Hamilton Square, NJ, United States


Hamilton Square, NJ, United States
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Ucisik-Akkaya E.,HUMIGEN LLC | Davis C.F.,HUMIGEN LLC | Do T.N.,HUMIGEN LLC | Morrison B.A.,HUMIGEN LLC | And 3 more authors.
Molecular Human Reproduction | Year: 2010

Success rate in human pregnancies is believed to be very low and sex-specific mechanisms may operate in prenatal loss. Assuming a sex-differential in prenatal loss exists, we examined genetic markers in biologically plausible targets in the HLA complex, other immune system-related and iron-regulatory genes in 388 healthy newborns from Wales (UK) using one sex as a control group for the other. Genotyping of 333 single nucleotide polymorphisms (SNPs) from 107 genes was achieved mainly by TaqMan assays. Twenty-two of autosomal SNPs showed frequency differences between 187 male and 201 female newborns either individually or as part of a haplotype. Of these, six markers (RXRB rs2076310, HLA complex haplotype HLA-DQA1 rs1142316-HLA-DRA rs7192-HSPA1B rs1061581, HIST1H1T rs198844, IFNG rs2069727, NKG2D rs10772266 and IRF4 heterozygosity) showed statistically robust differences between male and female newborns and multivariable modeling confirmed their independence. There were fewer males homozygote for combined wildtype genotypes of LIF rs929271, TP53 rs1042522 and MDM2 rs2279744 compared with females [OR = 0.3, 95% confidence interval (CI) = 0.1-0.8; P < 0.01] although these SNPs did not show any association individually. It is unlikely that SNPs have clinical utility as single markers in any trait with complex etiology but polygenic predictive models remain a possibility. If their validity is confirmed in larger studies of different populations and functional mechanisms of these preliminary associations are elucidated, these markers from the HLA complex, NKG2D region and cytokines may cumulatively have sufficient predictive value for susceptibility to prenatal selection in each sex. © The Author 2010. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.

Morrison B.A.,HUMIGEN LLC | Ucisik-Akkaya E.,HUMIGEN LLC | Flores H.,Institute Diagnostico y Referencia Epidemiologicos InDRE | Alaez C.,Institute Diagnostico y Referencia Epidemiologicos InDRE | And 2 more authors.
Autoimmunity | Year: 2010

Previous epidemiologic studies showed four times increased risk of acute lymphoblastic leukemia (ALL) in children of women with multiple sclerosis (MS). MS shows a risk association with Human leukocyte antigens (HLA)-DRA single nucleotide polymorphism (SNP) rs3135388, which is a proxy marker for DRB1*1501. We examined the relevance of rs3135388 in childhood ALL risk along with two other HLA-DRA SNPs in two casecontrol groups: 114 cases and 388 controls from South Wales (UK) and 100 Mexican Mestizo cases and 253 controls. We first confirmed the correlation between rs3135388 and DRB1*1501 in HLA-typed reference cell lines. We noted a female-specific risk association in childhood ALL (pooled odds ratio (OR) =2.6, 95% confidence interval (CI) =1.5-4.5, MantelHaenszel P =0.0009) similar to the stronger association of DRB1*1501 in females with MS. Examination of an HLA-C 5′ flanking region SNP rs9264942, known to correlate with HLA-C expression, showed a protective association in girls (OR 0.4, 95% CI =0.2-0.7, MantelHaenszel P 0.0003) similar to the protective HLA-Cw*05 association in MS. In a reference cell line panel, HLA-Cw5 homozygous samples (n 8) were also homozygous for the minor allele of the SNP. Likewise, the male-specific protective association of interferon-gamma (IFNG) SNP rs2069727 in MS was replicated with the same sex specificity in childhood ALL (OR 0.6, 95% CI =0.4-1.0, MantelHaenszel P =0.03). Two other SNPs in superkiller viralicidic activity 2-like and tenascin XB that are markers for systemic lupus erythematosus susceptibility showed female-specific associations but due to linkage disequilibrium with HLA-DRB1*15. Our observations supported the epidemiologic link between MS and childhood ALL and added the sex effect to this connection. It appears that only girls born to mothers with MS may have an increased risk of ALL. Investigating the mechanism of these sex-specific associations may help understand the pathogenesis of MS and ALL. © 2010 Informa UK, Ltd.

Ucisik-Akkaya E.,HUMIGEN LLC | Davis C.F.,HUMIGEN LLC | Gorodezky C.,Institute Diagnostico y Referencia Epidemiologicos InDRE | Alaez C.,Institute Diagnostico y Referencia Epidemiologicos InDRE | Dorak M.T.,HUMIGEN LLC
Cell Stress and Chaperones | Year: 2010

Three heat shock protein 70 (HSP70) genes, HSPA1L, HSPA1A, and HSPA1B, are located within the human leukocyte antigen (HLA) class III region. HSPs act as stress signals and regulate natural killer cell response to cancer. HSP70 gene polymorphisms show disease associations partly due to their linkage disequilibrium with HLA alleles. To systematically evaluate their associations with childhood acute lymphoblastic leukemia (ALL), we examined the three functional single nucleotide polymorphisms (SNPs) rs2227956 (T493M) in HSPA1L, rs1043618 in HSPA1A 5′ UTR, and rs1061581 (Q351Q) in HSPA1B by TaqMan assays or polymerase chain reaction-restriction fragment length polymorphism in 114 ALL cases and 414 controls from Wales (UK), in 100 Mexican Mestizo ALL cases and 253 controls belonging to the same ethnic group, and in a panel of 82 HLA-typed reference cell line samples. Homozygosity for HSPA1B rs1061581 minor allele G was associated with protection (odds ratio (OR)∈=∈0.37, 95% confidence interval (CI)∈=∈0.16-0.78; P∈=∈0.007) with gene-dosage effect (additive model) reaching significance (P∈=∈0.0001) in the Welsh case-control group. This association was replicated in the second case-control group from Mexico (OR (recessive model)∈=∈0.49, 95% CI∈=∈0.24-0.96; P∈=∈0.03), and the pooled analysis yielded a strong association (Mantel-Haenszel OR∈=∈0.43, 95% CI∈=∈0.27-0.69, P∈=∈0.0004). The association was stronger in males in each group and in the pooled analysis. A three-SNP haplotype including the major allele A of rs1061581 showed a highly significant increase in Welsh cases compared with respective controls (6.7% vs 1.8%; P∈=∈0.0003) due to the difference between male cases and controls. The protective allele of rs1061581 occurred more frequently on the HLA-DRB3 haplotypes (especially DRB1 03) in the cell line panel, but the HSPA1B association was independent from the HLA-DRB4 association previously detected in the same case-control group from Wales (adjusted P∈=∈0.001). Given the cancer promoting roles played by HSPs intracellularly as well as roles in immune surveillance when expressed on the cell surface and the known correlations between expression levels and the HSP polymorphisms, these results are likely to indicate a primary association and warrant detailed assessment in childhood ALL development. © 2009 Cell Stress Society International.

Journal of Immunology | Year: 2015

The human IL23R gene single nucleotide polymorphism rs11209026 A allele confers protection against inflammatory diseases. However, although this difference has been associated with reductions in IL-23-induced IL-17A production and STAT3 phosphorylation, the molecular mechanism underlying these changes remains undefined. Th17 cell maturation depends on IL-23 signaling. Multiple splice forms of the human IL23R transcript exist, and one, Δ9, encodes a soluble form of the receptor. In this study, we asked whether this protective allele was associated with mRNA splicing. Using mini-gene constructs and competitive oligonucleotide binding, we showed that the A allele alters IL-23R α-chain mRNA splicing and favors exon 9 skipping by reducing the binding of the splicing enhancer SF2. This enhances expression of the Δ9 mRNA and consequently diminishes IL-23 signaling. Thus, the presence of the A allele increases expression of the soluble form of IL23R mRNA (which then functions as a decoy receptor) and lowers the ability to develop a Th17 phenotype upon IL-23 stimulation. We further showed that antisense oligonucleotides targeting the SF2 binding site could efficiently induce exon 9 skipping in the presence of the G allele, and thereby replicate the effect of the A allele. Antisense oligonucleotide treatment caused dose-responsive induction of the IL23RD9 mRNA and interfered with in vitro differentiation of human Th17 cells, reducing their expression of the signature Th17 cytokines IL-17A and IL-17F. This may represent a novel approach to therapy of Th17-mediated diseases by elevating soluble IL-23R while simultaneously reducing the remaining cell surface receptor density. Copyright © 2015 by The American Association of Immunologists, Inc.

Biochimica et Biophysica Acta - Molecular Basis of Disease | Year: 2010

The interferon regulatory factor (IRF) family of DNA-binding proteins regulates expression of interferon-inducible genes with roles in the immune response and carcinogenesis. IRF4 is involved in the differentiation of B and T cells and is overexpressed in B-cell malignancies as a result of c-REL (NF-κB) hyperactivation. IRF4 polymorphisms are associated with susceptibility to chronic lymphoid leukemia (CLL) and non-Hodgkin lymphoma (NHL). We examined 13 IRF4 SNPs in 114 cases of childhood acute lymphoblastic leukemia (ALL) and 388 newborn controls from Wales (U.K.) using TaqMan assays. IRF4 intron 4 SNP rs12203592 showed a male-specific risk association (OR = 4.4, 95% CI = 1.5 to 12.6, P = 0.007). Functional consequences of the C > T substitution at this SNP were assessed by cell-based reporter assays using three different cell lines. We found a repressive effect of the rs12203592 wildtype allele C on IRF4 promoter activity (P < 0.001) but no repression by the variant allele in any cell line tested. Thus, homozygosity for the rs12203592 variant allele would result in increased IRF4 expression. This increase would be compounded by high levels of NF-κB activity in males due to the absence of estrogen. IRF4 differs from other IRFs in its anti-interferon activity which interferes with immune surveillance. We propose that a detailed study of IRF4 can provide information on the mechanism of the sex effect and the role of immune surveillance in childhood ALL development. © 2009 Elsevier B.V. All rights reserved.

Megjugorac N.J.,HUMIGEN LLC | Gallagher G.E.,HUMIGEN LLC | Gallagher G.,HUMIGEN LLC
Blood | Year: 2010

The type-III interferon (IFN) family is composed of 3 molecules in humans: IFN-λ1 (interleukin-29 [IL-29]), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B), each of which signals through the same receptor complex. Plasmacytoid dendritic cells (pDCs) are major IFN-λ producers among peripheral lymphocytes. Recently, it has been shown that IFN-λ1 exerts a powerful inhibitory effect over the T-helper 2 (Th2) response by antagonizing the effect of IL-4 on CD4+ T cells and inhibiting the production of Th2-associated cytokines. Here, we asked whether Th2 cytokines exert reciprocal control over IFN-λ production. IL-4 treatment during stimulation of human peripheral lymphocytes significantly elevated IFN-λ1 transcription and secretion. However, pDCs were not directly responsive to IL-4. Using depletion and reconstitution experiments, we showed that IL-4-responsive monocytes are an intermediary cell, responding to IL-4 by elevating their secretion of IL-1 receptor antagonist (IL-Ra); this IL-1Ra acts on pDCs to elevate their IFN-λ1 output. Thus, our experiments revealed a novel mechanism for regulation of both IFN-λ1 production and pDC function, and suggests an expanded immunomodulatory role for Th2-associated cytokines. © 2010 by The American Society of Hematology.

Swider A.,HUMIGEN LLC | Siegel R.,HUMIGEN LLC | Eskdale J.,HUMIGEN LLC | Gallagher G.,HUMIGEN LLC
Cytokine | Year: 2014

The efficient regulation of intestinal immune responses is critical to colon health. Viruses, for example noraviruses, are key pathogens of the intestine. The lambda interferons (comprising three ligands: IFNL1, L2 and L3 - the so-called "Type III" interferons) constitute the most recently discovered IFN family and are known to be important in intestinal anti-viral defense. A fourth family member, IFNL4, was recently described. Expression of the IFN-lambda receptor is restricted to epithelial and immune cells; together, these ligands and their receptor represent an important anti-viral and immunoregulatory component of the immune/epithelial inteface. We investigated control of IFNL1 expression in human colon epithelial cells. We used the TLR3 agonist poly I:C to drive expression of IFNL1 in SW480 cells, and small interfering RNA (siRNA) to knockdown target transcription factors. We identified ZEB1 and BLIMP-1 as transcription factors that strongly inhibited IFNL1 expression in SW480 cells. Interestingly, while BLIMP-1 inhibited both type-III and type-I interferons (IFN-β), the inhibitory action of ZEB1 was specific for IFNL1. We also defined the NF-κB family member, p65 as a key activator of IFNL1 and NF-κB p50 as a key inhibitor. Finally, we demonstrated that siRNA targeting of ZEB1 or NF-κB p50 resulted in a significant elevation of secreted IFN-λ1 protein and expression of the anti-viral gene OAS1, while knockdown of p65 inhibited these events. Our data provide insight to the regulation of IFNL1 expression in the human colon and suggest novel therapeutic approaches to elevate IFNλ-1 protein where required. © 2013 Elsevier Ltd.

The most common mutation of the HFE gene C282Y has shown a risk association with childhood acute lymphoblastic leukemia (ALL) in Welsh and Scottish case- control studies. This finding has not been replicated outside Britain. Here, we present a thorough analysis of the HFE gene in a panel of HLA homozygous reference cell lines and in the original population sample from South Wales (117 childhood ALL cases and 414 newborn controls). The 21 of 24 variants analyzed were from the HFE gene region extending 52 kb from the histone gene HIST1H1C to HIST1H1T. We identified the single-nucleotide polymorphism (SNP) rs807212 as a tagging SNP for the most common HFE region haplotype, which contains wild-type alleles of all HFE variants examined. This intergenic SNP rs807212 yielded a strong male-specific protective association (per allele OR=0.38, 95% CI=0.22-0.64, P (trend)= 0.0002; P=0.48 in females), which accounted for the original C282Y risk association. In the HapMap project data, rs807212 was in strong linkage disequilibrium with 25 other SNPs spanning 151 kb around HFE. Minor alleles of these 26 SNPs characterized the most common haplotype for the HFE region, which lacked all disease-associated HFE variants. The HapMap data suggested positive selection in this region even in populations where the HFE C282Y mutation is absent. These results have implications for the sex-specific associations observed in this region and suggest the inclusion of rs807212 in future studies of the HFE gene and the extended HLA class I region. © Springer-Verlag 2009.

Gallagher G.,HUMIGEN LLC | Megjugorac N.J.,HUMIGEN LLC | Yu R.Y.,HUMIGEN LLC | Eskdale J.,HUMIGEN LLC | And 3 more authors.
Journal of Interferon and Cytokine Research | Year: 2010

The type-III interferons (IFNs) are the most recently discovered IFNs in the human immune system and have important, but as yet poorly characterized, functions in innate and adaptive immunity that complement their antiviral functions. It is now becoming clear that these type-III IFNs have a functional niche where epithelial surfaces interact with the adaptive immune system, that their antiviral capability is not as highly developed as that of the type-I IFNs, and that they have their own profile of immunomodulatory functions; specifically, they are key modulators of the T-helper (Th)2 response. Copyright 2010, Mary Ann Liebert, Inc.

Type: Journal Article | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2011

The type III () IFNs (IFN-1, IFN-2, and IFN-3) and their receptor are the most recently discovered IFN family. They are induced by viruses and mediate antiviral activity, but type III IFNs have an important, specific functional niche at the immune/epithelial interface, as well as in the regulation of Th2 cytokines. Their expression appears diminished in bronchial epithelial cells of rhinovirus-infected asthmatic individuals. We investigated the regulation of IFN-1 expression in human airway epithelial cells using reporter genes analysis, chromatin immunoprecipitation, small interfering RNA knockdown, and DNase footprinting. In this article, we define the c-REL/p65 NF-B heterodimer and IRF-1 as key transcriptional activators and ZEB1, B lymphocyte-induced maturation protein 1, and the p50 NF-B homodimer as key repressors of the IFN-1 gene. We further show that ZEB1 selectively regulates type III IFNs. To our knowledge, this study presents the first characterization of any type III IFN promoter in its native context and conformation in epithelial cells and can now be applied to understanding pathogenic dysregulation of IFN-1 in human disease.

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