Atopic dermatitis in West Highland white terriers is associated with a 1.3-Mb region on CFA 17

Canine atopic dermatitis (AD) is an allergic inflammatory skin disease that shares similarities with AD in humans. Canine AD is likely to be an inherited disease in dogs and is common in West Highland white terriers (WHWTs). We performed a genome-wide association study using the Affymetrix Canine SNP V2 array consisting of over 42,800 single nucleotide polymorphisms, on 35 atopic and 25 non-atopic WHWTs. A gene-dropping simulation method, using SIB-PAIR, identified a projected 1.3 Mb area of association (genome-wide P = 6 × 10⁻⁵ to P = 7 × 10⁻⁴) on CFA 17. Nineteen genes on CFA 17, including 1 potential candidate gene (PTPN22), were located less than 0.5 Mb from the interval of association identified on the genome-wide association analysis. Four haplotypes within this locus were differently distributed between cases and controls in this population of dogs. These findings suggest that a major locus for canine AD in WHWTs may be located on, or in close proximity to an area on CFA 17.     

Variants of C-C motif chemokine 22 (CCL22) are associated with susceptibility to atopic dermatitis: case-control studies

Atopic dermatitis (AD) is a common inflammatory skin disease caused by multiple genetic and environmental factors. AD is characterized by the local infiltration of T helper type 2 (Th2) cells. Recent clinical studies have shown important roles of the Th2 chemokines, CCL22 and CCL17 in the pathogenesis of AD. To investigate whether polymorphisms of the CCL22 gene affect the susceptibility to AD, we conducted association studies and functional studies of the related variants. We first resequenced the CCL22 gene and found a total of 39 SNPs. We selected seven tag SNPs in the CCL22 gene, and conducted association studies using two independent Japanese populations (1(st) population, 916 cases and 1,032 controls; 2(nd) population 1,034 cases and 1,004 controls). After the association results were combined by inverse variance method, we observed a significant association at rs4359426 (meta-analysis, combined P?=?9.6×10??; OR, 0.74; 95% CI, 0.65-0.85). Functional analysis revealed that the risk allele of rs4359426 contributed to higher expression levels of CCL22 mRNA. We further examined the allelic differences in the binding of nuclear proteins by electrophoretic mobility shift assay. The signal intensity of the DNA-protein complex derived from the G allele of rs223821, which was in absolute LD with rs4359426, was higher than that from the A allele. Although further functional analyses are needed, it is likely that related variants play a role in susceptibility to AD in a gain-of-function manner. Our findings provide a new insight into the etiology and pathogenesis of AD.     

The chemokine RANTES is a crucial mediator of the progression from acute to chronic colitis in the rat

Chemokines have well characterized proinflammatory actions, including the ability to induce extravasation of leukocytes that participate in chronic inflammation. In this study, we evaluated the role of a C-C chemokine, RANTES, in the chronic phase of a rat model of colitis. Colitis was induced by intracolonic administration of trinitrobenzene sulfonic acid. At various timepoints thereafter (2 h to 14 days), colonic tissue levels of several chemokines were measured. Unlike the expression of monocyte chemoattractant protein-1, macrophage inflammatory protein-2, and cytokine-induced neutrophil chemoattractant, the expression of RANTES was significantly elevated during the chronic phase of colitis (> or =7 days after induction). Colonic RANTES mRNA expression was also significantly elevated during the chronic phase of colitis. The numbers of macrophages and monocytes in the colonic mucosa increased substantially during the chronic phase, as did expression of two of the receptors (CCR1 and CCR5) to which RANTES is known to bind. Administration on days 7 through 14 after trinitrobenzene sulfonic acid administration of a CCR1/CCR5 receptor antagonist, Met-RANTES, resulted in a significant reduction of both macroscopic and microscopic colonic damage, as well as reducing the recruitment into the colon of monocytes, mast cells, and neutrophils. In some rats, treatment with Met-RANTES resulted in a near-complete resolution of colonic damage and inflammation. These results suggest a crucial role of RANTES in the progression from acute to chronic inflammation in a rat model of colitis.     

Angiogenic properties of the chemokine RANTES/CCL5

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.     

A potentially functional polymorphism in the promoter region of let-7 family is associated with survival of hepatocellular carcinoma

Background: The let-7 family plays a vital role in the normal cellular activity of liver cells and the carcinogenesis of hepatocellular carcinoma (HCC). In the previous study, we have detected the association between single nucleotide polymorphisms (SNPs) in the promoter region of let-7 and susceptibility to HCC. However, it is still unknown whether these polymorphisms are associated with HCC prognosis. Methods: We investigated the effect of two potentially functional SNPs in the promoter region of let-7 family, rs10877887 (T > C) and rs13293512 (T > C), on the overall survival of 331 HCC patients. Log-rank test and Cox proportional hazard models were used for the survival analyses. Results: We found that HCC patients carrying the C allele of rs10877887 had a significantly increased death risk (adjusted HR = 1.22, 95%CI = 1.02–1.47, P = 0.03 in the additive model), compared to those with T allele. In the stratified analysis, the risk effect was evident in HCC patients with Barcelona Clinic Liver Cancer (BCLC) stage B (adjusted HR = 1.24, 95%CI = 1.02–1.51, P = 0.03) and in those who received chemotherapy or intervention (adjusted HR = 1.25, 95%CI = 1.02–1.53, P = 0.04). Conclusions: Our results suggest that rs10877887 in the promoter region of let-7 may be a prognostic biomarker for HCC patients, which need the validation from other larger studies in different populations.     

Retrovirally mediated IFN-beta transduction of macrophages induces resistance to HIV, correlated with up-regulation of RANTES production and down-regulation of C-C chemokine receptor-5 expression

Constitutive expression of IFN-beta by HIV target cells may be an alternative or complementary therapeutic approach for the treatment of AIDS. We show that macrophages derived from CD34+ cells from umbilical cord blood can be efficiently transduced by a retroviral vector carrying the IFN-beta coding sequence. This results in resistance to infection by a macrophage-tropic HIV type 1, as shown by the drastic reduction in the HIV DNA copy number per cell and in p24 release. Moreover, IFN-beta transduction totally blocked secretion of proinflammatory cytokines after HIV infection. The constitutive IFN-beta production also resulted in an increased production of IL-12 and IFN-gamma Th1-type cytokines and of the beta-chemokines macrophage-inflammatory protein-1alpha, macrophage-inflammatory protein-1beta, and RANTES. RANTES was found to be involved in the HIV resistance observed, and this was correlated with a down-regulation of the CCR-5 HIV entry coreceptor. These results demonstrate the feasibility and the efficacy of such IFN-beta-mediated gene therapy. In addition to inhibiting HIV replication, IFN-beta transduction could have beneficial immune effects in HIV-infected patients by favoring cellular immune responses.     

Localization of marginal zone macrophages is regulated by C-C chemokine ligands 21/19

The marginal zone (MZ) of the spleen is an important site for the capture of blood-borne pathogens and a gateway for lymphocytes entering the white pulp. We have recently reported that Leishmania donovani infection results in a remarkably selective loss of MZ macrophages (MZM) from the MZ. To understand the basis of this observation, we have investigated how MZM maintain their anatomical distribution in the steady state in uninfected mice. We now report that plt/plt mice, which lack functional CCL19 and CCL21, have significantly reduced numbers of MZM compared with normal C57BL/6 (B6) mice. Similarly, in B6.CD45.1-->plt/plt chimeras, donor-derived MZM were rare compared with the number observed in reciprocal plt/plt-->B6.CD45.1 chimeras. Moreover, we show that administration of pertussis toxin, an inhibitor of chemokine receptor signaling, to B6 mice results in exit of MZM from the MZ, that MZM can migrate in response to CCL19 and CCL21 in vitro, and that MZM colocalize with CD31+CCL21+ endothelial cells. Collectively, these data indicate that CCL21 and, to a lesser extent, CCL19 play significant roles in the distinctive localization of MZM within the splenic MZ. Deficiency of CCL19 and CCL21, as also previously observed in mice infected with L. donovani, may thus account for the selective loss of MZM seen during this infection.     

Identification and functional analysis of a caveolin-3 mutation associated with familial hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are caused by mutations in 14 and 15 different disease genes, respectively, in a part of the patients and the disease genes for cardiomyopathy overlap in part with that for limb-girdle muscular dystrophy (LGMD). In this study, we examined an LGMD gene encoding caveolin-3 (CAV3) for mutation in the patients with HCM or DCM. A Thr63Ser mutation was identified in a sibling case of HCM. Because the mutation was found at the residue that is involved in the LGMD-causing mutations, we investigate the functional change due to the Thr63Ser mutation as compared with the LGMD mutations by examining the distribution of GFP-tagged CAV3 proteins. It was observed that the Thr63Ser mutation reduced the cell surface expression of caveolin-3, albeit the change was mild as compared with the LGMD mutations. These observations suggest that HCM is a clinical spectrum of CAV3 mutations.     

Progressive retinal atrophy in Shetland sheepdog is associated with a mutation in the CNGA1 gene

Progressive retinal atrophy (PRA) is the collective name of a class of hereditary retinal dystrophies in the dog and is often described as the equivalent of retinitis pigmentosa in humans. PRA is characterized by visual impairment due to degeneration of the photoreceptors in the retina, usually leading to blindness. PRA has been reported in dogs from more than 100 breeds and can be genetically heterogeneous both between and within breeds. The disease can be subdivided by age at onset and rate of progression. Using genome‐wide association with 15 Shetland Sheepdog (Sheltie) cases and 14 controls, we identified a novel PRA locus on CFA13 (P_raw = 8.55 × 10^?7, P_genome = 1.7 × 10^?4). CNGA1, which is known to be involved in human cases of retinitis pigmentosa, was located within the associated region and was considered a likely candidate gene. Sequencing of this gene identified a 4‐bp deletion in exon 9 (c.1752_1755delAACT), leading to a frameshift and a premature stop codon. The study indicated genetic heterogeneity as the mutation was present in all PRA‐affected individuals in one large family of Shelties, whereas some other cases in the studied Sheltie population were not associated with this CNGA1 mutation. To our knowledge, this is the first report of a mutation in CNGA1 causing PRA in dogs.     

LIPG promoter polymorphism is associated with ischemic stroke in Korean population

Endothelial lipase (LIPG) is a member of the triglyceride lipase family which includes hepatic lipase and lipoprotein lipase. Its activity is related to clinically important parameters like blood lipid levels, hypertension, and obesity. In this work, we investigated the association of a LIPG promoter polymorphism, rs9958947C>T, with susceptibility to ischemic stroke in a Korean population. A total of 1,144 subjects (656 cerebral infarction patients and 488 controls) were enrolled on a voluntary basis. The rs9958947C>T polymorphism was genotyped using the single-base extension method. The association of rs9958947C>T with disease status was evaluated by statistical analyses. The frequencies of the rs9958947 C and T alleles were significantly different between the stroke patient group and control group (OR [95% CI], 1.300 [1.000?C1.691], P=0.0449). A significantly higher frequency of the CT+TT genotype was observed in the patient group compared to the control group (CC/CT+TT, OR [95% CI], 1.632 [1.094?C2.435], P=0.0164). The results suggest that the T allele of the LIPG promoter polymorphism rs9958947C>T should be considered as a genetic risk factor for ischemic stroke. Further association studies in other ethnic populations would help to generalize this hypothesis.     

Expression of the C-C chemokine receptor CCR3 in human airway epithelial cells

Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-alpha stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-gamma potentiated the TNF-alpha-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca(2+) flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, (125)I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.     

Time- and temperature-dependent acetylation of the chemokine RANTES produced in recombinant Escherichia coli

The S24F mutant of the chemokine RANTES was found to be partly acetylated when produced in recombinant Escherichia coli BL21(DE3)(pDIA17)(CCL5-S24F-pET-26b). Mass spectrometry and Edman sequencing of peptides generated by lys-C endopeptidase indicated that Lys-26, Lys-34, Lys-46, and Lys-57 were susceptible to acetylation. The extent of acetylation of the RANTES S24F polypeptide increased with temperature and with the time during which the culture was incubated after adding the inducer isopropyl-beta-D-thiogalactoside (IPTG). These findings suggest that induction at low temperature and for a short period of time should be preferred when spurious acetylation is a problem for the production of genuine recombinant polypeptides. Acetylation of the polypeptide was not affected by deleting acs, yfiQ, or speG, which encode acetyl-CoA synthetase, acetyl-CoA synthetase acetylase, and spermidine acetyl transferase, respectively, nor by the presence or absence of the pDIA17 plasmid, which harbours the cat gene encoding chloramphenicol acetyl transferase. By contrast, spontaneous acetylation of RANTES could be demonstrated by incubating either the purified polypeptide or inclusion bodies derived from an induced culture in the presence of acetyl-CoA.