishmaniasis.     

Acetylsalicylic acid-induced release of HSP70 from mast cells results in cell activation through TLR pathway

OBJECTIVE: Mast cells are considered major players in IgE-mediated allergic responses, but have also recently been recognized as active participants in innate as well as specific immune responses. Heat stress can modulate innate immunity by inducing stress proteins such as heat shock proteins (HSPs). It has been reported that HSPs are capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system. In the current study, we explored whether the stress response induces HSPs and affects the signaling pathways of mast cells. METHODS: In mouse mast cells, derived from a culture of bone marrow cells of male BALB/cBy and null HSF-1(-/-) mice, responsiveness to exogenous and endogenous HSP70 was monitored by measuring cytokine release. RESULTS: Using BMMC, we show that treatment with heat shock or acetylsalicylic acid results in a selective induction of HSPs, and leads to release of HSP70 into the extracellular environment. The release of HSP70 from mast cells may be of functional importance. We found that after induction of HSP70, the production of TNF-alpha and IL-6 was increased. In a number of experiments, we demonstrated that exogenous/secreted HSP70 is most likely responsible for the activation of mast cells to produce cytokines. Extracellular HSP70 induced production of TNF-alpha and IL-6 through the activation of the TLR4 receptor pathway, which was evidenced by an abrogation of the response in mast cells cultured from TLR4(null) or HSF-1(-/-) mice. CONCLUSION: Our experiments suggest that stress conditions can induce pro-inflammatory cytokine production by mast cells through an autocrine or paracrine stimulation of TLR receptors after a heat shock response. The recognition that heat shock proteins induce mast cell activation suggests an involvement of these cells in the immunological processes induced by heat shock response.