Systemic Treatment with Fas-Blocking Peptide Attenuates Apoptosis in Brain Ischemia.

Apoptosis plays a crucial role in neuronal injury, with substantial evidence implicating Fas-mediated cell death as a key factor in ischemic strokes. To address this, inhibition of Fas-signaling has emerged as a promising strategy in preventing neuronal cell death and alleviating brain ischemia. However, the challenge of overcoming the blood-brain barrier (BBB) hampers the effective delivery of therapeutic drugs to the central nervous system (CNS). In this study, we employed a 30 amino acid-long leptin peptide to facilitate BBB penetration. By conjugating the leptin peptide with a Fas-blocking peptide (FBP) using polyethylene glycol (PEG), we achieved specific accumulation in the Fas-expressing infarction region of the brain following systemic administration. Notably, administration in leptin receptor-deficient db/db mice demonstrated that leptin facilitated the delivery of FBP peptide. We found that the systemic administration of leptin-PEG-FBP effectively inhibited Fas-mediated apoptosis in the ischemic region, resulting in a significant reduction of neuronal cell death, decreased infarct volumes, and accelerated recovery. Importantly, neither leptin nor PEG-FBP influenced apoptotic signaling in brain ischemia. Here, we demonstrate that the systemic delivery of leptin-PEG-FBP presents a promising and viable strategy for treating cerebral ischemic stroke. Our approach not only highlights the therapeutic potential but also emphasizes the importance of overcoming BBB challenges to advance treatments for neurological disorders.

The requirement of IRE1 and XBP1 in resolving physiological stress during Drosophila development.

IRE1 mediates the unfolded protein response (UPR) in part by regulating XBP1 mRNA splicing in response to endoplasmic reticulum (ER) stress. In cultured metazoan cells, IRE1 also exhibits XBP1-independent biochemical activities. IRE1 and XBP1 are developmentally essential genes in Drosophila and mammals, but the source of the physiological ER stress and the relative contributions of XBP1 activation versus other IRE1 functions to development remain unknown. Here, we employed Drosophila to address this question. Explicitly, we find that specific regions of the developing alimentary canal, fat body and the male reproductive organ are the sources of physiological stress that require Ire1 and Xbp1 for resolution. In particular, the developmental lethality associated with an Xbp1 null mutation was rescued by transgenic expression of Xbp1 in the alimentary canal. The domains of IRE1 that are involved in detecting unfolded proteins, cleaving RNAs and activating XBP1 splicing were all essential for development. The earlier onset of developmental defects in Ire1 mutant larvae compared to in Xbp1-null flies supports a developmental role for XBP1-independent IRE1 RNase activity, while challenging the importance of RNase-independent effector mechanisms of Drosophila IRE1 function.

Delivery of Hypoxia-Inducible Heme Oxygenase-1 Gene for Site-Specific Gene Therapy in the Ischemic Stroke Animal Model.

PURPOSE: To reduce side effects due to non-specific expression, the heme oxygenase-1 (HO-1) gene under control of a hypoxia-inducible erythropoietin (Epo) enhancer (pEpo-SV-HO-1) was developed for site-specific gene therapy of ischemic stroke. METHODS: pEpo-SV-HO-1 was constructed by insertion of the Epo enhancer into pSV-HO-1. Dexamethasone-conjugated polyamidoamine (PAMAM-Dexa) was used as a gene carrier. In vitro transfection assays were performed in the Neuro2A cells. In vivo efficacy of pEpo-SV-HO-1 was evaluated in the transient middle cerebral artery occlusion (MCAO) model. RESULTS: In vitro transfection assay with the PAMAM-Dexa/pEpo-SV-HO-1 complex showed that pEpo-SV-HO-1 had higher HO-1 gene expression than pSV-HO-1 under hypoxia. In addition, pEpo-SV-HO-1 reduced the level of apoptosis more efficiently than pSV-HO-1 in Neuro2A cells under hypoxia. For in vivo evaluation, the PAMAM-Dexa/pEpo-SV-HO-1 complex was injected into the ischemic brain of the transient MCAO model. pEpo-SV-HO-1 increased HO-1 expression and reduced the number of apoptotic cells in the ischemic brain, compared with the pSV-HO-1 injection group. As a result, the infarct volume was more efficiently decreased by pEpo-SV-HO-1 than by pSV-HO-1. CONCLUSIONS: pEpo-SV-HO-1 induced HO-1 gene expression and therapeutic effect in the ischemic brain. Therefore, pEpo-SV-HO-1 may be useful for site-specific gene therapy of ischemic stroke.

Conditioned medium of adipose-derived stromal cell culture in three-dimensional bioreactors for enhanced wound healing.

BACKGROUND: It was previously shown that human adipose-derived stromal cell (hADSC)-conditioned medium (CM) promotes wound healing. An essential part of the wound healing process is neovascularization in the wound bed. MATERIALS AND METHODS: We hypothesized that CM prepared from hADSCs cultured as spheroids in three-dimensional suspension bioreactors (spheroid CM) would contain much higher concentrations of angiogenic growth factors secreted by hADSCs, induce a higher extent of neovascularization in the wound bed, and improve wound healing as compared with CM prepared by conventional monolayer culture (monolayer CM). RESULTS: The concentrations of angiogenic growth factors (i.e., vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor) in spheroid CM were 20- to 145-fold higher than those in monolayer CM. Either fresh medium, monolayer CM, or spheroid CM was administered to full-thickness wounds created on the dorsal aspects of athymic mice. The monolayer CM promoted wound healing as compared with fresh medium or no treatment. Importantly, wound closure was faster, and dermal and epidermal regeneration was improved in the spheroid CM-treated mice compared with that in the monolayer CM-treated mice. CONCLUSIONS: The improved wound healing by spheroid CM may be attributed, at least in part, to enhanced neovascularization in the wound beds. The spheroid-based CM approach showed potential as a therapy for skin wound repair.

Proteomic analysis in pterygium; upregulated protein expression of ALDH3A1, PDIA3, and PRDX2.

PURPOSE: To identify differentially expressed proteins in the pterygium compared to healthy conjunctiva using a proteomic analysis. METHODS: Pterygial and healthy conjunctival tissues were obtained from 24 patients undergoing pterygium excision. Total proteins of the pterygia and healthy conjunctiva were analyzed with one-dimensional electrophoresis, and protein bands of interest were excised and subjected to liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-MS/MS) using Thermo's Finnigan ProteomeX workstation LTQ linear ion trap MS/MS. Using bioinformatics, differentially expressed proteins were classified, and three proteins closely involved in the response to oxidative stress were selected for further validation. Differential expression of these proteins was confirmed with western blot and immunohistochemistry. RESULTS: A web-based gene ontology program, DAVID, was used to classify 230 proteins that were differentially expressed in pterygial tissues. Among these genes, we chose three proteins, aldehyde dehydrogenase, dimeric NADP-preferring (ALDH3A1), protein disulfide-isomerase A3 (PDIA3), and peroxiredoxin-2 (PRDX2), that were significantly upregulated in pterygium and further increased in recurrent pterygium. Immunohistochemistry and western blot analysis confirmed that these three proteins were mainly detected in the basal epithelial layer, and their expression was significantly increased in the pterygium compared to normal conjunctiva. CONCLUSIONS: This study reported increased expression of ALDH3A1, PDIA3, and PRDX2 in pterygia using a proteomic approach. These proteins are presumed to have a protective role against oxidative stress-induced apoptosis. This result is consistent with the hypothesis that oxidative stress is a significant factor in the pathogenesis of pterygia.

Calnexin as a dual-role biomarker: antibody-based diagnosis and therapeutic targeting in lung cancer.

Lung cancer carries one of the highest mortality rates among all cancers. It is often diagnosed at more advanced stages with limited treatment options compared to other malignancies. This study focuses on calnexin as a potential biomarker for diagnosis and treatment of lung cancer. Calnexin, a molecular chaperone integral to N-linked glycoprotein synthesis, has shown some associations with cancer. However, targeted therapeutic or diagnostic methods using calnexin have been proposed. Through 1D-LCMSMS, we identified calnexin as a biomarker for lung cancer and substantiated its expression in human lung cancer cell membranes using Western blotting, flow cytometry, and immunocytochemistry. Anti-calnexin antibodies exhibited complement-dependent cytotoxicity to lung cancer cell lines, resulting in a notable reduction in tumor growth in a subcutaneous xenograft model. Additionally, we verified the feasibility of labeling tumors through in vivo imaging using antibodies against calnexin. Furthermore, exosomal detection of calnexin suggested the potential utility of liquid biopsy for diagnostic purposes. In conclusion, this study establishes calnexin as a promising target for antibody-based lung cancer diagnosis and therapy, unlocking novel avenues for early detection and treatment. [BMB Reports 2024; 57(3): 155-160].

Tumor-specific cytolysis by peptide-conjugated echogenic polymer micelles.

Interest in multifunctional polymer nanoparticles for targeted delivery of anti-cancer drugs has grown significantly in recent years. In this study, tumor-targeting echogenic polymer micelles were prepared from poly(ethylene glycol) methyl ether-alkyl carbonate (mPEG-AC) derivatives, and their potential in cancer therapy was assessed. Various mPEG derivatives with carbonate linkages were synthesized via an alkyl halide reaction between mPEG and alkyl chloroformate. Micelle formation using polymer amphiphiles in aqueous media and the subsequent carbon dioxide (CO2) gas generation from the micelles was confirmed. Their ability to target neuroblastoma was substantially enhanced by incorporating the rabies virus glycoprotein (RVG) peptide. RVG-modified gas-generating micelles significantly inhibited tumor growth in a tumor-bearing mouse model owing to CO2 gas generation within tumor cells and resultant cytolytic effects, showing minimal side effects. The development of multifunctional polymer micelles may offer a promising therapeutic approach for various diseases, including cancer.

Intranasal Delivery of Anti-Apoptotic siRNA Complexed with Fas-Signaling Blocking Peptides Attenuates Cellular Apoptosis in Brain Ischemia.

Ischemic stroke-induced neuronal cell death leads to the permanent impairment of brain function. The Fas-mediating extrinsic apoptosis pathway and the cytochrome c-mediating intrinsic apoptosis pathway are two major molecular mechanisms contributing to neuronal injury in ischemic stroke. In this study, we employed a Fas-blocking peptide (FBP) coupled with a positively charged nona-arginine peptide (9R) to form a complex with negatively charged siRNA targeting Bax (FBP9R/siBax). This complex is specifically designed to deliver siRNA to Fas-expressing ischemic brain cells. This complex enables the targeted inhibition of Fas-mediating extrinsic apoptosis pathways and cytochrome c-mediating intrinsic apoptosis pathways. Specifically, the FBP targets the Fas/Fas ligand signaling, while siBax targets Bax involved in mitochondria disruption in the intrinsic pathway. The FBP9R carrier system enables the delivery of functional siRNA to hypoxic cells expressing the Fas receptor on their surface-a finding validated through qPCR and confocal microscopy analyses. Through intranasal (IN) administration of FBP9R/siCy5 to middle cerebral artery occlusion (MCAO) ischemic rat models, brain imaging revealed the complex specifically localized to the Fas-expressing infarcted region but did not localize in the non-infarcted region of the brain. A single IN administration of FBP9R/siBax demonstrated a significant reduction in neuronal cell death by effectively inhibiting Fas signaling and preventing the release of cytochrome c. The targeted delivery of FBP9R/siBax represents a promising alternative strategy for the treatment of brain ischemia.

Drug delivery systems for the treatment of ischemic stroke.

Stroke is the third leading cause of death in the United States. Reduced cerebral blood flow causes acute damage to the brain due to excitotoxicity, reactive oxygen species (ROS), and ischemia. Currently, the main treatment for stroke is to revive the blood flow by using thrombolytic agents. Reviving blood flow also causes ischemia-reperfusion (I/R) damage. I/R damage results from inflammation and apoptosis and can persist for days to weeks, increasing the infarct size. Drugs can be applied to stroke to intervene in the sub-acute and chronic phases. Chemical, peptide, and genetic therapies have been evaluated to reduce delayed damage to the brain. These drugs have different characteristics, requiring that delivery carriers be developed based on these characteristics. The delivery route is another important factor affecting the efficiency of drug delivery. Various delivery routes have been developed, such as intravenous injection, intranasal administration, and local direct injection to overcome the blood-brain-barrier (BBB). In this review, the delivery carriers and delivery routes for peptide and gene therapies are discussed and examples are provided. Combined with new drugs, drug delivery systems will eventually provide useful treatments for ischemic stroke.

Rapid Assessment of Di(2-ethylhexyl) Phthalate Migration from Consumer PVC Products.

Poly(vinyl chloride) (PVC) is widely used to produce various consumer goods, including food packaging, toys for children, building materials, and cosmetic products. However, despite their widespread use, phthalate plasticizers have been identified as endocrine disruptors, which cause adverse health effects, thus leading to increasing concerns regarding their migration from PVC products to the environment. This study proposed a method for rapidly measuring the migration of phthalates, particularly di(2-ethylhexyl) phthalate (DEHP), from PVC products to commonly encountered liquids. The release of DEHP under various conditions, including exposure to aqueous and organic solvents, different temperatures, and household microwaves, was investigated. The amount of DEHP released from both laboratory-produced PVC films and commercially available PVC products was measured to elucidate the potential risks associated with its real-world applications. Furthermore, tests were performed to evaluate cytotoxicity using estrogen-dependent and -independent cancer cell lines. The results revealed a dose-dependent impact on estrogen-dependent cells, thus emphasizing the potential health implications of phthalate release. This comprehensive study provides valuable insights into the migration patterns of DEHP from PVC products and forms a basis for further research on the safety of PVC and plasticizers.

Adipocytolytic Polymer Nanoparticles for Localized Fat Reduction.

The demand for body fat reduction is increasing. However, conventional lipolytic approaches fail to control adipose tissue reduction and cause severe side effects in adjacent nonadipose tissues. A strategy to specifically reduce subcutaneous fat using adipocytolytic polymer nanoparticles in a minimally invasive manner is reported here. The polymer nanoparticles are designed to generate carbon dioxide gas when selectively absorbed by adipocytes. The carbon dioxide gas generated within late endosomes/lysosomes induces adipocytolysis, thereby reducing the number of cells. Localized injection of the adipocytolytic nanoparticles substantially reduces subcutaneous fat in a high-fat diet-induced obese mouse model, without significant changes in hematological or serum biochemical parameters. The adipocytolytic efficacy of the nanoparticles is also evaluated in a porcine model. This strategy addresses the need to develop safe and effective adipocytolytic agents using functional polymer nanoparticles.

T cell-specific siRNA delivery using antibody-conjugated chitosan nanoparticles.

The intracellular delivery of small interfering RNA (siRNA) plays a key role in RNA interference (RNAi) and provides an emerging technique to treat various diseases, including infectious diseases. Chitosan has frequently been used in gene delivery applications, including siRNA delivery. However, studies regarding the modification of chitosan with antibodies specifically targeting T cells are lacking. We hypothesized that chitosan nanoparticles modified with T cell-specific antibodies would be useful for delivering siRNA to T cells. CD7-specific single-chain antibody (scFvCD7) was chemically conjugated to chitosan by carbodiimide chemistry, and nanoparticles were prepared by a complex coacervation method in the presence of siRNA. The mean diameter and zeta potential of the scFvCD7-chitosan/siRNA nanoparticles were approximately 320 nm and +17 mV, respectively, and were not significantly influenced by the coupling of antibody to chitosan. The cellular association of antibody-conjugated nanoparticles to CD4+ T cell lines as well as gene silencing efficiency in the cells was significantly improved compared to nonmodified chitosan nanoparticles. This approach to introducing T cell-specific antibody to chitosan nanoparticles may find useful applications for the treatment of various infectious diseases.

Identification and validation of SAA as a potential lung cancer biomarker and its involvement in metastatic pathogenesis of lung cancer.

Lung cancer is recently regarded as an overhealed inflammatory disease. Serum amyloid A (SAA) is known as an acute phase protein, but it is likely involved in the cancer pathogenesis. We identified both SAA1 and SAA2 in the pooled sera of lung cancer patients but not in the healthy control, by LC-MS/MS analysis. We found that about 14-fold higher levels of SAA in lung cancer patients' sera and plasma compared to healthy controls by ELISA using total 350 samples (13.89 ± 37.18 vs 190.49 ± 234.70 ug/mL). The SAA levels were also significantly higher than in other pulmonary disease or other cancers. An immunohistochemical study using tissue microarray showed that, unlike other cancer tissues, lung cancer tissues highly express SAA. Further in vitro experiments showed that SAA is induced from lung cancer cells by the interaction with THP-1 monocytes and this, in return, induces MMP-9 from THP-1. In in vivo animal models, overexpressed SAA promoted Lewis lung carcinoma (LLC) cells to metastasize and colonize in the lung. Our data suggest that a higher concentration of SAA can serve as an indicator of lung adenocarcinoma and represents a therapeutic target for the inhibition of lung cancer metastasis.

Vacuolar (H+)-ATPases in Caenorhabditis elegans: what can we learn about giant H+ pumps from tiny worms?

Vacuolar (H(+))-ATPases, also called V-ATPases, are ATP-driven proton pumps that are highly phylogenetically conserved. Early biochemical and cell biological studies have revealed many details of the molecular mechanism of proton pumping and of the structure of the multi-subunit membrane complex, including the stoichiometry of subunit composition. In addition, yeast and mouse genetics have broadened our understanding of the physiological consequences of defective vacuolar acidification and its related disease etiologies. Recently, phenotypic investigation of V-ATPase mutants in Caenorhabditis elegans has revealed unexpected new roles of V-ATPases in both cellular function and early development. In this review, we discuss the functions of the V-ATPases discovered in C. elegans.

Application of proteomics in asthma research.

Bronchial asthma is caused by allergic airway inflammation, resulting in reversible airway obstruction, characterized by airway hyper-responsiveness, bronchoconstriction, increased mucus secretion and an increase in lung vessel permeability. The pathophysiological changes in asthma have been attributed to the altered expression of biologically plausible proteins associated with transcriptional pathways, inflammatory mediators, chemokines, cytokines, apoptosis and cell proliferation. Such multifactorial diseases characteristically involve an interplay of many genetic variations of molecular and biochemical pathways and their interactions with environmental factors. The complex nature of the asthma phenotype, together with genetic heterogeneity and environmental influences, has made it difficult to uncover the aspects that underlie this common disease. Recently, genomic and proteomic technologies have been developed to identify associations between genes, proteins and disease. This approach, called 'omics biology', aims to recognize early onset of disease, institute preventive treatment and identify new molecular targets for novel drugs in multifactorial diseases. This article reviews examples of how proteomic technology can be used to find asthma marker proteins (from the cell model to clinical samples). Identification of protein changes in different stages of asthma could provide further insights into the complex molecular mechanisms involved in this disease. These studies provide new insights for finding novel pathological mediators and biomarkers of asthma.

Skin regeneration with fibroblast growth factor 2 released from heparin-conjugated fibrin.

Fibroblast growth factor 2 (FGF2) stimulates skin wound healing but does long-term delivery of FGF2 enhance skin regeneration compared to short-term delivery? Heparin-conjugated fibrin (HCF) was used as a vehicle for long-term delivery of FGF2. Fibrin, HCF, FGF2-loaded fibrin, and FGF2-loaded HCF were implanted into full-thickness skin defects of mice. The neoepidermis thickness was significantly larger in the FGF2-loaded HCF group than in the other groups, except for the FGF2-loaded fibrin group. Suprabasal cytokeratin differentiation in squamous neoepithelium was greatest in the FGF2-loaded HCF group. The enhanced skin regeneration accompanying the long-term delivery of FGF2 could be mediated, at least partially, by enhanced neovascularization and cell proliferation in the neodermis.

Targeted delivery of Chil3/Chil4 siRNA to alveolar macrophages using ternary complexes composed of HMG and oligoarginine micelles.

Cell-type-specific genes involved in disease can be effective therapeutic targets; therefore, the development of a cell-type-specific gene delivery system is essential. In this study, targeted delivery of Chil3 and Chil4 siRNA to activated macrophages was developed using a ligand called high mobility group (HMG) and oligoarginine (OR) micelles. HMG binds to TLR4 and RAGE located on the surface of activated macrophages. Since HMG is positively charged, it binds to the negatively charged siRNA by charge interaction. However, the stable formation of the siRNA/HMG complex requires an additional molecule to act as a carrier. In this study, OR micelles were used as the carrier. Gel retardation assays showed that siRNA, HMG, and OR micelles formed stable siRNA/HMG/OR micelle ternary complexes. In vitro transfection showed that the ternary complexes selectively delivered siRNA to TLR4 expressing macrophages. In addition, intratracheal administration of siRNA/HMG/OR ternary complexes delivered Chil3 and Chil4 siRNA specifically to alveolar macrophages. Furthermore, the siRNA that was delivered using ternary complexes reduced Chil3 and Chil4 expression and suppressed the symptoms of asthma, such as airway inflammation and mucin secretion.

Genetic effect of CysLTR2 polymorphisms on its mRNA synthesis and stabilization.

BACKGROUND: We previously demonstrated that single nucleotide polymorphism (SNP) and haplotypes were associated with aspirin hypersensitivity in asthmatics. We investigated the genetic effects of the SNPs and haplotypes on the expression of the CysLTR2 gene. METHODS: We measured CysLTR2 protein and mRNA expression in EB virus-infected B cell lines from asthmatics having ht1+/+ and ht2+/+. A gel retardation assay was used to identify nuclear protein binding to the c.-819 promoter site. The function of promoter and 3'-UTR were assessed using pGL3 luciferase and pEGFP reporter system, respectively. RESULTS: We found that the expression of CysLTR2 protein was higher in B cell lines of asthmatics having ht2+/+ than in those having ht1+/+. PMA/ionomycin induced higher mRNA expression of CysLTR2 in B cell lines from ht2+/+ asthmatics than those from ht1+/+ asthmatics. A nuclear protein from the B cell lines showed stronger DNA binding affinity with a probe containing c.-819T than one containing c.-819G. The luciferase activity of the c.-819T type of CysLTR2 promoter was higher than that of the c.-819G type. EGFP expression was higher in the EGFP-c.2078T 3'-UTR fusion construct than in the c.2078C construct. CONCLUSION: The sequence variants of CysLTR2 may affect its transcription and the stability of its mRNA, resulting in altered expression of CysLTR2 protein, which in turn causes some asthmatics to be susceptible to aspirin hypersensitivity.

The fast skeletal muscle myosin light chain is differentially expressed in smooth muscle cells of OVA-challenged mouse trachea.

In a search for new molecular pathways associated with asthma, we performed an mRNA differential display analysis using total RNA extracted from the tracheal tissues of ovalbumin (OVA)-challenged mice and sham controls. cDNAs corresponding to mRNAs for which expression levels were altered by OVA-challenge were isolate and sequenced. Twenty-eight genes differentially expressed in sham and OVA challenged mice were identified. A GenBank BLAST homology search revealed that they were related to cytoskeleton remodeling, transcription, protein synthesis and modification, energy production, and cell growth and differentiation. Two were selected for further characterization. Up-regulation of both the perinatal skeletal myosin heavy chain (skMHC) and fast skeletal muscle myosin light chain (skMLC) genes was confirmed by RT-PCR of trachea tissue from OVA challenged mice. Overexpression of skMLC protein was observed in the smooth muscle layers of OVA-challenged mice by immunohistochemistry, and the surface areas stained with skMLC antibody increased in the OVA-challenged mice. The overexpression of skMLC in murine asthma may be associated with the changes of bronchial smooth muscle.

Genetic effect of CCR3 and IL5RA gene polymorphisms on eosinophilia in asthmatic patients.

BACKGROUND: Eosinophilic infiltration and peripheral blood eosinophilia in asthma require the cooperation of eosinophil-specific cytokines and chemokines and their receptors. OBJECTIVE: We investigated the association of polymorphisms in CCR3 and IL5RA with asthma susceptibility or peripheral blood eosinophilia and the effects of the polymorphisms on receptor expression. METHODS: Polymorphisms in CCR3 and IL5RA were identified and genotyped in 576 asthmatic patients and 180 healthy control subjects. CCR3 and IL-5 receptor alpha (IL-5R alpha) protein expression on eosinophils was measured by means of flow cytometry. RESULTS: Although polymorphisms in CCR3 were not associated with asthma susceptibility, the CCR3 haplotype ht2 showed a negative gene dose effect on the eosinophil count (P = .003-.009). IL5RA c.-5091G>A was weakly associated with eosinophil count. The effects of ht2 were greater when paired with IL5RA c.-5091A (P = .001-.002). CCR3 protein expression was higher on eosinophils of asthmatic patients without ht2 than in those with ht2. Asthmatic patients with the IL5RA c.-5091A allele showed higher IL-5R alpha expression than those who were homozygous for the G allele. CONCLUSION: The genetic association between CCR3 polymorphisms and the number of circulating eosinophils was revealed as a novel finding. These associations were more pronounced when the CCR3 polymorphisms were paired with polymorphisms in IL5RA. The protein expression levels of CCR3 and IL-5R alpha on peripheral blood eosinophils are associated with the polymorphisms on their own genes. CLINICAL IMPLICATIONS: The identification of single nucleotide polymorphisms and haplotypes of CCR3 and IL5RA might be useful in developing markers for intermediate phenotypes of eosinophil number and in designing strategies to control diseases related to hypereosinophilia.