Despite strong evidence that high-density lipoproteins (HDLs) modulate the immune response, the role of HDL in allergies is still poorly understood. Many patients with allergic rhinitis (AR) develop a late-phase response, characterized by infiltration of monocytes and eosinophils into the nasal submucosa. Functional impairment of HDL in AR-patients may insufficiently suppress inflammation and cell infiltration, but the effect of AR on the composition and function of HDL is not understood. We used apolipoprotein (apo) B-depleted serum as well as isolated HDL from AR-patients (n = 43) and non-allergic healthy controls (n = 20) for detailed compositional and functional characterization of HDL. Both AR-HDL and apoB-depleted serum of AR-patients showed decreased anti-oxidative capacity and impaired ability to suppress monocyte nuclear factor-κB expression and pro-inflammatory cytokine secretion, such as interleukin (IL)-4, IL-6, IL-8, tumor necrosis factor alpha and IL-1 beta. Sera of AR-patients showed decreased paraoxonase and cholesteryl-ester transfer protein activities, increased lipoprotein-associated phospholipase A2 activity, while lecithin-cholesterol acyltransferase activity and cholesterol efflux capacity were not altered. Surprisingly, apoB-depleted serum and HDL from AR-patients showed an increased ability to suppress eosinophil effector responses upon eotaxin-2/CCL24 stimulation. Mass spectrometry and biochemical analyses showed reduced levels of apoA-I and phosphatidylcholine, but increased levels of apoA-II, triglycerides and lyso-phosphatidylcholine in AR-HDL. The changes in AR-HDL composition were associated with altered functional properties. In conclusion, AR alters HDL composition linked to decreased anti-oxidative and anti-inflammatory properties but improves the ability of HDL to suppress eosinophil effector responses.

Therapeutic proteins can induce immune responses that affect their safety and efficacy. Product aggregates and innate immune response modulating impurities (IIRMI) are risk factors of product immunogenicity. In this study, we use Intravenous Immunoglobulin (IVIG), Avastin, and Human Serum Albumin (HSA) to explore whether increased aggregates activate innate immune cells or modify the response to IIRMI. We show that increased aggregates (shaken or stirred) in IVIG and Avastin, but not HSA, induced activation of MAPKs (pp38, pERK and pJNK) and transcription of immune-related genes including IL8, IL6, IL1β, CSF1, CCL2, CCL7, CCL3, CCL24, CXCL2, IRAK1, EGR2, CEBPβ, PPARg and TNFSF15 in human PBMC. The immunomodulatory effect was primarily mediated by FcγR, but not by TLR. Interestingly, increased aggregates in IVIG or Avastin magnified innate immune responses to TLR2/4 agonists, but diminished responses to TLR3/9 agonists. This study shows that IIRMI and aggregates can modify the activity of immune cells potentially modifying the milieu where the products are delivered highlighting the complex interplay of different impurities on product immunogenicity risk. Further, we show that aggregates could modify the sensitivity of PBMC-based assays designed to detect IIRMI. Understanding and managing immunogenicity risk is a critical component of product development and regulation.

OBJECTIVE

The previously reported Japanese clinical scoring study (JESREC) suggests that chronic rhinosinusitis (CRS) can be divided into 4 subtypes according to the degree of eosinophilic CRS (ECRS) and offers the information regarding the prognosis of CRS to clinicians. However, this scoring system has not yet been validated by an immunological study and needs to provide treatment guidelines based on underlying immunologic profiles. We investigated the immunologic profile of each CRS subgroup according to the JESREC classification and suggest its clinical application.

METHODS

A total of 140 CRS patients and 20 control subjects were enrolled. All patients were classified into 4 groups according to the JESREC (non-, mild, moderate and severe ECRS). Nasal tissues were analyzed for mRNA expression of major cytokines (IL-5, IL-10, IL-13, IL-17A, IL-22, IL-23p19, IFN-γ, periostin, thymic stromal lymphopoietin [TSLP] and ST2), major chemokines (CCL11, CCL24, CXCL1 and CXCL2), transcription factors (T-bet, GATA3, RORC and FOXP3) and COL1A1 for type I collagen. Protein levels of 3 major cytokines (IL-5, IL-17A and IFN-γ) were also measured by multiplex immunoassay. Principal component analysis (PCA) was conducted to investigate the overall profile of multiple mediators.

RESULTS

The moderate/severe ECRS showed up-regulation of type 2-related mediators (IL-5, IL-13, periostin, TSLP and ST-2), whereas INF-γ (type 1 cytokine) and CXCL1 (neutrophil chemokine) expressions were increased in non-/mild ECRS compared with moderate/severe ECRS. The JESREC classification reflected an immunological endotype. In PCA data, PCA1 indicates a relative type 2 profile, whereas PCA2 represents a type 1/type 17-related profile. In this analysis, mild ECRS was indistinguishable from non-ECRS, whereas moderate to severe ECRS showed a distinct distribution compared with non-ECRS. The JESREC classification could be divided into 2 categories, non-/mild vs. moderate/severe ECRS based on underlying immunological analyses.

CONCLUSIONS

The CRS clinical scoring system from the JESREC study reflects an inflammatory endotype. However, the immunologic profile of mild ECRS was similar to that of non-ECRS. Therefore, we propose type 2-targeted medical treatment for moderate to severe ECRS and type 1/type 17-targeted for non-ECRS and mild ECRS as the first treatment option.

CCL24 is one chemotactic factor extensively studied in airway inflammation and colorectal cancer but less studied in hepatocellular carcinoma (HCC) retrospectively. So HCC tissue microarray (TMA) was used to estimate relationship between CCL24 and prognosis, cell experiments were conducted to study its influence for HCC cell biological behavior. CCL24 was injected to nude mice to monitor tumor formation and pulmonary metastasis; qRT-PCR, western blot and Immunohistochemistry were used to explore potential mechanism. CCL24 plays roles in target cells via its downstream CCR3, or it is regulated by Type 2 helper T cells (Th2 cell) factors, so immune related experiments were conducted. Meanwhile, Rho GTPase family have close relation not only with T cell priming, but with neovascularization; CCL24 contributes to neovascularization in age-related macular degeneration via CCR3, so Rho GTPase family, Th2 cell factors, Human Umbilical Vein Endothelial Cells were used to uncover their trafficking. Ultimate validation was confirmed by small interfering RNA. Results showed CCL24 expression was higher in caner tissues than adjacent normal tissues, it could contribute to proliferation, migration, and invasion in HCCs, could accelerate pulmonary metastasis, promote HUVECs tube formation. Th2 cell factors were irrelevant with CCL24 in HCCs; and RhoB, VEGFA, and VEGFR2 correlated with CCL24 in both mRNA and protein level. Downstream RhoB-VEGFA signaling pathway was validated by siRhoB and siVEGFA inhibition. In a word, CCL24 contributes to HCC malignancy via RhoB-VEGFA-VEGFR2 angiogenesis pathway and indicates poor prognosis, which urges us to study further CCL24 effects on diagnosis and potential therapy for HCC.

Rhinovirus (RV) infections in asthmatic patients are often associated with asthma exacerbation, characterized by worsened airways hyperreactivity and increased immune cell infiltration to the airways. The C-X-C chemokines, CXCL3 and CXCL5, regulate neutrophil trafficking to the lung via CXCR2, and their expression in the asthmatic lung is associated with steroid-insensitive type 2 inflammatory signatures. Currently, the role of CXCL3 and CXCL5 in regulating neutrophilic and type 2 responses in viral-induced asthma exacerbation is unknown. Inhibition of CXCL3 or CXCL5 with silencing RNAs in a mouse model of RV-induced exacerbation of asthma attenuated the accumulation of CXCR2⁺ neutrophils, eosinophils, and innate lymphoid cells in the lung and decreased production of type 2 regulatory factors IL-25, IL-33, IL-5, IL-13, CCL11, and CCL24. Suppression of inflammation was associated with decreased airways hyperreactivity, mucus hypersecretion, and collagen deposition. Similar results were obtained by employing RC-3095, which has been shown to bind to CXCR2, or by depletion of neutrophils. Our data demonstrate that CXCL3 and CXCL5 may be critical in the perpetuation of RV-induced exacerbation of asthma through the recruitment of CXCR2-positive neutrophils and by promoting type 2 inflammation. Targeting the CXCL3/CXCL5/CXCR2 axis may provide a new therapeutic approach to attenuating RV-induced exacerbations of asthma.

BACKGROUND

During bacterial infections of the airways, a Th1-profiled inflammation promotes the production of several host defense proteins and peptides with antibacterial activities including β-defensins, ELR-negative CXC chemokines, and the cathelicidin LL-37. These are downregulated by Th2 cytokines of the allergic response. Instead, the eosinophil-recruiting chemokines eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 are expressed. This study set out to investigate whether these chemokines could serve as innate host defense molecules during allergic inflammation.

METHODS

Antibacterial activities of the eotaxins were investigated using viable count assays, electron microscopy, and methods assessing bacterial permeabilization. Fragments generated by mast cell proteases were characterized, and their potential antibacterial, receptor-activating, and lipopolysaccharide-neutralizing activities were investigated.

RESULTS

CCL11, CCL24, and CCL26 all showed potent bactericidal activity, mediated through membrane disruption, against the airway pathogens Streptococcus pneumoniae, Staphylococcus aureus, Nontypeable Haemophilus influenzae, and Pseudomonas aeruginosa. CCL26 retained bactericidal activity in the presence of salt at physiologic concentrations, and the region holding the highest bactericidal activity was the cationic and amphipathic COOH-terminus. Proteolysis of CCL26 by chymase and tryptase, respectively, released distinct fragments of the COOH- and NH2 -terminal regions. The COOH-terminal fragment retained antibacterial activity while the NH2 -terminal had potent LPS-neutralizing properties in the order of CCL26 full-length protein. An identical fragment to NH2 -terminal fragment generated by tryptase was obtained after incubation with supernatants from activated mast cells. None of the fragments activated the CCR3-receptor.

CONCLUSIONS

Taken together, the findings show that the eotaxins can contribute to host defense against common airway pathogens and that their activities are modulated by mast cell proteases.

Macrophages are the most abundant cells in tumor stroma and their polarization within tumor microenvironment exert the key roles in tumorigenesis. Astragaloside IV is a natural extract from traditional Chinese herbal Radix Astragali, and fulfills pleiotropic function in several cancers. Nevertheless, its function in ovarian cancer microenvironment remains elusive. In the present research, astragaloside IV exhibited little cytotoxicity within a certain dose range in THP-1 cells. Moreover, astragaloside IV suppressed the ratio of CD14⁺CD206⁺ cells in IL-4/IL-13-treated THP-1 macrophages and transcripts of M2 macrophage markers (including CD206, CCL24, PPARγ, Arg-1, IL-10), indicating the inhibitory effects of astragaloside IV on IL-4/IL-13-induced macrophage M2 polarization. Intriguingly, astragaloside IV antagonized M2 macrophages coculture-evoked cell proliferation, invasion and migration in ovarian cancer cells. During this process, administration with astragaloside IV restrained the high expression of high-mobility group box1 (HMGB1) and TLR4 in macrophages co-cultured with ovarian cancer cells, concomitant with decreases in release of M2 marker TGF-β, MMP-9 and IL-10. Moreover, targeting the HMGB1 signaling reversed M2 macrophages-induced ovarian cancer cell proliferation, invasion and migration. Noticeably, exogenous HMGB1 overturned the inhibitory efficacy of astragaloside IV against macrophage M2 polarization-evoked malignant potential in ovarian cancer cells. Together, these findings suggest that astragaloside IV may protect against M2 macrophages-evoked malignancy in ovarian cancer cells by suppressing the HMGB1-TLR4 signaling. Therefore, astragaloside may alleviate the progression of ovarian cancer by regulating macrophage M2 polarization within tumor microenvironment, implying a promising therapeutic strategy against ovarian cancer.

Keywords: Astragaloside IV; HMGB1 signaling; Macrophages; Ovarian cancer; Tumor microenvironment.

The outcome of many infections depends on the initial interactions between agent and host. Aiming at elucidating the effect of the M. tuberculosis Mce1 protein complex on host transcriptional and immunological responses to infection with M. tuberculosis, RNA from murine macrophages at 15, 30, 60 min, 4 and 10 hrs post-infection with M. tuberculosis H37Rv or Δ-mce1 H37Rv was analyzed by whole-genome microarrays and RT-QPCR. Immunological responses were measured using a 23-plex cytokine assay. Compared to uninfected controls, 524 versus 64 genes were up-regulated by 15 min post H37Rv- and Δ-mce1 H37Rv-infection, respectively. By 15 min post-H37Rv infection, a decline of 17 cytokines combined with up-regulation of Ccl24 (26.5-fold), Clec4a2 (23.2-fold) and Pparγ (10.5-fold) indicated an anti-inflammatory response initiated by IL-13. Down-regulation of Il13ra1 combined with up-regulation of Il12b (30.2-fold), suggested switch to a pro-inflammatory response by 4 hrs post H37Rv-infection. Whereas no significant change in cytokine concentration or transcription was observed during the first hour post Δ-mce1 H37Rv-infection, a significant decline of IL-1b, IL-9, IL-13, Eotaxin and GM-CSF combined with increased transcription of Il12b (25.1-fold) and Inb1 (17.9-fold) by 4 hrs, indicated a pro-inflammatory response. The balance between pro-and anti-inflammatory responses during the early stages of infection may have significant bearing on outcome.

BACKGROUND

Compounds which activate the innate immune system, such as lipopolysaccharide, are significant components of ambient air, and extremely difficult to remove from the environment. It is currently unclear how prior inhalation of endotoxin affects allergen sensitization. We examined whether lung-specific endotoxin tolerance induction prior to sensitization can modulate the response to allergen.

METHODS

Endotoxin tolerance was induced by repeated intratracheal exposure to endotoxin. All mice were then sensitized and challenged by direct intratracheal instillation of cockroach allergen.

RESULTS

After allergen sensitization and challenge, endotoxin tolerant mice had significantly decreased airways hyperresponsiveness to methacholine challenge, which was confirmed by invasive lung function tests. Decreased goblet cell hyperplasia and mucus production were also found by histological assessment. Tolerant mice were protected from airway eosinophilia through the mechanism of reduced CCL11 and CCL24. Interestingly, endotoxin tolerant mice had only a modest reduction in cockroach-specific IgE; however, total IgE was significantly reduced.

CONCLUSIONS

These data show that induction of endotoxin tolerance prior to sensitization protects against the hallmark features of asthma-like inflammation, and that transient modulation of innate immunity can have long-lasting effects on adaptive responses.

This study investigated the clinical usefulness of a multiple tear cytokine/chemokine test by simultaneously determining tear levels of CC chemokine ligand 17 (CCL17)/thymus and activation-regulated chemokine (TARC), CCL24/eotaxin-2, and interleukin-16 (IL-16) for assessing acute and chronic allergic inflammation in allergic conjunctival disorders (ACDs).

This study included 37 patients with ACD and 11 healthy adults (controls). Patients with ACDs were divided into the following three groups; patients with allergic conjunctivitis (AC group, n = 17), patients with atopic keratoconjunctivitis (AKC group, n = 6), and patients with vernal keratoconjunctivitis (VKC group, n = 14). Tear samples were collected using the Schirmer I method with a filter paper. Tear levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 were determined by performing a magnetic bead assay (tear cytokine/chemokine test). Tear levels of eosinophil cationic protein (ECP) were determined by performing enzyme immunoassay. In patients with AC, clinical scores of objective findings and results of the tear cytokine/chemokine test at baseline were compared with those at 7 days after treatment with the histamine H1 receptor antagonist (epinastine) ophthalmic solution.

Tear positive rates of CCL17/TARC, CCL24/eotaxin-2, and IL-16 were higher in patients with AC, AKC, and VKC compared with controls. Tear levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 in patients with AKC and VKC were significantly higher than those in patients with AC. Moreover, tear levels of IL-16 in patients with AC that showed improvement of their clinical score by treatment with epinastine ophthalmic solution decreased significantly after 7 days of the treatment compared with those at baseline. In patients with AKC and VKC, a significant correlation was observed between the tear levels of CCL24/eotaxin-2 and ECP.

Simultaneous measurement of the tear levels of CCL17/TARC, CCL24/eotaxin-2, and IL-16 may be a useful test for assessing acute and chronic allergic inflammation in ACDs.

Chemokines and their cognate receptors play important role in the control of leukocyte chemotaxis, HIV entry and other inflammatory diseases. Developing an effcient method to investigate the functional expression of chemokines and its interactions with specific receptors will be helpful to asses the structural and functional characteristics as well as the design of new approach to therapeutic intervention.

By making systematic optimization study of expression conditions, soluble and functional production of chemokine C-C motif ligand 8 (CCL8) in Escherichia coli (E. coli) has been achieved with approx. 1.5 mg protein/l culture. Quartz crystal microbalance (QCM) analysis exhibited that the purified CCL8 could bind with C-C chemokine receptor type 3 (CCR3) with dissociation equilibrium constant (K D) as 1.2 × 10-7 M in vitro. Obvious internalization of CCR3 in vivo could be detected in 1 h when exposed to 100 nM of CCL8. Compared with chemokine C-C motif ligand 11 (CCL11) and chemokine C-C motif ligand 24 (CCL24), a weaker chemotactic effect of CCR3 expressing cells was observed when induced by CCL8 with same concentration.

This study delivers a simple and applicable way to produce functional chemokines in E. coli. The results clearly confirms that CCL8 can interact with chemokine receptor CCR3, therefore, it is promising area to develop drugs for the treatment of related diseases.

Peptidoglycan recognition protein (Pglyrp) 1 is a pattern-recognition protein that mediates antibacterial host defense. Because we had previously shown that Pglyrp1 expression is increased in the lungs of house dust mite (HDM)-challenged mice, we hypothesized that it might modulate the pathogenesis of asthma. Wild-type and Pglyrp1(-/-) mice on a BALB/c background received intranasal HDM or saline, 5 days/week for 3 weeks. HDM-challenged Pglyrp1(-/-) mice showed decreases in bronchoalveolar lavage fluid eosinophils and lymphocytes, serum IgE, and mucous cell metaplasia, whereas airway hyperresponsiveness was not changed when compared with wild-type mice. T helper type 2 (Th2) cytokines were reduced in the lungs of HDM-challenged Pglyrp1(-/-) mice, which reflected a decreased number of CD4(+) Th2 cells. There was also a reduction in C-C chemokines in bronchoalveolar lavage fluid and lung homogenates from HDM-challenged Pglyrp1(-/-) mice. Furthermore, secretion of CCL17, CCL22, and CCL24 by alveolar macrophages from HDM-challenged Pglyrp1(-/-) mice was markedly reduced. As both inflammatory cells and airway epithelial cells express Pglyrp1, bone marrow transplantation was performed to generate chimeric mice and assess which cell type promotes HDM-induced airway inflammation. Chimeric mice lacking Pglyrp1 on hematopoietic cells, not structural cells, showed a reduction in HDM-induced eosinophilic and lymphocytic airway inflammation. We conclude that Pglyrp1 expressed by hematopoietic cells, such as alveolar macrophages, mediates HDM-induced airway inflammation by up-regulating the production of C-C chemokines that recruit eosinophils and Th2 cells to the lung. This identifies a new family of innate immune response proteins that promotes HDM-induced airway inflammation in asthma.

The host immune response plays an important role in viral clearance in patients who are chronically infected with hepatitis C virus (HCV) and are treated with interferon and ribavirin. Activation of the immune system involves the release of pro and anti-inflammatory molecules that can be measured in plasma samples. The present study aimed to evaluate the association between pretreatment plasma levels of chemokines and soluble tumor necrosis factor receptors (sTNF-R) and the virological response in treated patients with chronic hepatitis C infection. Forty-one chronically-infected HCV patients that were being treated with interferon-α (IFN-α) plus ribavirin were included in the study. Socio-demographic, clinical and laboratory data were collected and pretreatment plasma levels of chemokine CCL2, CCL3, CCL11, CCL24, chemokine CXCL9, CXCL10, sTNF-R1 and sTNF-R2 were measured. The virological response was assessed at treatment week 12, at the end of treatment and 24 weeks after treatment. Pretreatment CXCL10 levels were significantly higher in patients without an early virological response (EVR) or sustained virological response (SVR) compared to responders [512.9 pg/mL vs. 179.1 pg/mL (p = 0.011) and 289.9 pg/mL vs. 142.7 pg/mL (p = 0.045), respectively]. The accuracy of CXCL10 as a predictor of the absence of EVR and SVR was 0.79 [confidence interval (CI) 95%: 0.59-0.99] and 0.69 (CI 95%: 0.51-0.87), respectively. Pretreatment plasma levels of the other soluble inflammatory markers evaluated were not associated with a treatment response. Pretreatment CXCL10 levels were predictive of both EVR and SVR to IFN-α and ribavirin and may be useful in the evaluation of candidates for therapy.

BACKGROUND

M2 macrophages play a critical role in the recruitment of T helper 2 (Th2) regulatory T cells (Treg).

OBJECTIVE

To study the role of M2 macrophages and Treg cells in eosinophilic celulitis.

METHODS

We employed immunohistochemical staining for CD163( )and CD206 (macrophages) as well as FoxP3 (Treg), in lesional skin of four cases of eosinophilic cellulitis.

RESULTS

CD163(+) CD206(+) M2 macrophages, which were previously reported to produce CCL17 to induce Th2 cells and Treg cells, were predominantly infiltrating the subcutaneous tissues and interstitial area of the dermis. M2 macrophages derived from PBMC showed significantly increased expression of CCL11, CCL17, CCL24 and CCL26 mRNA and production of CCL17 and CCL24, when stimulated by IL-4 or IL- 13. In addition, CCL17-producing cells and CCL24-producing cells were prominent in the lesional skin of EC.

CONCLUSIONS

Our study sheds light on one of the possible immunological mechanisms of eosinophilic cellulitis.

BACKGROUND

Chemokine receptors (CCRs) are expressed on airway smooth muscle (ASM) cells. As their ligands are present in the airways in asthma, we hypothesized that ASM CCR activation could promote the increase in ASM mass seen in patients with chronic asthma.

OBJECTIVE

To determine which CCRs are expressed by ASM cells and their potential functional relevance to the chronic airway changes seen in asthma.

METHODS

CCR expression in primary ASM cell cultures and airway biopsies from patients with and without asthma was examined by RT-PCR, fluorescence-activated cell sorting and immunohistochemistry. ASM p42/44 MAPK activity, proliferation, migration and apoptosis were examined by western blotting, thymidine incorporation, transwell assay and TUNEL assay respectively.

RESULTS

CCR3 was the most frequently expressed CCR protein and was present on 79 ± 14% of cells. CX3CR1 and CXCR6 were present on 6% and 11% of cells respectively. CCR3 ligands CCL11 and CCL24 caused rapid activation of p42/44 MAPK but not Akt. CCR3 activation did not affect ASM proliferation, migration or VEGF secretion. DNA fragmentation detected by TUNEL staining could be induced by staurosporine and Fas activation although only Fas activation resulted in caspase 3 cleavage. CCL11 and CCL24 protected ASM cells against DNA fragmentation dependent upon p42/44 MAPK activity only via caspase 3 independent pathways. CCR3 was expressed in the smooth muscle and epithelium in the airways of patients with and without asthma. Smooth muscle cell DNA fragmentation in the airways of patients with stable asthma and controls was very uncommon.

CONCLUSIONS

CCR3 is strongly expressed by ASM cells in vitro and in vivo. Protection against cell death by CCR3 activation is dependent on p42/44 MAPK but does not affect caspase 3 mediated apoptosis.

OBJECTIVE

To evaluate the plasma levels of CCL2, CCL3, CCL11, CCL24, tumor necrosis factor alpha, sTNFR1, and sTNFR2 in subjects exposed to silica (SES) with and without silicosis compared with unexposed reference control group, and their associations with the radiological severity and duration of exposure to silica.

METHODS

Fifty-seven SES; 36 with silicosis and 22 subjects in control group, were included in the study.

RESULTS

CCL3, CCL24, sTNFR1, and sTNFR2 were increased in SES and in SES with silicosis than in controls. There were no differences in the levels of CCL2, CCL11, or tumor necrosis factor alpha. The sTNFR2 level was greater in SES with silicosis than in SES without silicosis. There was a positive correlation between sTNFR1 and sTNFR2 and the radiological severity and time of exposure to silica. sTNFR2 was associated with all categories of radiological severity.

CONCLUSIONS

sTNFR2 is associated with silicosis severity and early exposure to silica.

OBJECTIVE

To develop a predictive model based on inflammatory gene mRNA expression in conjunctival cells of graft versus host disease (GvHD)-associated dry eye (DE) patients, as well as to find meaningful correlations between gene signals and clinical signs.

METHODS

Twenty GvHD-DE patients and 14 healthy controls were recruited. Patients discontinued medications for 1 week before examination. Dry eye-related symptoms and signs were recorded, and conjunctival epithelial cells were collected by impression cytology after spending 20 minutes under standard conditions within a Controlled Environmental Research Laboratory. Gene expression of inflammatory molecules was determined by polymerase chain reaction, and the results were correlated with clinical signs. Shrinkage discriminant analysis, support vector machine, and k-nearest neighbor classifier methods were used to develop predictive models that were validated considering accuracy, calibration, and discriminant capability.

RESULTS

Out of the 84 genes analyzed, 34 showed significant differences in expression. IL-6, IL-9, CCL24, CCL18, IL-10, IFN-γ, and CCL2 were highly increased (>6-fold); 26 genes were moderately upregulated (2- to 6-fold), whereas EGFR was downregulated (2.63 fold) in GvHD-DE samples. A panel based on EGFR, IL-6, IL-9, and NAMPT had an area under the receiver operating characteristic curve of 0.994, a sensitivity of 100%, and a specificity of 92.9%. EGFR expression correlated negatively with ocular surface damage markers, while IL-6, IL-9, and NAMPT correlated positively with these tests.

CONCLUSIONS

EGFR, IL-6, IL-9, and NAMPT have the greatest potential as diagnostic biomarkers, with excellent sensitivity, specificity, and clinical relevance to the ocular surface status of GvHD.

We studied the expression of some CC chemokines and their receptors in the synovium of patients with rheumatoid arthritis, osteoarthrosis, and a history of joint injury. In patients with rheumatoid arthritis, the levels mRNA for some angiogenic and proinflammatory chemokines (CCL5/RANTES, CCL11/eotaxin, CCL24/eotaxin-2, and CCL26/eotaxin-3) and their receptors (CCR1, CCR2, CCR3, CCR4, and CCR5) was elevated. mRNA expression correlated with activity, stage, and serological status of rheumatoid arthritis. Obtained data confirm the importance of CC chemokines as mediators of angiogenesis and inflammation in the synovium in rheumatoid arthritis.

When a human leukemic cell line, HT93 was incubated with all-trans retinoic acid (ATRA), IL-5, or both, this cell line was differentiated into eosinophic lineage, in that an eosinophilic specific granule proteins, major basic protein (MBP) and eosinophil peroxidase (EPO) appeared. Both CD11b and CC chemokine receptor, CCR3 expression were upregulated, while CD71 expression was downregulated by ATRA or ATRA+IL-5. Concomitantly, marked production of eotaxin-2/CCL24 was observed, but no production of eotaxin-1/CCL11 and eotaxin-3/CCL26 was detected. Since only 20 to 30% cells incubated with ATRA became positive for CCR3, CCR3(+) population was enriched by a magnetic activated cell sorter (MACS). Enriched CCR3(+) population produced higher eotaxin-2/CCL24 than the CCR3(-) population, indicating that differentiated eosinophils are capable of producing eotaxin-2/CCL24. During the ATRA-induced differentiation, expression of a transcriptional factor, GATA-1 was significantly increased. Introduction of siRNA against GATA-1 markedly reduced the ATRA-induced differentiation markers including CD11b and CCR3, as well as reduced eotaxin-2/CCL24 production. Finally, ATRA-induced differentiation and eotaxin-2/CCL24 production were greatly enhanced in the GATA-1-overexpressed clones. These results indicate that the ability to produce eotaxin-2/CCL24 is acquired during the differentiation into eosinophilic lineage which is dependent on GATA-1 expression.

Nasopharyngeal carcinoma (NPC) is a fast-growing cancer characterized by high occurrences of nodal and distant metastases and poor prognosis. It is therefore important to identify new serum biomarkers for the early diagnosis and prognostic prediction of this disease. The present study identifies biomarkers in NPC patient serum using a solid-phase antibody array detecting the expression profiles of 174 cytokines in a single experiment. ELISA was performed to validate the array results. The levels of TIMP-2, SELL, CCL24, MMP-1, MMP-3, IGF-I and IL-8 were significantly higher in serum from NPC patients, while the levels of MSP-alpha and HCC-4 were lower. Furthermore, the validation results were identical to those obtained from the antibody array. These results indicate that these cytokines might serve as novel biomarkers for the diagnosis and prognostic prediction of NPC.

Viruses have long been studied not only for their pathology and associated disease but also as model systems for understanding cellular and immunological processes. Rodent herpesvirus Peru (RHVP) is a recently characterized rhadinovirus related to murine gammaherpesvirus 68 (MHV68) and Kaposi's sarcoma-associated herpesvirus (KSHV) that establishes acute and latent infection in laboratory mice. RHVP encodes numerous unique proteins that we hypothesize might facilitate host immune evasion during infection. We report here that open reading frame (ORF) R17 encodes a high-affinity chemokine binding protein that broadly recognizes human and murine CC and C chemokines. The interaction of R17 with chemokines is generally characterized by rapid association kinetics, and in the case of CCL3, CCL4, CCL5, CCL24, and XCL1, extremely stable complexes are formed. Functionally, R17 potently inhibited CCL2-driven chemotaxis of the human monocytic cell line THP-1, CCL3-driven chemotaxis of peripheral blood mononuclear cells, and CCL2-mediated calcium flux. Our studies also reveal that R17 binds to glycosaminoglycans (GAGs) in a process dependent upon two BBXB motifs and that chemokine and GAG binding can occur simultaneously at distinct sites. Collectively, these studies suggest that R17 may play a role in RHVP immune evasion through the targeted sabotage of chemokine-mediated immune surveillance.

Asthma is affecting more than 300 million people worldwide, which represents the most common chronic disease among children. We previously found that mesenchymal stem cells (MSCs) derived from induced pluripotent stem cells (iPSCs) modulated the immune response on Th2-mediated asthma in vivo and in vitro. This study further evaluated the immunomodulatory effects of MSCs from human embryonic stem cells (hESCs) on asthma.

Multipotent hESC-MSCs were obtained using a feeder-free method. The hESC-MSCs were analysed for the expression of stem cell surface markers by flow cytometry, their differentiation potentials were analysed using in vitro trilineage differentiation methods hESC-MSCs were transplanted into the murine model with ovalbumin (OVA)-induced airway allergic inflammation. The expression levels of allergic related genes were measured by the mRNA PCR arrays.

The hESC-MSCs expressed classical MSC markers and held the capability of differentiation into multiple mesoderm-type cell lineages. hESC-MSCs were able to suppress allergic inflammation by modulating Th2 cells and eosinophils in the mice, and reversed the reduction of regulatory T cells. By using PCR array, 5 mRNAs- chemokine (C-C motif) ligand 11 (Ccl11), Ccl24, interleukin13 (Il13), Il33 and eosinophil-associated, ribonuclease A family, member 11 (Ear11) were identified the most relevant in murine airway allergic inflammation and hESC-MSCs treatment.

The therapeutic effects of hESC-MSCs were identified in the murine model of airway allergic inflammation with key mRNAs involved. This study will provide a better understanding regarding the mechanisms underlying hESC-MSCs therapeutic application in airway allergic inflammation.

Mansonella perstans (Mp) filariasis is present in large populations in sub-Saharan Africa, and to what extent patent Mp infection modulates the expression of immunity in patients, notably their cellular cytokine and chemokine response profile, remains not well known. We studied the spontaneous and inducible cellular production of chemokines (C-X-C motif) ligand 9 (CXCL9) [monokine induced by interferon (IFN)-γ (MIG)], CXCL-10 [inducible protein (IP)-10], chemokine (C-C motif) ligand 24 (CCL24) (eotaxin-2), CCL22 [macrophage-derived chemokine (MDC)], CCL13 [monocyte chemotactic protein-4 (MCP-4)], CCL18 [pulmonary and activation-regulated chemokine (PARC)], CCL17 [thymus- and activation-regulated chemokine (TARC)] and interleukin (IL)-27 in mansonelliasis patients (Mp-PAT) and mansonelliasis-free controls (CTRL). Freshly isolated peripheral mononuclear blood cells (PBMC) were stimulated with helminth, protozoan and bacterial antigens and mitogen [phytohaemagglutinin (PHA)]. PBMC from Mp-PAT produced spontaneously (without antigen stimulation) significantly higher levels of eotaxin-2, IL-27, IL-8, MCP-4 and MDC than cells from CTRL, while IFN-γ-IP-10 was lower in Mp-PAT. Helminth antigens activated IL-27 and MCP-4 only in CTRL, while Ascaris antigen, Onchocerca antigen, Schistosoma antigen, Entamoeba antigen, Streptococcus antigen, Mycobacteria antigen and PHA stimulated MIG release in CTRL and Mp-PAT. Notably, Entamoeba antigen and PHA strongly depressed (P < 0·0001) eotaxin-2 (CCL24) production in both study groups. Multiple regression analyses disclosed in Mp-PAT and CTRL dissimilar cellular chemokine and cytokine production levels being higher in Mp-PAT for CCL24, IL-27, IL-8, MCP-4, MDC and PARC (for all P < 0·0001), at baseline (P < 0·0001), in response to Entamoeba histolytica strain HM1 antigen (EhAg) (P < 0·0001), Onchocerca volvulus adult worm-derived antigen (OvAg) (P = 0·005), PHA (P < 0·0001) and purified protein derivative (PPD) (P < 0·0001) stimulation. In Mp-PAT with hookworm co-infection, the cellular chemokine production of CXCL10 (IP-10) was diminished. In summary, the chemokine and cytokine responses in Mp-PAT were in general not depressed, PBMC from Mp-PAT produced spontaneously and selectively inducible inflammatory and regulatory chemokines and cytokines at higher levels than CTRL and such diverse and distinctive reactivity supports that patent M. perstans infection will not polarize innate and adaptive cellular immune responsiveness in patients.