Chemokine profiling of melanoma-macrophage crosstalk identifies CCL8 and CCL15 as prognostic factors in cutaneous melanoma.

During cancer evolution, tumor cells attract and dynamically interact with monocytes/macrophages. To find biomarkers of disease progression in human melanoma, we used unbiased RNA sequencing and secretome analyses of tumor-macrophage co-cultures. Pathway analysis of genes differentially modulated in human macrophages exposed to melanoma cells revealed a general upregulation of inflammatory hallmark gene sets, particularly chemokines. A selective group of chemokines, including CCL8, CCL15, and CCL20, was actively secreted upon melanoma-macrophage co-culture. Because we previously described the role of CCL20 in melanoma, we focused our study on CCL8 and CCL15 and confirmed that in vitro both chemokines contributed to melanoma survival, proliferation, and 3D invasion through CCR1 signaling. In vivo, both chemokines enhanced primary tumor growth, spontaneous lung metastasis, and circulating tumor cell survival and lung colonization in mouse xenograft models. Finally, we explored the clinical significance of CCL8 and CCL15 expression in human skin melanoma, screening a collection of 67 primary melanoma samples, using multicolor fluorescence and quantitative image analysis of chemokine-chemokine receptor content at the single-cell level. Primary skin melanomas displayed high CCR1 expression, but there was no difference in its level of expression between metastatic and nonmetastatic cases. By contrast, comparative analysis of these two clinically divergent groups showed a highly significant difference in the cancer cell content of CCL8 (p = 0.025) and CCL15 (p < 0.0001). Kaplan-Meier curves showed that a high content of CCL8 or CCL15 in cancer cells correlated with shorter disease-free and overall survival (log-rank test, p < 0.001). Our results highlight the role of CCL8 and CCL15, which are highly induced by melanoma-macrophage interactions in biologically aggressive primary melanomas and could be clinically applicable biomarkers for patient profiling. © 2024 The Pathological Society of Great Britain and Ireland.

Single-cell RNA-sequencing of human eosinophils in allergic inflammation in the esophagus.

BACKGROUND: Eosinophils are elusive cells involved in allergic inflammation. Single-cell RNA-sequencing (scRNA-seq) is an emerging approach to deeply characterize cellular properties, heterogeneity, and functionality. OBJECTIVES: We sought to comprehensively characterize the transcriptome and biological functions of human eosinophils at a site of severe allergic inflammation in the esophagus (ie, eosinophilic esophagitis [EoE]). METHODS: We employed a gravity-based scRNA-seq methodology to sequence blood eosinophils from patients with EoE and control individuals compared to a reanalyzed public scRNA-seq dataset of human esophageal eosinophils of EoE patients. We used flow cytometry, immunostaining, and a stimulation assay to verify mRNA findings. RESULTS: In total, scRNA-seq was obtained from 586 eosinophils (188 from blood [n = 6 individuals] and 398 from esophagus [n = 6 individuals]). The esophageal eosinophils were composed of a population of activated eosinophils (enriched in 659 genes compared with peripheral blood-associated eosinophils) and a small population of eosinophils resembling peripheral blood eosinophils (enriched in 62 genes compared with esophageal eosinophils). Esophageal eosinophils expressed genes involved in sensing and responding to diverse stimuli, most notably IFN-γ, IL-10, histamine and leukotrienes, and succinate. Esophageal eosinophils were most distinguished from other esophageal populations by gene expression of the receptors CCR3, HRH4, SUCNR1, and VSTM1; transcription factors CEBPE, OLIG1, and OLIG2; protease PRSS33; and the hallmark eosinophil gene CLC. A web of bidirectional eosinophil interactions with other esophageal populations was derived. Comparing esophageal eosinophils and mast cells revealed that esophageal eosinophils expressed genes involved in DNAX-activation protein-12 (also known as TYROBP) interactions, IgG receptor-triggered events, immunoregulation, and IL-10 signaling. CONCLUSIONS: In EoE, esophageal eosinophils exist as 2 populations, a minority population resembling blood eosinophils and the other population characterized by high de novo transcription of diverse sensing receptors and inflammatory mediators readying them to potentially intersect with diverse cell types.

IgE directly affects eosinophil migration in chronic rhinosinusitis with nasal polyps through CCR3 and predicts the efficacy of omalizumab.

BACKGROUND: Whether IgE affects eosinophil migration in chronic rhinosinusitis with nasal polyps (CRSwNP) remains largely unclear. Moreover, our understanding of local IgE, eosinophils, and omalizumab efficacy in CRSwNP remains limited. OBJECTIVE: We investigated whether IgE acts directly on eosinophils and determined its role in omalizumab therapy. METHODS: Eosinophils and their surface receptors were detected by hematoxylin and eosin staining and flow cytometry. IgE and its receptors, eosinophil peroxidase (EPX), eosinophilic cationic protein, and CCR3 were detected by immunohistochemistry and immunofluorescence. Functional analyses were performed on blood eosinophils and polyp tissues. Logistic regression was performed to screen for risk factors. Receiver operating characteristic curve was generated to evaluate the accuracy. RESULTS: Both FcεRI and CD23 were expressed on eosinophils. The expression of FcεRI and CD23 on eosinophil in nasal polyp tissue was higher than in peripheral blood (both P < .001). IgE and EPX colocalized in CRSwNP. IgE directly promoted eosinophil migration by upregulating CCR3 in CRSwNP but not in healthy controls. Omalizumab and lumiliximab were found to be effective in restraining this migration, indicating CD23 was involved in IgE-induced eosinophil migration. Both IgE+ and EPX+ cells were significantly reduced after omalizumab treatment in those who experienced response (IgE+ cells, P = .001; EPX+ cells, P = .016) but not in those with no response (IgE+ cells, P = .060; EPX+ cells, P = .151). Baseline IgE+ cell levels were higher in those with response compared to those without response (P = .024). The baseline local IgE+ cell count predicted omalizumab efficacy with an accuracy of 0.811. CONCLUSIONS: IgE directly promotes eosinophil migration, and baseline local IgE+ cell counts are predictive of omalizumab efficacy in CRSwNP.

Genetic fusion of CCL11 to antigens enhances antigenicity in nucleic acid vaccines and eradicates tumor mass through optimizing T-cell response.

Nucleic acid vaccines have shown promising potency and efficacy for cancer treatment with robust and specific T-cell responses. Improving the immunogenicity of delivered antigens helps to extend therapeutic efficacy and reduce dose-dependent toxicity. Here, we systematically evaluated chemokine-fused HPV16 E6/E7 antigen to improve the cellular and humoral immune responses induced by nucleotide vaccines in vivo. We found that fusion with different chemokines shifted the nature of the immune response against the antigens. Although a number of chemokines were able to amplify specific CD8 + T-cell or humoral response alone or simultaneously. CCL11 was identified as the most potent chemokine in improving immunogenicity, promoting specific CD8 + T-cell stemness and generating tumor rejection. Fusing CCL11 with E6/E7 antigen as a therapeutic DNA vaccine significantly improved treatment effectiveness and caused eradication of established large tumors in 92% tumor-bearing mice (n = 25). Fusion antigens with CCL11 expanded the TCR diversity of specific T cells and induced the infiltration of activated specific T cells, neutrophils, macrophages and dendritic cells (DCs) into the tumor, which created a comprehensive immune microenvironment lethal to tumor. Combination of the DNA vaccine with anti-CTLA4 treatment further enhanced the therapeutic effect. In addition, CCL11 could also be used for mRNA vaccine design. To summarize, CCL11 might be a potent T cell enhancer against cancer.

Strategies for acquisition of resonance assignment spectra of highly dynamic membrane proteins: a GPCR case study.

In protein nuclear magnetic resonance (NMR), chemical shift assignment provides a wealth of information. However, acquisition of high-quality solid-state NMR spectra depends on protein-specific dynamics. For membrane proteins, bilayer heterogeneity further complicates this observation. Since the efficiency of cross-polarization transfer is strongly entwined with protein dynamics, optimal temperatures for spectral sensitivity and resolution will depend not only on inherent protein dynamics, but temperature-dependent phase properties of the bilayer environment. We acquired 1-, 2-, and 3D homo- and heteronuclear experiments of the chemokine receptor CCR3 in a 7:3 phosphatidylcholine:cholesterol lipid environment. 1D direct polarization, cross polarization (CP), and T2' experiments indicate sample temperatures below - 25 °C facilitate higher CP enhancement and longer-lived transverse relaxation times. T1rho experiments indicate intermediate timescales are minimized below a sample temperature of - 20 °C. 2D DCP NCA experiments indicated optimal CP efficiency and resolution at a sample temperature of - 30 °C, corroborated by linewidth analysis in 3D NCACX at - 30 °C compared to - 5 °C. This optimal temperature is concluded to be directly related the lipid phase transition, measured to be between - 20 and 15 °C based on rINEPT signal of all-trans and trans-gauche lipid acyl conformations. Our results have critical implications in acquisition of SSNMR membrane protein assignment spectra, as we hypothesize that different lipid compositions with different phase transition properties influence protein dynamics and therefore the optimal acquisition temperature.

C-C Motif Chemokine Receptor 3-Mediated Extracellular Signal-Regulated Kinase 1/2 and p38 Mitogen-Activated Protein Kinase Signaling: Promising Targets for Human Airway Epithelial Mucin 5AC Induction by Eotaxin-2 and Eotaxin-3.

INTRODUCTION: Eotaxin-2 and -3 of the C-C chemokine subfamily function as potent chemoattractant factors for eosinophil recruitment and various immune responses in allergic and inflammatory airway diseases. Mucin 5AC (MUC5AC), a major gel-forming secretory mucin, is overexpressed in airway inflammation. However, the association between mucin secretion and eotaxin-2/3 expression in the upper and lower airway epithelial cells has not been fully elucidated. Therefore, in this study, we investigated the effects of eotaxin-2/3 on MUC5AC expression and its potential signaling mediators. METHODS: We analyzed the effects of eotaxin-2 and -3 on NCI-H292 human airway epithelial cells and primary human nasal epithelial cells (HNEpCs) via reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. Along with immunoblot analyses with specific inhibitors and small interfering RNA (siRNA), we explored the signaling pathway involved in MUC5AC expression following eotaxin-2/3 treatment. RESULTS: In HCI-H292 cells, eotaxin-2/3 activated the mRNA expression and protein production of MUC5AC. A specific inhibitor of C-C motif chemokine receptor 3 (CCR3), SB328437, suppressed eotaxin-2/3-induced MUC5AC expression at both the mRNA and protein levels. Eotaxin-2/3 induced the phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 and p38, whereas pretreatment with a CCR3 inhibitor significantly attenuated this effect. Induction of MUC5AC expression with eotaxin-2/3 was decreased by U0126 and SB203580, specific inhibitors of ERK1/2 and p38 mitogen-activated protein kinase (MAPK), respectively. In addition, cell transfection with ERK1/2 and p38 siRNAs inhibited eotaxin-2/3-induced MUC5AC expression. Moreover, specific inhibitors (SB328437, U0126, and SB203580) attenuated eotaxin-2/3-induced MUC5AC expression in HNEpCs. CONCLUSION: Our results imply that CCR3-mediated ERK1/2 and p38 MAPK are involved in the signal transduction of eotaxin-2/3-induced MUC5AC overexpression.

CD193 (CCR3) expression by B cells correlates with reduced IgE production in paediatric schistosomiasis.

We demonstrate that CD193, the eotaxin receptor, is highly expressed on circulating B cells in paediatric schistosomiasis mansoni. CD193 plays a role in directing granulocytes into sites of allergic-like inflammation in the mucosa, but little is known about its functional significance on human B cells. We sought to characterize CD193 expression and its relationship with S. mansoni infection. We found that CD193+ B cells increased with the intensity of schistosome infection. In addition, a significant negative association was observed between CD193 expression by B cells and IgE production. Decreased IgE levels are generally associated with susceptibility to re-infection. B cell stimulation with eotaxin-1 increased CD193 levels whereas IL-4 led to a reduction. This was supported by plasma levels of eotaxin-1 correlating with CD193 levels on B cells and other cells. In contrast, CD193 expression was induced on naive B cells with a combination of IL-10 and schistosome antigens. Whereas T cells had a modest increase in CD193 expression, only B cell CD193 appeared functionally chemotactic to eotaxin-1. Thus, CD193+ B cells, which co-express CXCR5, may be enroute to sites with allergic-like inflammation, such as gastrointestinal follicles, or even to Th2 granulomas, which develop around parasite eggs. Overall, our results suggest that schistosome infection may promote CD193 expression and suppress IgE via IL-10 and other undefined mechanisms related to B cell trafficking. This study adds to our understanding of why young children may have poor immunity. Nonetheless, praziquantel treatment was shown to reduce percentages of circulating CD193+ B cells lending hope for future vaccine efforts.

Keras/TensorFlow in Drug Design for Immunity Disorders.

Homeostasis of the host immune system is regulated by white blood cells with a variety of cell surface receptors for cytokines. Chemotactic cytokines (chemokines) activate their receptors to evoke the chemotaxis of immune cells in homeostatic migrations or inflammatory conditions towards inflamed tissue or pathogens. Dysregulation of the immune system leading to disorders such as allergies, autoimmune diseases, or cancer requires efficient, fast-acting drugs to minimize the long-term effects of chronic inflammation. Here, we performed structure-based virtual screening (SBVS) assisted by the Keras/TensorFlow neural network (NN) to find novel compound scaffolds acting on three chemokine receptors: CCR2, CCR3, and one CXC receptor, CXCR3. Keras/TensorFlow NN was used here not as a typically used binary classifier but as an efficient multi-class classifier that can discard not only inactive compounds but also low- or medium-activity compounds. Several compounds proposed by SBVS and NN were tested in 100 ns all-atom molecular dynamics simulations to confirm their binding affinity. To improve the basic binding affinity of the compounds, new chemical modifications were proposed. The modified compounds were compared with known antagonists of these three chemokine receptors. Known CXCR3 compounds were among the top predicted compounds; thus, the benefits of using Keras/TensorFlow in drug discovery have been shown in addition to structure-based approaches. Furthermore, we showed that Keras/TensorFlow NN can accurately predict the receptor subtype selectivity of compounds, for which SBVS often fails. We cross-tested chemokine receptor datasets retrieved from ChEMBL and curated datasets for cannabinoid receptors. The NN model trained on the cannabinoid receptor datasets retrieved from ChEMBL was the most accurate in the receptor subtype selectivity prediction. Among NN models trained on the chemokine receptor datasets, the CXCR3 model showed the highest accuracy in differentiating the receptor subtype for a given compound dataset.

Overexpression of VEGF in the MOPC 315 Plasmacytoma Induces Tumor Immunity in Mice.

Vascular endothelial growth factor (VEGF) has important effects on hematopoietic and immune cells. A link between VEGF expression, tumor progression, and metastasis has been established in various solid tumors; however, the impact of VEGF expression by hematopoietic neoplasias remains unclear. Here, we investigated the role of VEGF in plasma cell neoplasia. Overexpression of VEGF in MOPC 315 tumor cells (MOPCSVm) had no effect on their growth in vitro. However, constitutive ectopic expression of VEGF dramatically reduced tumorigenicity of MOPC 315 when implanted subcutaneously into BALB/c mice. Mice implanted with MOPCSVm effectively rejected tumor grafts and showed strong cytotoxic T lymphocyte (CTL) activity against parental MOPC 315 cells. MOPCSVm implants were not rejected in nude mice, suggesting the process is T-cell-dependent. Adoptive transfer of splenocytes from recipients inoculated with MOPCSVm cells conferred immunity to naïve BALB/c mice, and mice surviving inoculation with MOPCSVm rejected the parental MOPC 315 tumor cells following a second inoculation. Immunohistochemical analysis showed that MOPCSVm induced a massive infiltration of CD3+ cells and MHC class II+ cells in vivo. In addition, exogenous VEGF induced the expression of CCR3 in T cells in vitro. Together, these data are the first to demonstrate that overexpression of VEGF in plasmacytoma inhibits tumor growth and enhances T-cell-mediated antitumor immune response.

Expression of the Chemokine Receptor CCR1 in Burkitt Lymphoma Cell Lines Is Linked to the CD10-Negative Cell Phenotype and Co-Expression of the EBV Latent Genes EBNA2, LMP1, and LMP2.

Chemokines and their receptors regulate the migration of immune cells and the dissemination of cancer cells. CCR1, CCR2, CCR3, and CCR5 all belong to a single protein homology cluster and respond to the same inflammatory chemokines. We previously reported that CCR1 and CCR2B are induced upon Epstein-Barr virus (EBV) infection of B cells in vitro. EBV is present in almost all cases of endemic Burkitt lymphoma (BL); however, the contribution of EBV in the pathogenesis of the disease is not fully understood. Here, we analyzed the relation of the expression of CCR1, CCR2, CCR3, and CCR5, the EBV DNA load and expression of EBV latent genes in nine EBV-carrying and four EBV-negative BL cell lines. We revealed that CCR1 is expressed at high mRNA and protein levels in two CD10-negative BL cell lines with co-expression of the EBV latent genes EBNA2, LMP1, and LMP2. Low levels of CCR2 transcripts were found in three BL cell lines. CCR3 and CCR5 transcripts were hardly detectable. Our data suggest that in vivo, CCR1 may be involved in the dissemination of BL cells and in the selection of BL cells with restricted EBV gene expression programs.

Potent CCR3 Receptor Antagonist, SB328437, Suppresses Colonic Eosinophil Chemotaxis and Inflammation in the Winnie Murine Model of Spontaneous Chronic Colitis.

Eosinophils and their regulatory molecules have been associated with chronic intestinal inflammation and gastrointestinal dysfunctions; eosinophil accumulation in the gut is prominent in inflammatory bowel disease (IBD). The chemokine receptor CCR3 plays a pivotal role in local and systemic recruitment and activation of eosinophils. In this study, we targeted CCR3-ligand interactions with a potent CCR3 receptor antagonist, SB328437, to alleviate eosinophil-associated immunological responses in the Winnie model of spontaneous chronic colitis. Winnie and C57BL/6 mice were treated with SB328437 or vehicle. Clinical and histopathological parameters of chronic colitis were assessed. Flow cytometry was performed to discern changes in colonic, splenic, circulatory, and bone marrow-derived leukocytes. Changes to the serum levels of eosinophil-associated chemokines and cytokines were measured using BioPlex. Inhibition of CCR3 receptors with SB328437 attenuated disease activity and gross morphological damage to the inflamed intestines and reduced eosinophils and their regulatory molecules in the inflamed colon and circulation. SB328437 had no effect on eosinophils and their progenitor cells in the spleen and bone marrow. This study demonstrates that targeting eosinophils via the CCR3 axis has anti-inflammatory effects in the inflamed intestine, and also contributes to understanding the role of eosinophils as potential end-point targets for IBD treatment.

Menthone Inhalation Alleviates Local and Systemic Allergic Inflammation in Ovalbumin-Sensitized and Challenged Asthmatic Mice.

Menthone is rich in Mentha × Piperita L. essential oil and it has anti-inflammatory properties; research shows that it is useful, via percutaneous absorption, in treating inflammation-related diseases. However, anti-allergic inflammatory effects of volatile menthone have not yet been used to treat allergic asthma, in vivo. We hypothesized that menthone inhalation may have anti-inflammatory and anti-allergic effects in patients with allergic asthma. Therefore, in our study, menthone inhalation was used to treat ovalbumin (OVA)-sensitized and challenged asthmatic mice. Allergic inflammation mediator changes in the lungs and airways, sera, splenocytes, and peritoneal macrophages of the mice were measured. Relative expression amounts of six receptor genes related to allergic inflammation of the lungs and airways were quantitated using a two-step real time quantitative polymerase chain reaction (qPCR). Results showed that menthone inhalation increased serum OVA-specific IgG2a/IgG1 and IgG2a/IgE ratios, increased Th1-type cytokine production in the bronchoalveolar lavage fluid, and decreased nitric oxide, protein, and eotaxin levels. Menthone inhalation inhibited mast cell and eosinophil degranulation, and chemokine (C-C motif) receptor 3 (Ccr3) gene expression amounts, but (relatively) increased Th1 cytokine secretion by splenocytes. Our results evidence that menthone inhalation alleviates local and systemic allergic inflammation in asthmatic mice.

Platelets Independently Recruit into Asthmatic Lungs and Models of Allergic Inflammation via CCR3.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

Asthma-related inflammation promotes lung metastasis of breast cancer cells through CCL11-CCR3 pathway.

BACKGROUND: Mechanisms that preclude lung metastasis are still barely understood. The possible consequences of allergic airways inflammation on cancer dissemination were studied in a mouse model of breast cancer. METHODS: Balb/c mice were immunized and daily exposed to ovalbumin (OVA) from day 21. They were subcutaneously injected with 4T1 mammary tumor cells on day 45 and sacrificed on day 67. Lung metastases were measured by biophotonic imaging (IVIS® 200 Imaging System) and histological measurement of tumor area (Cytomine software). Effects of CCL11 were assessed in vivo by intratracheal instillations of recCCL11 and in vitro using Boyden chambers. CCR3 expression on cell surface was assessed by flow cytometry. RESULTS: The extent of tumor metastases was significantly higher in lungs of OVA-exposed mice and increased levels of CCL11 expression were measured after OVA exposure. Migration of 4T1 cells and neutrophils was stimulated in vitro and in vivo by recCCL11. 4T1 cells and neutrophils express CCR3 as shown by flow cytometry and a selective CCR3 antagonist (SB-297006) inhibited the induction of 4T1 cells migration and proliferation in response to recCCL11. CONCLUSIONS: Allergic inflammation generated by exposure to allergens triggers the implantation of metastatic cells from primary breast tumor into lung tissues plausibly in a CCL11-CCR3-dependent manner. This indicates that asthma related inflammation in lungs might be a risk factor for lung metastasis in breast cancer patients.

CCR3 gene overexpression in patients with Down syndrome.

Chromosome 21 trisomy or Down syndrome (DS) is the most common genetic cause of intellectual disability (ID). DS is also associated with hypotonia, muscle weakness, autoimmune diseases, and congenital heart disease. C-C chemokine receptor type 3 (CCR3) plays a role in inflammatory, autoimmune, and neuronal migration mechanisms. The present study aimed to evaluate the expression of the CCR3 gene by NGS and qRT-PCR in patients with DS and normal controls (NC). The CCR3 gene was over-expressed in DS patients compared to NC. These data suggest that an over-expression of the CCR3 gene is associated with the phenotype of patients with DS.

The Chemokine Receptor CCR3 Is Potentially Involved in the Homing of Prostate Cancer Cells to Bone: Implication of Bone-Marrow Adipocytes.

Bone metastasis remains the most frequent and the deadliest complication of prostate cancer (PCa). Mechanisms leading to the homing of tumor cells to bone remain poorly characterized. Role of chemokines in providing navigational cues to migrating cancer cells bearing specific receptors is well established. Bone is an adipocyte-rich organ since 50 to 70% of the adult bone marrow (BM) volume comprise bone marrow adipocytes (BM-Ads), which are likely to produce chemokines within the bone microenvironment. Using in vitro migration assays, we demonstrated that soluble factors released by human primary BM-Ads are able to support the directed migration of PCa cells in a CCR3-dependent manner. In addition, we showed that CCL7, a chemokine previously involved in the CCR3-dependent migration of PCa cells outside of the prostate gland, is released by human BM-Ads. These effects are amplified by obesity and ageing, two clinical conditions known to promote aggressive and metastatic PCa. In human tumors, we found an enrichment of CCR3 in bone metastasis vs. primary tumors at mRNA levels using Oncomine microarray database. In addition, immunohistochemistry experiments demonstrated overexpression of CCR3 in bone versus visceral metastases. These results underline the potential importance of BM-Ads in the bone metastatic process and imply a CCR3/CCL7 axis whose pharmacological interest needs to be evaluated.

Analysis of CCR3 expression in corneal neovascularization in a murine model and human corneas.

The aim of this study was to examine the expression of the cytokines and chemokines receptor-3 (CCR3) molecule in endothelial cells and vascular structures in a murine model of corneal neovascularization and in samples of neovascularized human corneas. An immunofluorescence assay using the murine model showed a greater proportion and intensity of CCR3 in the epithelium and corneal subepithelial regions in corneas with neovascularization. In the absence of vascularization, no CCR3 was found. Of the 32 studied tissues, eight were vascularized and 24 were avascular. In the human corneas, vascularized corneas showed positive labeling for CD31 in all the analzedtissues, as well as positive labeling for CCR3. Therefore, all vascularized tissues showed positive coexpression of CCR3 and CD31, whereas none of the avascular corneas showed immunolabeling for either of these receptors. These results suggest that CCR3 could be a possible marker for corneal neovascularization with potential to be a therapeutic target.

Bioinformatic analysis of eosinophil activity and its implications for model and target species.

Eosinophils are important immune cells that have been implicated in resistance to gastrointestinal nematode (GIN) infections in both naturally and experimentally infected sheep. Proteins of particular importance appear to be IgA-Fc alpha receptor (FcαRI), C-C chemokine receptor type 3 (CCR3), proteoglycan 3 (PRG3, major basic protein 2) and EPX (eosinophil peroxidase). We used known human nucleotide sequences to search the ruminant genomes, followed by translation to protein and sequence alignments to visualize differences between sequences and species. Where a sequence was retrieved for cow, but not for sheep and goat, this was used additionally as a reference sequence. In this review, we show that eosinophil function varies among host species. Consequently, investigations into the mechanisms of ruminant immune responses to GIN should be conducted using the natural host. Specifically, we address differences in protein sequence and structure for eosinophil proteins.

Novel peptide nanoparticle-biased antagonist of CCR3 blocks eosinophil recruitment and airway hyperresponsiveness.

BACKGROUND: Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason might be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. OBJECTIVE: We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G protein signaling but enable or promote receptor internalization. METHODS: Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by means of dynamic light scattering and nuclear magnetic resonance. Inhibitory activity on CCR3 signaling was assessed in vitro by using flow cytometry, confocal microscopy, and Western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed by using a triple-allergen mouse asthma model. RESULTS: R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. Half-maximal inhibitory concentration values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of extracellular signal-regulated kinase 1/2 activation and not the late phase generally associated with ß-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo R321 effectively blocks eosinophil recruitment into the blood, lungs, and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. CONCLUSIONS: R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism might hold significant therapeutic promise by eluding the formation of drug tolerance.

The basophil activation test differentiates between patients with alpha-gal syndrome and asymptomatic alpha-gal sensitization.

BACKGROUND: Galactose-alpha-1,3-galactose (alpha-gal) syndrome is characterized by the presence of serum specific IgE antibodies to alpha-gal and delayed type I allergic reactions to the carbohydrate alpha-gal after consumption of mammalian (red) meat products and drugs of mammalian origin. Diagnostics currently rely on patient history, skin tests, determination of serum specific IgE antibodies, and oral food or drug challenges. OBJECTIVE: We sought to assess the utility of different basophil parameters (basophil reactivity and sensitivity, the ratio of the percentage of CD63+ basophils induced by the alpha-gal-containing allergen to the percentage of CD63+ basophils after stimulation with anti-FcεRI antibody [%CD63+/anti-FcεRI], and area under the dose-response curve [AUC]) as biomarkers for the clinical outcome of patients with alpha-gal syndrome compared with subjects with asymptomatic alpha-gal sensitization. METHODS: In addition to routine diagnostics, a basophil activation test (Flow CAST) with different concentrations of alpha-gal-containing allergens (eg, commercially available alpha-gal-carrying proteins and pork kidney extracts) was performed in 21 patients with alpha-gal syndrome, 12 alpha-gal-sensitized subjects, and 18 control subjects. RESULTS: Alpha-gal-containing allergens induced strong basophil activation in a dose-dependent manner in patients. Basophil reactivity at distinct allergen concentrations, the %CD63+/anti-FcεRI ratio across most allergen concentrations, the AUC of dose-response curves, and basophil allergen threshold sensitivity (CD-sens) with pork kidney extract were significantly higher in patients with alpha-gal syndrome compared with those in sensitized subjects. All parameters were negative in control subjects. CONCLUSION: The basophil activation test should be considered as an additional diagnostic test before performing time-consuming and potentially risky oral provocation tests. The %CD63+/anti-FcεRI ratio for all allergens and AUCs for pork kidney were the best parameters for distinguishing patients with alpha-gal syndrome from subjects with asymptomatic alpha-gal sensitization.