Charcot-Leyden Crystal Protein Research Articles

Presence of sputum IgG against eosinophilic inflammatory proteins in asthma.

Sputum immunoglobulin G (Sp-IgG) has been discovered to induce cytolytic extracellular trap cell death in eosinophils, suggesting a potential autoimmune mechanism contributing to asthma. This study aimed to explore the potential origin of Sp-IgG and identify clinically relevant subtypes of Sp-IgG that may indicate autoimmune events in asthma. This study included 165 asthmatic patients and 38 healthy volunteers. We measured Sp-IgG and its five subtypes against eosinophil inflammatory proteins (Sp-IgGEPs), including eosinophil peroxidase, eosinophil major basic protein, eosinophil-derived neurotoxin, eosinophil cationic protein, and Charcot-Leyden Crystal protein in varying asthma severity. Clinical and Mendelian randomization (MR) analyses were conducted. A positive Sp-IgGEPs signature (Sp-IgGEPs+) was defined when any of the five Sp-IgGEPs values exceeded the predefined cutoff thresholds, calculated as the mean values of healthy controls plus twice the standard deviation. The levels of Sp-IgG and Sp-IgGEPs were significantly elevated in moderate/severe asthma than those in mild asthma/healthy groups (all p < 0.05). Sp-IgG levels were positively correlated with airway eosinophil and Sp-IgGEPs. MR analysis showed causality between eosinophil and IgG (OR = 1.02, 95%CI = 1.00-1.04, p = 0.020), and elevated IgG was a risk factor for asthma (OR = 2.05, 95%CI = 1.00-4.17, p = 0.049). Subjects with Sp-IgGEPs+ exhibited worse disease severity and served as an independent risk factor contributing to severe asthma (adjusted-OR = 5.818, adjusted-95% CI = 2.193-15.431, adjusted-p < 0.001). Receiver operating characteristic curve analysis demonstrated that the combination of Sp-IgGEPs+ with non-allergic status, an ACT score < 15, and age ≥ 45 years, effectively predicted severe asthma (AUC = 0.84, sensitivity = 86.20%, specificity = 67.80%). This study identifies a significant association between airway eosinophilic inflammation, Sp-IgG, and asthma severity. The Sp-IgGEPs panel potentially serves as the specific biomarker reflecting airway autoimmune events in asthma.

Chronic rhinosinusitis with nasal polyposis (CRSwNP): the correlation between expression of Galectin-10 and Clinical-Cytological Grading (CCG).

Chronic rhinosinusitis with nasal polyps (CRSwNP) is typically characterized by Type 2 inflammation. Several biomarkers of eosinophilic inflammation, including Galectin-10, also known as Charcot-Leyden crystal protein (CLCP), have been identified to establish eosinophilic infiltration of polyps, a reliable predictor of recurrence.Objective: We aimed to evaluate the Galectin-10 expression in nasal polyps of patients with CRSwNP and to assess the correlation of Charcot-Leyden crystals expression to the severity of CRSwNP according to Clinical-Cytological Grading (CCG). A double-label immunofluorescence was performed to evaluate the expression of Gal-10, CD15, Tryptase, and CD63 and their eventual co-localization on histological samples of 18 patients with CRSwNP. Double-positive Gal-10+CD15+ and Galectin-10+Tryptase+ inflammatory cells were counted by confocal microscopy. Galectin-10 was detectable in all examined tissues from CRSwNP patients, and its expression increased as low, medium and high CCG tissues were examined, respectively. Galectin-10 was extensively present in inflammatory cells, while limited Galectin-10 deposits were detected around mucosal epithelial cells. We showed the strong correlation between CCG and Galectin-10 expression, mainly colocalized with infiltrating eosinophils and mast-cells, in patients affected by CRSwNP.

Predictive Significance of Charcot-Leyden Crystal Protein in Nasal Secretions in Recurrent Chronic Rhinosinusitis with Nasal Polyps

Introduction: The recurrence occurs frequently among patients with chronic rhinosinusitis with nasal polyps (CRSwNP), and predictors that could be conveniently detected during practice in outpatient service are needed. Objective: We aimed to illustrate that the concentration of Charcot-Leyden crystal (CLC) in nasal secretions can effectively and noninvasively predict polyp recurrence. Methods: 108 patients with CRSwNP were divided into recurrence (n = 68) and recurrence-free (n = 40) groups. Preoperative CLC concentrations in nasal secretions were collected and detected by ELISA. Polyp tissues were harvested during biopsy or endoscopic sinus surgery and were evaluated for inflammatory cells by histopathological staining. Demographic information and the clinical characteristics of each patient were reviewed for associations with recurrence. Binary logistic regression analysis was performed to determine predictive factors for polyp recurrence. Receiver operating characteristic (ROC) curves and the Youden index were performed to determine their predictive values. Survival analysis was performed to compare recurrence risk of patients with different CLC concentrations. Results: Sixty-eight (62.96%) patients developed recurrence during a 12- to 33-month postoperative follow-up. CLC concentrations in nasal secretions were positively correlated with eosinophil percent in polyp tissue and peripheral blood and were significantly higher in patients of the recurrence group than in the patients of the recurrence-free group (p < 0.001). Binary logistic regression and ROC curve demonstrated that CLC protein in nasal secretions is predictive of polyp recurrence. According to the Youden index, a CLC concentration of 34.24 ng/mL can predict postoperative polyp recurrence with 92.6% sensitivity and 87.5% specificity. Patients with CLC concentrations higher than the cutoff value yielded a higher risk of recurrence (p < 0.001, HR = 11.31, 95% CI: 6.41–19.98). Conclusions: CLC protein in nasal secretions may serve as a promising noninvasive biomarker to predict CRSwNP recurrence.

Charcot-Leyden Crystal Protein in Nasal Secretions of Patients with Nonallergic Rhinitis with Eosinophilia Syndrome

Introduction: Charcot-Leyden crystal (CLC) protein has been regarded as a hallmark of eosinophilic inflammation. Objective: The purpose of this study was to investigate the role and levels of CLC protein in patients with nonallergic rhinitis with eosinophilia syndrome (NARES). Methods: Overall, 39 NARES patients and 19 controls were recruited. The severity of nasal symptoms was measured by visual analogue scale and serum and local specific immunoglobulin E were determined in all patients. Nasal eosinophilia was assessed by semiquantitative analysis of eosinophils in nasal scrapings. Nasal secretion CLC protein concentrations were evaluated by ELISA. Results: CLC protein concentrations were significantly higher in NARES patients than in controls (p < 0.0001). Nasal secretion CLC protein levels were significantly correlated with the degree of eosinophilia in nasal scrapings (r_s = 0.331; p = 0.04) in NARES patients. Patients with high CLC protein concentrations displayed more severe nasal symptoms than patients with low CLC protein concentrations (p = 0.0080), particularly, nasal itching (p = 0.0029). Pilot study in 8 NARES patients demonstrated that treatment for 1 month with intranasal fluticasone propionate significantly decreased the nasal secretion CLC protein concentrations from baseline levels (p = 0.0335) and markedly attenuated the degree of swelling of inferior turbinate. Conclusions: CLC protein levels are significantly higher in nasal secretions of NARES patients and associated with the degree of nasal eosinophilia and the severity of nasal symptoms. Significantly, nasal secretion CLC protein levels obviously decreased after treatment with intranasal corticosteroids, suggesting its possible role in evaluating the medical treatment.

The Charcot-Leyden crystal protein revisited-A lysopalmitoylphospholipase and more.

The Charcot-Leyden crystal protein (CLC-P), a constituent of human and not mouse eosinophils, is one of the most abundant proteins within human eosinophils. It has a propensity to form crystalline structures, Charcot-Leyden crystals, which are hallmarks in their distinctive extracellular crystalline forms as markers of eosinophilic inflammation. The functions of CLC-P within eosinophils have been uncertain. Although the action of CLC-P as a lysophospholipase has been questioned, assays of chromatographically purified CLC-P and crystal-derived CLC-P as well as studies of transfected recombinant CLC-P have consistently documented that CLC-P endogenously expresses lysophospholipase activity, releasing free palmitate from substrate lysopalmitoylphosphatidylcholine. Rather than acting solely as a hydrolytic enzyme to release palmitate from a lysolipid substrate, some other lysophospholipases function more dominantly as acyl-protein thioesterases (APTs), enzymes that catalyze the removal of thioester-linked, long chain fatty acids, such as palmitate, from cysteine residues of proteins. As such APTs participate in palmitoylation, a post-translational modification that can affect membrane localization, vesicular transport, and secretion. CLC-P has attributes of an APT. Thus, whereas CLC-P expresses inherent lysophospholipase activity, like some other lysophospholipase enzymes, it likely also functions in regulating the dynamic palmitoylation cycle, including, given its dominant subplasmalemmal location, at the human eosinophil's plasma membrane.

Galectin-10, the protein that forms Charcot-Leyden crystals, is not stored in granules but resides in the peripheral cytoplasm of human eosinophils.

A predominant protein of human eosinophils is galectin-10 (Gal-10), also known as Charcot-Leyden crystal protein (CLC-P) because of its remarkable ability to form Charcot-Leyden crystals (CLCs), which are frequently found in tissues from patients with eosinophilic disorders. CLC-P/Gal-10 is highly expressed in human eosinophils and considered a biomarker of eosinophil involvement in inflammation. However, the intracellular sites where large pools of CLC-P/Gal-10 constitutively reside are still unclear, and whether this protein is derived or not from eosinophil granules remains to be established. Here, we applied pre-embedding immunonanogold transmission electron microscopy combined with strategies for optimal antigen and cell preservation and quantitative imaging analysis to investigate, for the first time, the intracellular localization of CLC-P/Gal-10 at high resolution in resting and activated human eosinophils. We demonstrated that CLC-P/Gal-10 is mostly stored in the peripheral cytoplasm of human eosinophils, being accumulated within an area of ∼250nm wide underneath the plasma membrane and not within specific (secretory) granules, a pattern also observed by immunofluorescence. High-resolution analysis of single cells revealed that CLC-P/Gal-10 interacts with the plasma membrane with immunoreactive microdomains of high CLC-P/Gal-10 density being found in ∼60% of the membrane area. Eosinophil stimulation with CCL11 or TNF-α, which are known inducers of eosinophil secretion, did not change the peripheral localization of CLC-P/Gal-10 as observed by both immunofluorescence and immuno-EM (electron microscopy). Thus, in contrast to other preformed eosinophil proteins, CLC-P/Gal-10 neither is stored within secretory granules nor exported through classical degranulation mechanisms (piecemeal degranulation and compound exocytosis).

Charcot-Leyden crystal protein/galectin-10 interacts with cationic ribonucleases and is required for eosinophil granulogenesis

The human eosinophil Charcot-Leyden crystal (CLC) protein is a member of the Galectin superfamily and is also known as galectin-10 (Gal-10). CLC/Gal-10 forms the distinctive hexagonal bipyramidal crystals that are considered hallmarks of eosinophil participation in allergic responses and related inflammatory reactions; however, the glycan-containing ligands of CLC/Gal-10, its cellular function(s), and its role(s) in allergic diseases are unknown. We sought to determine the binding partners of CLC/Gal-10 and elucidate its role in eosinophil biology. Intracellular binding partners were determined by ligand blotting with CLC/Gal-10, followed by coimmunoprecipitation and coaffinity purifications. The role of CLC/Gal-10 in eosinophil function was determined by using enzyme activity assays, confocal microscopy, and short hairpin RNA knockout of CLC/Gal-10 expression in human CD34+ cord blood hematopoietic progenitors differentiated to eosinophils. CLC/Gal-10 interacts with both human eosinophil granule cationic ribonucleases (RNases), namely, eosinophil-derived neurotoxin (RNS2) and eosinophil cationic protein (RNS3), and with murine eosinophil-associated RNases. The interaction is independent of glycosylation and is not inhibitory toward endoRNase activity. Activation of eosinophils with INF-γ induces the rapid colocalization of CLC/Gal-10 with eosinophil-derived neurotoxin/RNS2 and CD63. Short hairpin RNA knockdown of CLC/Gal-10 in human cord blood-derived CD34+ progenitor cells impairs eosinophil granulogenesis. CLC/Gal-10 functions as a carrier for the sequestration and vesicular transport of the potent eosinophil granule cationic RNases during both differentiation and degranulation, enabling their intracellular packaging and extracellular functions in allergic inflammation.

Decreased Expression of Innate Immunity-Related Genes in Peripheral Blood Mononuclear Cells from Patients with IgG4-Related Disease.

BackgroundIgG4-related disease (IgG4-RD) is a new clinical entity of unknown etiology characterized by elevated serum IgG4 and tissue infiltration by IgG4-positive plasma cells. Although aberrancies in acquired immune system functions, including increases in Th2 and Treg cytokines observed in patients with IgG4-RD, its true etiology remains unclear. To investigate the pathogenesis of IgG4-RD, this study compared the expression of genes related to innate immunity in patients with IgG4-RD and healthy controls.Materials and MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from patients with IgG4-RD before and after steroid therapy and from healthy controls. Total RNA was extracted and DNA microarray analysis was performed in two IgG4-RD patients to screen for genes showing changes in expression. Candidate genes were validated by real-time RT-PCR in 27 patients with IgG4-RD and 13 healthy controls.ResultsDNA microarray analysis identified 21 genes that showed a greater than 3-fold difference in expression between IgG4-RD patients and healthy controls and 30 genes that showed a greater than 3-fold change in IgG4-RD patients following steroid therapy. Candidate genes related to innate immunity, including those encoding Charcot–Leyden crystal protein (CLC), membrane-spanning 4-domain subfamily A member 3 (MS4A3), defensin alpha (DEFA) 3 and 4, and interleukin-8 receptors (IL8R), were validated by real-time RT-PCR. Expression of all genes was significantly lower in IgG4-RD patients than in healthy controls. Steroid therapy significantly increased the expression of DEFA3, DEFA4 and MS4A3, but had no effect on the expression of CLC, IL8RA and IL8RB.ConclusionsThe expression of genes related to allergy or innate immunity, including CLC, MS4A3, DEFA3, DEFA4, IL8RA and IL8RB, was lower in PBMCs from patients with IgG4-RD than from healthy controls. Although there is the limitation in the number of patients applied in DNA microarray, impaired expression of genes related to innate immunity may be involved in the pathogenesis of IgG4-RD as well as in abnormalities of acquired immunity.

The Enigmatic Charcot-Leyden Crystal Protein (Galectin-10): Speculative Role(s) in the Eosinophil Biology and Function

Eosinophilic inflammation in peripheral tissues is typically marked by the deposition of a prominent eosinophil protein, Galectin-10, better known as Charcot-Leyden crystal protein (CLC). Unlike the eosinophil’s four distinct toxic cationic proteins and enzymes [major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO)], there is a paucity of information on the precise role of the crystal protein in the biology of the eosinophil. While its clinical significance at inflammatory foci remains highly speculative, its relative abundance (~10% of total eosinophil protein), as well as its dual nuclear and cytosolic localization is, however, suggestive of its biological and functional significance. In this article, we present a short review of the Charcot-Leyden crystal protein, specifically highlighting its most recently delineated modulatory role in regulatory T lymphocytes, and its speculative intracellular and extracellular role(s) in eosinophil function or associated inflammatory responses.

Transcriptional changes between uninflamed ulcerative colitis and familial adenomatous polyposis pouch mucosa can be attributed to an altered immune response.

A total proctocolectomy with ileal pouch-anal anastomosis (IPAA) is considered the surgery of choice for definitive management of familial adenomatous polyposis (FAP) and some patients with ulcerative colitis (UC). However, this surgical treatment is often associated with pouchitis, a long-term complication that occurs mostly in UC patients. The purpose of this study was to better define the molecular background of pouchitis. A microarray-based survey was performed using pouch mucosal samples collected from 28 and 8 patients undergoing surgery for UC and FAP, respectively. There were 4,770 genes that significantly differentiated uninflamed from inflamed mucosal samples, and their functional features were represented mostly by metabolic and cell proliferation pathways. In contrast, functional analyses of aberrantly expressed genes between UC and FAP samples, irrespective of mucosal inflammation status, revealed multiple pathways and terms that were linked to changes in immune response. Interestingly, the comparison of uninflamed UC and FAP samples identified a set of 29 altered probe sets, including an inflammation-related transcript encoding a Charcot-Leyden crystal (CLC) protein. The most distinct changes in gene expression profiles differentiating uninflamed UC and FAP pouch mucosal samples were attributed to the Gene Ontology category innate immune response. Our study confirmed that alterations in immune responses can be found between patients who underwent surgery for UC and FAP, independent of the pouch inflammation status. This observation may be important when managing IPAA patients.

Mast Cells, Eosinophils and Eosinophilic Esophagitis

Eosinophilic esophagitis (EoE) is an increasingly recognized disease entity in adults. Studies show a role for eosinophils and mast cells in its pathogenesis. Upper and mid-esophageal biopsy slides from adult patients (n=36) with dysphagia were stained for eosinophil counts (H&E), and immunostained for eosinophil-derived neurotoxin (EDN) and eosinophil major basic protein (MBP). Gene expression was studied by microarray analysis. All patients were consented before the procedure. All research protocols were approved by the Mayo Clinic IRB. EoE patients (n=12; median age 36; 10 male, 2 female) had higher EDN and MBP tissue protein levels as quantified by immunofluorescence. The measured levels of intracellular and extracellular EDN and MBP correlated with eosinophil counts in the EoE patients (median eosinophil count 48±36 cells/high power field (HPF)) compared to controls (n=14; median age 47.2; median eosinophil count 2.8±4 cells/HPF). Genetic microarray analysis showed eotaxin-3 expression was 8-fold higher, eotaxin-2 was 1.3-fold higher, and Charcot-Leyden crystal protein was 2.4-fold higher than controls. For mast cell markers, cathepsin-G precursor expression was 6.2-fold higher, high affinity IgE receptor alpha chain was 2.1-fold higher, carboxypeptidase A3 was 4.5-fold higher, and both cathepsin C and tryptase were 3-fold higher than controls. All fold-changes reached statistical significance (p<0.002). Of note two control patients showed the control phenotype but matched the EoE genotype. The significant increase in eotaxin-3 gene expression in EoE patients is in agreement with previously reported expression levels in children. The increase in mast cell markers supports a role for mast cell involvement in EoE.

BetaSearch: a new method for querying β-residue motifs

BackgroundSearching for structural motifs across known protein structures can be useful for identifying unrelated proteins with similar function and characterising secondary structures such as β-sheets. This is infeasible using conventional sequence alignment because linear protein sequences do not contain spatial information. β-residue motifs are β-sheet substructures that can be represented as graphs and queried using existing graph indexing methods, however, these approaches are designed for general graphs that do not incorporate the inherent structural constraints of β-sheets and require computationally-expensive filtering and verification procedures. 3D substructure search methods, on the other hand, allow β-residue motifs to be queried in a three-dimensional context but at significant computational costs.FindingsWe developed a new method for querying β-residue motifs, called BetaSearch, which leverages the natural planar constraints of β-sheets by indexing them as 2D matrices, thus avoiding much of the computational complexities involved with structural and graph querying. BetaSearch exhibits faster filtering, verification, and overall query time than existing graph indexing approaches whilst producing comparable index sizes. Compared to 3D substructure search methods, BetaSearch achieves 33 and 240 times speedups over index-based and pairwise alignment-based approaches, respectively. Furthermore, we have presented case-studies to demonstrate its capability of motif matching in sequentially dissimilar proteins and described a method for using BetaSearch to predict β-strand pairing.ConclusionsWe have demonstrated that BetaSearch is a fast method for querying substructure motifs. The improvements in speed over existing approaches make it useful for efficiently performing high-volume exploratory querying of possible protein substructural motifs or conformations. BetaSearch was used to identify a nearly identical β-residue motif between an entirely synthetic (Top7) and a naturally-occurring protein (Charcot-Leyden crystal protein), as well as identifying structural similarities between biotin-binding domains of avidin, streptavidin and the lipocalin gamma subunit of human C8.

Proteomic Analysis of Colonic Mucosal Tissue from Tuberculous and Ulcerative Colitis Patients

Changes in the expression profiles of specific proteins leads to serious human diseases, including colitis. The proteomic changes related to colitis and the differential expression between tuberculous (TC) and ulcerative colitis (UC) in colon tissue from colitis patients has not been defined. We therefore performed a proteomic analysis of human TC and UC mucosal tissue. Total protein was obtained from the colon mucosal tissue of normal, TC, and UC patients, and resolved by 2-dimensional electrophoresis (2-DE). The results were analyzed with PDQuest using silver staining. We used matrix-assisted laser desorption ionization time-of-flight/time-of-flight spectrometry (MALDI TOF/TOF) to identify proteins differentially expressed in TC and UC. Of the over 1,000 proteins isolated, three in TC tissue and two in UC tissue displayed altered expression when compared to normal tissue. Moreover, two proteins were differentially expressed in a comparative analysis between TC and UC. These were identified as mutant β-actin, α-enolase and Charcot-Leyden crystal protein. In particular, the expression of α-enolase was significantly greater in TC compared with normal tissue, but decreased in comparison to UC, implying that α-enolase may represent a biomarker for differential diagnosis of TC and UC. This study therefore provides a valuable resource for the molecular and diagnostic analysis of human colitis.

CLC– a novel susceptibility gene for allergic rhinitis?

Studies of the nasal lavage fluid proteome have previously identified proteins differently expressed in patients with symptomatic allergic rhinitis, e.g. S100A7, prolactin-inducible protein (PIP), wingless-type MMTV integration site family, member 2B (WNT2B), Charcot-Leyden crystal protein (CLC) and palate lung nasal epithelial clone (PLUNC). The aim of the present study was to investigate if genetic variation associated with allergic rhinitis can be found in these genes. Peripheral blood was collected from 251 patients with birch and/or grass pollen-induced allergic rhinitis and 386 nonatopic healthy controls. A total of 39 single nucleotide polymorphisms (SNPs) distributed over the genes PIP, WNT2B, CLC and PLUNC were selected from dbSNP, genotyped and investigated for associations with allergic rhinitis. Twelve additional SNPs were subsequently analysed for CLC. All 22 investigated SNPs in CLC were polymorphic. Ten SNPs yielded significant differences between cases and controls with respect to genotype frequencies. Homozygotes for the minor allele were more common in allergic individuals compared to healthy controls. The minor alleles of these SNPs were all located on the same haplotype. Furthermore, homozygotes for the minor allele of two of the promoter SNPs had higher average scores for birch in skin prick test. In contrast, for seven SNPs within the gene, heterozygotes and homozygotes for the major allele had higher average scores for grass. None of the other three genes showed association. Genetic variation in CLC was found to be associated with allergic rhinitis. The pattern of variation is compatible with a recessive inheritance model and the previously observed altered protein levels detected in patients with allergic rhinitis.

Loss of detectability of Charcot–Leyden crystal protein transcripts in blood cells after treatment with dimethyl sulfoxide

Charcot–Leyden crystal protein (CLC) is a major secretory effector protein of eosinophils. In addition, CLC has been identified as marker for regulatory T-cells and differential expression of CLC has been described under diverse pathological conditions. By analysis of DNA microarray data from peripheral blood mononuclear cells (PBMC) we found differences for the expression of CLC between PBMC that had been cryopreserved or had been used for RNA isolation immediately after cell separation. Reverse transcriptase-polymerase chain reaction (RT-PCR) of separated cell populations indicated that contaminating granulocytes were the main source of CLC transcripts in PBMC. CLC was only detectable in fresh PBMC and not in cryopreserved material. Transcripts corresponding to CLC were also detectable by RT-PCR only in fresh PBMC and eosinophils. Loss of CLC transcripts in PBMC was induced by a short pulse with dimethyl sulfoxide (DMSO), indicating that the freezing medium was responsible for this phenomenon. We conclude that CLC transcripts are lost during cryopreservation in the presence of DMSO and can never be identified as differentially expressed in cryopreserved samples.

Whole blood gene expression in infants with respiratory syncytial virus bronchiolitis

BackgroundRespiratory syncytial virus (RSV) is a major cause of viral bronchiolitis in infants worldwide, and environmental, viral and host factors are all of importance for disease susceptibility and severity. To study the systemic host response to this disease we used the microarray technology to measure mRNA gene expression levels in whole blood of five male infants hospitalised with acute RSV, subtype B, bronchiolitis versus five one year old male controls exposed to RSV during infancy without bronchiolitis. The gene expression levels were further evaluated in a new experiment using quantitative real-time polymerase chain reaction (QRT-PCR) both in the five infants selected for microarray and in 13 other infants hospitalised with the same disease.ResultsAmong the 30 genes most differentially expressed by microarray nearly 50% were involved in immunological processes. We found the highly upregulated interferon, alpha-inducible protein 27 (IFI27) and the highly downregulated gene Charcot-Leyden crystal protein (CLC) to be the two most differentially expressed genes in the microarray study. When performing QRT-PCR on these genes IFI27 was upregulated in all but one infant, and CLC was downregulated in all 18 infants, and similar to that given by microarray.ConclusionThe gene IFI27 is upregulated and the gene CLC is downregulated in whole blood of infants hospitalised with RSV, subtype B, bronchiolitis and is not reported before. More studies are needed to elucidate the specificity of these gene expressions in association with host response to this virus in bronchiolitis of moderate severity.

A network-based analysis of the late-phase reaction of the skin

The late-phase reaction (LPR) of the skin is an in vivo model of allergic inflammation. We sought to identify disease-associated pathways in the LPR using a network-based analysis. The LPR was examined by means of DNA microarray analysis of skin biopsy specimens from 10 patients with allergic rhinitis and 10 healthy control subjects. The results were further analyzed in 2 different materials consisting of nasal fluids and allergen-challenged CD4(+) T cells from patients with allergic rhinitis. The DNA microarray analysis revealed several genes of known relevance to allergy. The eosinophil marker Charcot-Leyden crystal protein (CLC) that encodes Charcot-Leyden crystal protein differed most in expression. A network-based analysis showed upregulation of IL-4- and CCL4-dependent pathways and downregulation of a TGF-beta-induced pathway. CCL4 is expressed by CD4(+) T cells and chemotactic for eosinophils. We hypothesized that allergen induces release of CCL4 from T(H)2 cells and that this contributes to influx of eosinophils. Further analysis showed increase of CCL4 protein in nasal fluids from allergic patients during the season. Allergen challenge of PBMCs resulted in proliferation of T(H)2 cells and increased production of CCL4 in CD4(+) T cells from allergic patients. An analysis of the DNA microarray data revealed a significant correlation between CCL4 and the eosinophil marker CLC. A network-based analysis of the LPR showed increased activity of IL-4- and CCL4- dependent pathways and downregulation of the TGF-beta-induced pathway. Allergen-induced release of CCL4 from T(H)2 cells might contribute to influx of eosinophils during the LPR. Involvement of multiple interacting pathways indicates that it might be difficult to identify one single mediator as a biomarker or drug target in allergic inflammation.

Developmental and Differential Changes of Gene Expression in Erythroid Progenitor Cells.

During erythropoiesis and human development different globin genes (α, β, γ, δ and ε) are expressed as a result of globin gene switching. We investigated globin gene expression in comparison to the expression of other genes in erythroid progenitor cells (EPC) during ontogenesis using in-house produced microarrays containing 16,659 oligonucleotides. Human primitive CD34+ cells were isolated from fetal liver (FL), cord blood (CB), adult bone marrow (BM), peripheral blood (PB) and mobilized peripheral blood (mPB), and developed into EPC in the presence of erythropoietin and other cytokines. The differentiation to EPC was confirmed by flow cytometry as 100% cells were CD71+. In microarray studies, a total of 2996 genes were highly expressed in FL, 2673 genes in CB, 2580 in mPB, 1465 in PB and 1259 in BM derived EPC. 661 of these genes were common for all type of cells. The high level of expression, beside globin genes, was observed for the following genes: transferrin receptor, proteoglycans, ALAS2, Charcot-Leyden crystal protein, nucleophosmin, eosinophil peroxidase, myeloperoxidase and ribonucleases. Most of the analyzed genes demonstrated down-regulation during ontogenesis (elastase 2, glutathione peroxidase 1, SERPINB1, nudix, mitochondrial proteins, ribosomal proteins, enthoprotin, serine proteinase inhibitor), but some showed up-regulation (hexokinase, superoxide dismutase 2, spectrin). Besides developmental changes of globin gene expression during ontogenesis, we also analyzed changes in their expression during erythropoiesis in these different tissues by quantitative PCR. Beta-globin gene expression reached the maximum levels in cells of adult blood origin: BM (176 fmol/μg) and PB (110 fmol/μg). Gamma-globin gene expression, of FL origin, had steady levels during erythroid differentiation (20 fmol/μg), whereas cord blood derived EPC demonstrated consistent up-regulation (60 fmol/μg) in contrast to cells originated from adult blood (3–15 fmol/μg at day 14th). G protein related genes and histone deacetylases were elevated in CB derived EPC, concomitant with increased gamma-globin gene expression. We also analyzed the gamma-globin induction by hydroxyurea, a well known inducer, and established which G protein-coupled receptors involved pathways are activated in PB derived EPC: dopamine receptors D1, D2 and D5, beta 2 adrenergic receptor, human DP prostanoid receptor and prostaglandin E receptor 1, as well as genes activated by cAMP/PKA, PI-3 kinase, MAP and NO/cGMP pathways. This study establishes concomitant changes in expression of globin genes and other known and/or previously unrecognized genes, which appear to be involved in erythropoiesis.

ASRI2005‐92 Possible reproductive immunological importance of pp13 / galectin‐13, a galectin with principally placental expression

As previous sequential and structural analyses showed its conserved homology to members of the galectin superfamily, Placental Protein 13 (PP13) was designated galectin‐13. Similarly to eosinophil Charcot Leyden Crystal protein / galectin‐10, its weak lysophospholipase activity was proved. Sugar binding assays revealed that residues widely expressed in placenta had the strongest binding affinity to PP13, which expression was found to be restricted to placenta. With regard to its strong labeling of the syncythiotrophoblasts’ brush border membrane as well as its co‐localization and specific binding to annexin II, PP13 was assumed to be externalized to the cell surface like other galectins. In a comparative study we found, that in the 3rd trimester, expression of PP13 was significantly reduced in preeclamptic (n = 20) and HELLP syndrome (n = 5) placentas compared to gestational age matched healthy samples (n = 25). In the same pathologic cases, maternal PP13 serum levels were also lower than in controls. Recently, PP13 was also successfully tested as a new 1st and 2nd trimester placental marker for the prediction of preeclampsia and IUGR. With regard to these results, PP13 may have special immunobiological function at the lining of the common feto‐maternal blood‐paces. The possible clinical importance of PP13 in pathological pregnancies with immunological background have to be emphasized.

Charcot-Leyden Crystal Protein (Galectin-10) Is Not a Dual Function Galectin with Lysophospholipase Activity but Binds a Lysophospholipase Inhibitor in a Novel Structural Fashion

Charcot-Leyden crystal (CLC) protein, initially reported to possess weak lysophospholipase activity, is still considered to be the eosinophil's lysophospholipase, but it shows no sequence similarities to any known lysophospholipases. In contrast, CLC protein has moderate sequence similarity, conserved genomic organization, and near structural identity to members of the galectin superfamily, and it has been designated galectin-10. To definitively determine whether or not CLC protein is a lysophospholipase, we reassessed its enzymatic activity in peripheral blood eosinophils and an eosinophil myelocyte cell line (AML14.3D10). Antibody affinity chromatography was used to fully deplete CLC protein from eosinophil lysates. The CLC-depleted lysates retained their full lysophospholipase activity, and this activity could be blocked by sulfhydryl group-reactive inhibitors, N-ethylmaleimide and p-chloromercuribenzenesulfonate, previously reported to inhibit the eosinophil enzyme. In contrast, the affinity-purified CLC protein lacked significant lysophospholipase activity. X-ray crystallographic structures of CLC protein in complex with the inhibitors showed that p-chloromercuribenzenesulfonate bound CLC protein via disulfide bonds with Cys(29) and with Cys(57) near the carbohydrate recognition domain (CRD), whereas N-ethylmaleimide bound to the galectin-10 CRD via ring stacking interactions with Trp(72), in a manner highly analogous to mannose binding to this CRD. Antibodies to rat pancreatic lysophospholipase identified a protein in eosinophil and AML14.3D10 cell lysates, comparable in size with human pancreatic lysophospholipase, which co-purifies in small quantities with CLC protein. Ligand blotting of human and murine eosinophil lysates with CLC protein as probe showed that it binds proteins also recognized by antibodies to pancreatic lysophospholipase. Our results definitively show that CLC protein is not one of the eosinophil's lysophospholipases but that it does interact with eosinophil lysophospholipases and known inhibitors of this lipolytic activity.