BACKGROUND

To prospectively investigate patients with seasonal allergic conjunctivitis (SAC) during the pollen season and test associations between tears total IgE, eotaxin concentrations, and SAC severity.

METHODS

Enrolled patients presented ocular symptoms and clinical signs of SAC at the time of presentation. Ocular itching, hyperaemia, chemosis, eyelid swelling, and tearing were scored, and the sum of these scores was defined as the clinical score. Conjunctival papillae were separately graded. We measured eotaxin concentration in tears by an enzyme-linked immunosorbent assay (ELISA) and total tear IgE by Lacrytest strip.

RESULTS

Among thirty patients (30 eyes), 11 showed neither tear IgE nor tear eotaxin, while 15 out of 19 patients with positive IgE values presented a positive amount of eotaxin in their tears (Fisher's test: p < 0.001). The mean eotaxin concentration was 641 ± 154 (SEM) pg/ml. In patients with no amount of tear IgE, we observed a lower conjunctival papilla grade than in patients whose tears contained some amount of IgE (trend test: p = 0.032). In the 15 patients whose tear eotaxin concentration was null, tear IgE concentration was 5.3 ± 3.5 arbitrary units; in the other 15 patients whose eotaxin was positive, IgE reached 21 ± 4.3 arbitrary U (Mann-Whitney: p < 0.001). We measured 127 ± 47 pg/ml eotaxin in patients with no history of SAC but newly diagnosed as suffering from SAC, and 852 ± 218 pg/ml eotaxin in patients with a known SAC (p = 0.008). In contrast, tear IgE concentrations of both groups did not differ statistically significantly (p = 0.947).

CONCLUSIONS

If IgE and eotaxin secreted in tears are major contributors in SAC pathogenesis, they however act at different steps of the process.

OBJECTIVE

To analyze the role of chemokines and their receptors in the mechanism of allergic rhinitis (AR) by observing the expression of genes of chemokines and their receptors in nasal mucosa of AR through gene chip.

METHODS

The total RNAs were isolated from nasal mucosa of AR and purified to mRNAs, then reversely transcribed to cDNAs and incorporated with fluorescent-labled CY5-dUPT for probes preparion. CY3-dUTP probes prepared with normal nasal mucosa of normal for control. The chip contains cDNAs of chemokines and their receptors were used to hybridized with probes then screened with computer to study the expression of genes based on different density of fluorescent.

RESULTS

The chemokines of CCL11 (eotaxin-1), CCL24 (eotaxin-2), CCL7 (MCP-3), CCL13 (MCP-4), RANTES (CCL5) and receptors of CCR2, CCR3, CCR4, CCR5 were differential expressed on four samples, and most of the chemokines and their receptors has tendency regulation on T help 2 (Th2) lymphocytes and involved in the prodeeds such as inducement of allergic reaction,accumulation of inflammacytes and degranulation of sensitized cells.

CONCLUSIONS

A disorder exists in T helper immune system with AR. The chemokines and their receptors that polarized with Th2 lymphocytes perhaps play important roles in AR pathogenesis, and it represents a new approach to AR immunotherapy.

BACKGROUND

Eosinophil-associated RNases (EARs) are stored preformed in eosinophil cytoplasmic secretory granules and have a key role in eosinophil effector functions in host defence and inflammatory disorders. However, the secretion mechanisms of EARs are poorly understood.

OBJECTIVE

Our study aimed to understand the involvement of cytoskeleton machinery in EAR secretion.

METHODS

Fresh human and mouse eosinophils were stimulated with CCL11, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of cytoskeletal elements or microtubules was probed using specific inhibitors.

RESULTS

We found that dynamic polymerization of microtubules and cytoskeletal elements, such as Rho and Rac, is required for chemokine-mediated EAR secretion from human and mouse eosinophils. However, inhibition of ROCK (Rho-associated protein kinase) increased EAR secretion in human and mouse eosinophils even in the absence of chemokine stimulation, suggesting ROCK negatively regulates EAR secretion.

CONCLUSIONS

Collectively, these data suggest a cytoskeleton-dependent mechanism of EAR secretion from eosinophils, findings that are pertinent to host defence, allergy and other eosinophil-associated diseases.

Background: Neuropsychiatric disorders have been linked to immune mechanisms. Altered peripheral levels of eotaxin-1/CCL11; a cytokine implicated in allergic reactions and aging process; have been reported in bipolar disorder (BD). Several brain areas, especially the temporal lobe, seem to display volume loss and accelerated aging in BD. This study aimed at exploring potential associations between eotaxins and brain volumes in patients with BD compared to controls.

Methods: Twenty-two euthymic patients with BD and 22 controls were enrolled in this study. Serum levels of eotaxin-1/CCL11, eotaxin-2/CCL24 and eotaxin-3/CCL26 were determined alongside brain volumes.

Results: There were no differences in the levels of eotaxins between patients and controls. A negative correlation was found between eotaxin-1/CCL11 levels and left-hemisphere's superior-temporal volume only in BD patients, which persisted with covariate adjusted model.

Conclusion: This study corroborates the emerging evidence of association between inflammation and brain volumes in BD. Our preliminary results also support the hypothesis of a possible role of eotaxin-1/CCL11 in accelerated brain aging in BD.

Keywords: Accelerated aging; Bipolar disorder; CCL-11; Eotaxin; Superior-temporal volume.

BACKGROUND

Eosinophilia is a typical finding of the acute/juvenile form of paracoccidioidomycosis (PCM), a systemic mycosis endemic in Latin America. This clinical form is characterized by depressed cellular immune response and production of Th2 cytokines. Moreover, it has been shown that the increased number of eosinophils in peripheral blood of patients returns to normal values after antifungal treatment. However, the role of eosinophils in PCM has never been evaluated. This study aimed to assess the phenotypic and functional characteristics of eosinophils in PCM.

RESULTS

In 15 patients with the acute form of the disease, we detected expression of MBP, CCL5 (RANTES) and CCL11 (eotaxin) in biopsies of lymph nodes and liver. In addition, there were higher levels of chemokines and granule proteins in the peripheral blood of patients compared to controls. Isolation of eosinophils from blood revealed a higher frequency of CD69+ and TLR2+ eosinophils in patients compared to controls, and a lower population of CD80+ cells. We also evaluated the fungicidal capacity of eosinophils in vitro. Our results revealed that eosinophils from PCM patients and controls exhibit similar ability to kill P. brasiliensis yeast cells, although eosinophils of patients were less responsive to IL-5 stimulation than controls.

UNASSIGNED

In conclusion, we suggest that eosinophils might play a role in the host response to fungi and in the pathophysiology of PCM by inducing an intense and systemic inflammatory response in the initial phase of the infection.

Inappropriately activated eosinophils can contribute to disease pathogenesis and intracellular signalling pathways that regulate functional responses may represent a therapeutic target. Little is known about intracellular signalling in equine eosinophils and this study examined the role of phospholipase C (PLC) and a range of protein kinases on responses to histamine and CCL11. Histamine (10(-4) M) or CCL11 (5.6 x 10(-9) M)-induced actin polymerization, migration and superoxide production by eosinophils from healthy horses were compared in the presence and absence of selective kinase inhibitors. Inhibition of phosphatidylinositol-3 kinase (PI3K) significantly reduced the response in each assay. In contrast, whilst inhibition of PLC decreased actin polymerization and superoxide production, an increase in migration was observed; the latter effect was also seen when protein kinase C (PKC) was inhibited. With the exception of histamine-induced migration, which was significantly reduced by blocking extracellular regulated kinase (ERK)1/2, activation of ERK1/2, p38 MAPK and tyrosine kinase did not appear to play an important role in the responses studied. These results suggest that equine eosinophil activation by histamine and CCL11 is mediated through PI3K. Whilst PLC activation is required for actin polymerization and superoxide production, migration may be negatively regulated by PLC and PKC. These kinases represent potential targets for modulating eosinophil activation by multiple stimuli.

Extracellular acidification has been observed in allergic inflammatory diseases. Recently, we demonstrated that the proton-sensing receptor G protein-coupled receptor 65 (GPR65) regulates eosinophil survival in an acidic environment in vitro and eosinophil accumulation in an allergic lung inflammation model. For mast cells, another inflammatory cell type critical for allergic responses, it remains unknown whether GPR65 is expressed and/or regulates mast cell viability. Thus, in the present study, we employed in vitro experiments and an intestinal anaphylaxis model in which both mastocytosis and eosinophilia can be observed, particularly in the gastrointestinal tract, to enable us to directly compare the effect of GPR65 expression on these two cell types. We identified GPR65 expression on mast cells; however, unlike eosinophil viability, mast cell viability in vitro is not affected by acidification or GPR65 expression. Mechanistically, we determined that mast cells do not respond to extracellular acidification with increased cAMP levels. Furthermore, in the intestinal anaphylaxis model, we observed a significant reduction of eosinophils (59.1 ± 9.2% decrease) in the jejunum of allergen-challenged GPR65-deficient mice compared with allergen-challenged wild-type mice, despite the degree of antigen sensitization and the expression levels of Th2 cytokines (Il4, Il13) and eosinophil chemokines (Ccl11, Ccl24) in the jejunum being comparable. In contrast, the accumulation of mast cells in allergen-challenged mice was not affected by GPR65 deficiency. In conclusion, our study demonstrates differential regulation of eosinophils and mast cells in inflammatory tissue, with mast cell viability and accumulation being independent of GPR65.

Objective: To examine changes in plasma levels of CCL11, CCL2, and IL-10 after 10 controlled soccer headers.

Setting: Laboratory setting.

Participants: Thirty-nine healthy soccer players with at least 3 years of soccer heading experience, between 18 and 26 years old, and enrolled at a large public university.

Design: In this randomized clinical trial using a soccer heading model, participants were randomized into the heading (n = 22) or kicking-control (n = 17) groups to perform 10 headers or kicks.

Main measures: Plasma levels of CCL11, CCL2, and IL-10 at preintervention and 0, 2, and 24 hours postintervention.

Results: Mixed-effects regression models did not reveal any significant group differences in changes of plasma CCL11, CCL2, or IL-10 levels from preintervention. Within the heading group, there was a statistically significant time by years of heading experience interaction with 2.0-pg/mL increase in plasma CCL11 each year of prior experience at 24 hours postintervention (P = .001).

Conclusion: Findings from this study suggest that 10 soccer headers do not provoke an acute inflammatory response. However, the acute CCL11 response may be influenced by prior exposure to soccer headers, providing a precedent for future field studies that prospectively track head impact exposure and changes in CCL11.

Mesocestoides vogae is a cestode parasite of the family Mesocestoididae (order Cyclophyllidea). Its larvae, tetrathyridium, are approximately 1 mm long and 300 μm wide and infect a wide range of host species including humans. Tetrathyridium migrate through the intestinal wall to invade the peritoneal cavity. Despite intestinal penetration by such a large-sized parasite, symptomatic intestinal disorders are not common during the migration period. In this study, the dynamics of tetrathyridia migration and their pathogenicity towards intestinal tissues were examined in mice infected orally with these parasites. Most tetrathyridia were found to migrate through the intestinal wall, moving into the peritoneal cavity or liver 24 to 48 hours after the oral infections. Next, the pathogenicity of tetrathyridium in the intestinal wall was histopathologically evaluated, and tissue injury from tetrathyridium migration was confirmed. Inflammatory foci were observed as tetrathyridium migration tracks from 48 hours after oral infection; however, the number of inflammatory foci had decreased by half more than 48 hours later. Therefore, we examined the gene expression levels of the macrophage driving cytokine, IL-1β, and the eosinophil recruiting chemokine, CCL11, by quantitative reverse-transcriptase PCR. The expression levels of these genes in the infected group were significantly lower than those of the non-infected group at 48 hours post-infection. Although the immunomodulating ability of the excretory-secretory products released from tetrathyridium has been previously shown by in vitro assays, the significance of this ability in their lifecycle has remained unclear. In this study, we discovered that tetrathyridium causes temporal inflammation in the intestinal wall during penetration and large-scale migration in this organ, but tetrathyridium simultaneously suppresses the host's inflammatory gene expression, might to be a strategy that reduces inflammatory responses and increases survival of the parasite.

The immune response after transcatheter aortic valve implantation (TAVI) in comparison to that after surgical aortic valve replacement (SAVR) remains to be fully elucidated. In a 2-part study, we assessed laboratory data obtained before, immediately after, and 24 and 48 hours after SAVR (128 patients; age ≥80 [mean 82] years) or transfemoral TAVI (102 patients; age ≥80 [mean 86] years) performed for aortic stenosis. In-hospital mortalities were similar (3% vs 0%), but leukocyte counts and aspartate aminotransferase and creatine kinas concentrations were decreased immediately and 24 hours after surgery (all, p <0.001). We performed cytokine profiling in a SAVR group (11 patients; mean age, 77 years) and transfemoral TAVI group (12 patients; mean age, 84 years). By measuring normalized concentrations of 71 cytokines at 3 time points, we found a significant difference (defined as fold change >1.7 and p <0.05 [by Mann-Whitney U-test]) in 23 cytokines. The differentially expressed cytokines fell into 3 hierarchical clusters: cluster A (high increase after SAVR and suppressed increase after TAVI only immediately after surgery [CCL2, CCL4, and 2 others]), cluster B (high increase after SAVR and suppressed increase after TAVI at 2 time points [IL-1Ra, IL-6, IL-8, IL-10, and 5 others]), and cluster C (various patterns [TRAIL, CCL11, and 8 others]). Gene enrichment analysis identified multiple pathways associated with the inflammatory responses in SAVR and altered responses in TAVI, including cellular responses to tumor necrosis factor (p = 0.0035) and interleukin-1 (p = 0.0062). In conclusion, a robust inflammatory response follows SAVR, and a comparatively attenuated response follows TAVI.

Background: Early detection and prognostic prediction are crucial in improving the survival of patients with head and neck squamous cell carcinoma (HNSCC). Therefore, we provided potential molecular markers in this study for early diagnosis and prognosis of this cancer based on the The Cancer Genome Atlas (TCGA) database analysis and experimental validations.

Methods: Differentially expressed genes (DEGs) between HNSCC tumor and normal samples were identified by TCGA database-based analyses. Univariate and multivariate Cox regression analyses was applied respectively to identify survival-related DEGs and independent prognostic factors in HNSCC. Further, RT-qPCR was employed to verify expression of DEGs in cancer and adjacent tissues from HNSCC patients recruited in our hospital, in which we also clarified the correlation between candidate genes and clinicopathological characteristics and prognosis of HNSCC patients.

Results: TCGA data analyses yielded 59 DEGs. Cox analyses identified 13 candidate genes closely related to prognosis of HNSCC patients and established a five-gene signature comprising AC103702.2, LINC00941, RPL29, FOXL2 and CCL11. This five-gene signature could classify patients into high- and low-risk groups. The survival rate of the high-risk group was significantly lower than that of the low-risk group. Clinical tissue experiments further confirmed that AC103702.2, LINC00941, CCL11 and RPL29P19 genes were inversely associated with the prognosis of HNSCC patients, while CCL11 gene was positively associated. We also found that high-risk HNSCC patients presented a higher incidence of lymph-node metastasis.

Conclusion: Five prognostic marker genes (AC103702.2, LINC00941, CCL11, RPL29P19 and FOXL2) as a gene cluster may serve as prognostic marker genes in HNSCC.

Keywords: Differential gene analysis; Functional enrichment analysis; Head and neck squamous cell carcinoma; Kaplan-Meier survival curve; Pearson correlation coefficient; Prognostic marker genes; Prognostic risk assessment model; ROC curve; TCGA database; Univariate Cox analysis.

Background: The C-C motif chemokine ligand 11 (CCL11) has been receiving attention as a potential pro-aging factor. Accordingly, it may be involved in muscle metabolism and sarcopenia, a key component of aging phenotypes. To clarify this potential, we investigated the effects of CCL11 on in vitro muscle biology and its clinical relevance for sarcopenia parameters in older adults.

Methods: Myogenesis was induced in mouse C2C12 myoblasts with 2% horse serum. Human blood samples were collected from 79 participants who underwent a functional assessment. Thereafter, CCL11 level was measured using a quantikine ELISA kit. Sarcopenia was defined using the Asian-specific guideline.

Results: Recombinant CCL11 treatment significantly stimulated myogenesis in a dose-dependent manner, and consistently increased the expression of myogenic differentiation markers. Among the C-C chemokine receptors (CCRs), CCR5, not CCR2 and CCR3, was predominantly expressed in muscle cells. Further, the CCR5 inhibitor blocked recombinant CCL11-stimulated myogenesis. In a clinical study, serum CCL11 level was not significantly different according to the status of sarcopenia, low muscle mass, weak muscle strength, and poor physical performance, and was not associated with skeletal muscle index, grip strength, short physical performance battery score, gait speed, and time to complete 5 chair stands, after adjusting for sex, age, and body mass index.

Conclusion: Contrary to expectations, CCL11 exerted beneficial effects on muscle metabolism at least in vitro system. However, its impact on human muscle health was not evident, suggesting that circulating CCL11 may not be a useful biomarker for sarcopenia risk assessment in older adults.

Keywords: Aging; Biomarkers; Chemokine CCL11; Muscle development; Sarcopenia.

BACKGROUND

The aim of this study was to evaluate the modulatory effect of S-allyl cysteine against cyclophosphamide-induced changes in uroplakin IIIa, CCL11 and TNF-α.

METHODS

Mice were treated with cyclophosphamide (200mg/kg×7 d, ip). S-allyl cysteine (150mg/kg×7d, ip), and comparator compound mesna (40mg/kg×7d, ip) were administered 1h before and 4h after each cyclophosphamide dose. The urinary bladder was analysed for mRNA and protein changes in uroplakin IIIa (UPIIIa), CCL11 and TNF-α and histopathological findings.

RESULTS

Cyclophosphamide caused hemorrhagic cystitis formation and downregulation of UPIIIa. These changes were accompanied by upregulation of CCL11 and TNF-α. S-allyl cysteine attenuated these changes including protection at histological level. Mesna which was used as a comparator drug also showed protection. However, relatively S-allyl cysteine showed a stronger protective effect than mesna.

CONCLUSIONS

These findings highlight a correlation between downregulaion of UPIIIa and enhanced production of inflammatory biomarkers and protective effects of S-allyl cysteine which has been reported to be a potent uroprotective agent. The present study strengthens its role which could be clinically exploited in chemotherapy regimen.

BACKGROUND

Expression of murine calcium-activated chloride channel family member 3 (mCLCA3) has been reported to be increased in the airway epithelium of asthmatic mice challenged with ovalbumin (OVA). However, its role in asthmatic airway inflammation under no OVA exposure has not yet been clarified.

METHODS

mCLCA3 plasmids were transfected into the airways of normal BALB/c mice. mCLCA3 expression and airway inflammation in mouse lung tissue were evaluated. Cell differentials and cytokines in bronchoalveolar lavage fluid (BALF) were analyzed. The expression of mCLCA3 protein and mucus protein mucin-5 subtype AC (MUC5AC) were analyzed by Western blotting. The mRNA levels of mCLCA3, MUC5AC and interleukin-13 (IL-13) were determined quantitatively.

RESULTS

mCLCA3 expression was not detected in the control group while strong immunoreactivity was detected in the OVA and mCLCA3 plasmid groups, and was strictly localized to the airway epithelium. The numbers of inflammatory cells in lung tissue and BALF were increased in both mCLCA3 plasmid and OVA groups. The protein and mRNA levels of mCLCA3 and MUC5AC in the lung tissue were significantly increased in the mCLCA3 plasmid and OVA groups compared to the control group. The level of IL-13, but not IL-4, IL-5, IFN-γ, CCL2, CCL5 or CCL11, was significantly increased compared with control group in BALF in the mCLCA3 plasmid and OVA groups. The level of IL-13 in the BALF in the mCLCA3 plasmid group was much higher than that in the OVA group (P < 0.05). The level of mCLCA3 mRNA in lung tissue was positively correlated with the levels of MUC5AC mRNA in lung tissue, IL-13 mRNA in lung tissue, the number of eosinophils in BALF, and the content of IL-13 protein in BALF. The level of IL-13 mRNA in lung tissue was positively correlated with the number of eosinophils in BALF and the level of MUC5AC mRNA in lung tissue.

CONCLUSIONS

These findings suggest that increased expression of a single-gene, mCLCA3, could simulate an asthma attack, and its mechanism may involve mCLCA3 overexpression up-regulating IL-13 expression.

BACKGROUND

Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN), are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN binding to eosinophil-recruiting chemokines.

METHODS

Antibacterial activities of the chemokines were investigated using viable count assays and electron microscopy. Binding studies were performed by means of surface plasmon resonance. The potential interference of OPN with antibacterial, receptor-activating, and lipopolysaccharide-neutralizing abilities of these chemokines was investigated.

RESULTS

We found that OPN bound all eosinophil-recruiting chemokines with high affinity except for CCL5. The eosinophil-recruiting chemokines all displayed bactericidal activity against S. pneumoniae, but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride at physiologic concentrations. Preincubation of the chemokines with OPN strongly inhibited their antibacterial activity against S. pneumoniae but did not affect their ability to activate CCR3. All chemokines investigated showed LPS-neutralizing activity that was impaired by OPN only in the case of CCL24.

CONCLUSIONS

The data suggest that OPN may impair host defense activities of the chemokines without affecting their eosinophil-recruiting properties. This could be one mechanism explaining the increased vulnerability to acquire pneumococcal infection in parallel with sustained allergic inflammation in asthma.

Pro-inflammatory alterations of white adipose tissue (WAT) with increasing age play an important role in mammalian aging. WAT produced eotaxin-1 [CCL11--C-C motif chemokine ligand 11) and MCP-1 [CCL2 (C-C motif chemokine ligand 2] are elevated in old mammals. Obese and old adipose tissue produce excessive pro-inflammatory cytokines such as IL-6, CCL2 and IL1-beta that contribute to inflammaging. WAT-based inflammaging involves an altered homeostatic equilibrium between pro-inflammatory cells such as activated Type 1 macrophages, B (high IgJ) and T cells and anti-inflammatory eosinophils and Tregs. Specifically, young and lean individuals exhibit a high eosinophil-to-macrophage ratio with an enrichment of alternative activated tissue macrophages which is reduced in the WAT of aging mice. Eosinophils from young animals adoptively transferred to old mice, home to WAT and reverse many of the immunoinflammatory signatures associated with aging. Whether eosinophil-based therapies for inflammaging could be created remains an open question.

Eotaxin/CCL-11 is a major chemoattractant that contributes to eosinophilic inflammation in asthma. Glucocorticoids inhibit inflammation, but long-time exposure may cause paradoxical adverse effects by augmenting eotaxin/CCL-11production. The aim of this study was to determine if 7,4'-dihydroxyflavone (7,4'-DHF), the eotaxin/CCL11 inhibitor isolated from Glycyrrhiza uralensis, reduces in vitro eotaxin production induced by long-time dexamethasone (Dex) exposure, and if so, to elucidate the mechanisms of this inhibition. Human lung fibroblast-1 cells were used to identify the potency of 7,4'-DHF compared with other compounds from G. uralensis, to compare 7,4'-DHF with Dex on eotaxin production following 24-h short-time culture and 72-h longer-time (LT) culture, and to determine the effects of the 7,4'-DHF on Dex LT culture augmented eotaxin production and molecule mechanisms. 7,4'-DHF was the most potent eotaxin/CCL-11 inhibitor among the ten compounds and provided continued suppression. In contrast to short-time culture, Dex LT culture increased constitutively, and IL-4/TNF-α stimulated eotaxin/CCL11 production by human lung fibroblast-1 cells. This adverse effect was abrogated by 7,4'-DHF co-culture. 7,4'-DHF significantly inhibited Dex LT culture augmentation of p-STAT6 and impaired HDAC2 expression. This study demonstrated that 7,4'-DHF has the ability to consistently suppress eotaxin production and prevent Dex-paradoxical adverse effects on eotaxin production. Copyright © 2017 John Wiley & Sons, Ltd.

Eotaxin-1 (CCL11), an eosinophil-specific C-C chemokine, is a potent chemoattractant for mobilization of eosinophils into airways after allergic stimulation. Eotaxin-1 recruits eosinophils into inflammatory sites, and may play a role in the pathogenesis of asthma. Formoterol and salmeterol are two inhaled long acting beta(2) adrenoceptor agonists (LABAs), widely used for the local treatment of asthma. However, little is known about their effects on the eotaxin-1 expression of bronchial epithelial cells. BEAS-2B cells were stimulated by adding IL-4 with or without 2 h pre-treatment of formoterol or salmeterol. The protein and mRNA expression of eotaxin-1 were measured by ELISA assay and real-time PCR, respectively. Effects of formoterol and salmeterol on nuclear and cytosolic pSTAT-6 expression were evaluated by Western blot and immunofluorescence study. Formoterol and salmeterol (10(-7)-10(-10) m) significantly down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. A specific beta(2) adrenoceptor antagonist (ICI 118,551) reversed their suppression of eotaxin-1 production. Forskolin, an cAMP activator, could also suppress the expression of eotaxin-1 by IL-4 in a dose dependent manner (10(-7)-10(-10 )m). The western blot and immunofluorescence studies demonstrated that formoterol 10(-7 )m suppressed the nuclear expression of pSTAT-6. Formoterol and salmeterol, two inhaled long-acting beta(2) agonists, down-regulated IL-4- induced eotaxin-1 expression in BEAS-2B cells. The effect was mediated via the beta(2) adrenoceptor, and cAMP. Formoterol significantly down-regulated pSTAT6 at higher concentration, and further turned off the IL-4 signaling pathway.

The function of acetaldehyde dehydrogenase 1 (ALDH1) has been gradually elucidated in several diseases, especially in various cancers. However, the role of ALDH1 in skin-related diseases has been mostly unknown. Previously, we found that ALDH1 is involved in the pathogenesis of atopic dermatitis (AD). In this study, we used high-throughput screening (HTS) approaches to identify critical factors associated with ALDH1 in human keratinocytes to reveal its functions in skin. We overexpressed ALDH1 in human HaCaT keratinocytes and then conducted serial HTS studies, a DNA microarray and antibody array integrated with bioinformatics algorithms. Together, those tests identified several novel genes associated with the function of ALDH1 in keratinocytes, as well as AD, including CTSG and CCL11. In particular, GNB3, GHSR, TAS2R9, FFAR1, TAS2R16, CCL21, GPR32, NPFFR1, GPR15, FBXW12, CCL19, EDNRA, FFAR3, and RXFP3 proteins were consistently detected as hub proteins in the PPI maps. By integrating the datasets obtained from these HTS studies and using the strengths of each method, we obtained new insights into the functional role of ALDH1 in skin keratinocytes. The approach used here could contribute to the clinical understanding of ALDH1-associated applications for the treatment of AD.

The function of acetaldehyde dehydrogenase 1 (ALDH1) has been gradually elucidated in several diseases, especially in various cancers. However, the role of ALDH1 in skin-related diseases has been mostly unknown. Previously, we found that ALDH1 is involved in the pathogenesis of atopic dermatitis (AD). In this study, we used high-throughput screening (HTS) approaches to identify critical factors associated with ALDH1 in human keratinocytes to reveal its functions in skin. We overexpressed ALDH1 in human HaCaT keratinocytes and then conducted serial HTS studies, a DNA microarray and antibody array integrated with bioinformatics algorithms. Together, those tests identified several novel genes associated with the function of ALDH1 in keratinocytes, as well as AD, including CTSG and CCL11. In particular, GNB3, GHSR, TAS2R9, FFAR1, TAS2R16, CCL21, GPR32, NPFFR1, GPR15, FBXW12, CCL19, EDNRA, FFAR3, and RXFP3 proteins were consistently detected as hub proteins in the PPI maps. By integrating the datasets obtained from these HTS studies and using the strengths of each method, we obtained new insights into the functional role of ALDH1 in skin keratinocytes. The approach used here could contribute to the clinical understanding of ALDH1-associated applications for the treatment of AD.

The analysis of eosinophil shape change and mediator secretion is a useful tool in understanding how eosinophils respond to immunological stimuli and chemotactic factors. Eosinophils undergo dramatic shape changes, along with secretion of the granule-derived enzyme eosinophil peroxidase (EPX) in response to chemotactic stimuli including platelet-activating factor (PAF) and CCL11 (eotaxin-1). Here, we describe the analysis of eosinophil shape change by confocal microscopy analysis and provide an experimental approach for comparing unstimulated cells with those that have been stimulated to undergo chemotaxis. In addition, we illustrate two different degranulation assays for EPX using OPD and an ELISA technique and show how eosinophil degranulation may be assessed from in vitro as well as ex vivo stimulation.

Keywords: Chemokine; Chemotaxis; Confocal microscopy; Degranulation; Eosinophil peroxidase; Eotaxin; Platelet-activating factor; Secretion.

BACKGROUND

Epidemiological studies have implicated Asian sand dust (ASD) in the increased prevalence of respiratory disorders, including asthma. It has been observed that fungal elements such as β-glucan can be adsorbed onto ASD. In the present study, the exacerbating effect of the combined exposure to zymosan A (ZymA) containing yeast β-glucan and heat-inactivated ASD on ovalbumin (OVA)-induced murine lung eosinophilia was investigated.

METHODS

BALB/c mice were repeatedly instilled intratracheally with one of eight immunogenic formulations consisting of various combinations of (1) ZymA, (2) ASD that was briefly heated to remove organic substances (H-ASD), and (3) OVA in normal saline, or each of the above alone. Pathologic changes, cytological alterations in bronchoalveolar lavage fluid (BALF), changes in inflammatory cytokines and chemokines in BALF, and OVA-specific IgE and IgG1 antibodies in serum were investigated.

RESULTS

Exposure to ZymA with or without OVA had no effect on most indicators of lung inflammation. Exposure to H-ASD with OVA increased the recruitment of inflammatory cells to the lungs and the serum levels of OVA-specific IgE and IgG1. The combination OVA + ZymA + H-ASD induced a marked recruitment of eosinophils and upregulation of T helper 2 (Th2) cytokines (interleukin [IL]-4 and IL-13), IL-6, eotaxin/CCL11, and monocyte chemotactic protein (MCP)-3/CCL7 in BALF and OVA-specific IgE in serum. This treatment also induced the most severe pathological changes in the lungs of mice. ZymA was found to boost the effects of H-ASD, thereby exacerbating the OVA-induced allergic inflammation, even though ZymA alone did not have such effect.

CONCLUSIONS

The results suggest that fungal elements such as β-1,3-glucan aggravate the allergic inflammation caused by ASD. Our findings may facilitate prophylaxis of some allergic diseases in Asia.