BACKGROUND

IBC (Inflammatory Breast cancer) is a rare form of breast cancer with a particular phenotype. New molecular targets are needed to improve the treatment of this rapidly fatal disease. Given the role of NF-kappaB-related genes in cell proliferation, invasiveness, angiogenesis and inflammation, we postulated that they might be deregulated in IBC.

METHODS

We measured the mRNA expression levels of 60 NF-kappaB-related genes by using real-time quantitative RT-PCR in a well-defined series of 35 IBCs, by comparison with 22 stage IIB and III non inflammatory breast cancers. Twenty-four distant metastases of breast cancer served as "poor prognosis" breast tumor controls.

RESULTS

Thirty-five (58%) of the 60 NF-kappaB-related genes were significantly upregulated in IBC compared with non IBC. The upregulated genes were NF-kappaB genes (NFKB1, RELA, IKBKG, NFKBIB, NFKB2, REL, CHUK), apoptosis genes (MCL1L, TNFAIP3/A20, GADD45B, FASLG, MCL1S, IER3L, TNFRSF10B/TRAILR2), immune response genes (CD40, CD48, TNFSF11/RANKL, TNFRSF11A/RANK, CCL2/MCP-1, CD40LG, IL15, GBP1), proliferation genes (CCND2, CCND3, CSF1R, CSF1, SOD2), tumor-promoting genes (CXCL12, SELE, TNC, VCAM1, ICAM1, PLAU/UPA) or angiogenesis genes (PTGS2/COX2, CXCL1/GRO1). Only two of these 35 genes (PTGS2/COX2 and CXCL1/GRO1)were also upregulated in breast cancer metastases. We identified a five-gene molecular signature that matched patient outcomes, consisting of IL8 and VEGF plus three NF-kappaB-unrelated genes that we had previously identified as prognostic markers in the same series of IBC.

CONCLUSIONS

The NF-kappaB pathway appears to play a major role in IBC, possibly contributing to the unusual phenotype and aggressiveness of this form of breast cancer. Some upregulated NF-kappaB-related genes might serve as novel therapeutic targets in IBC.

A variety of cardiovascular, neurological, and neoplastic conditions have been associated with oxidative stress, i.e., conditions under which levels of reactive oxygen species (ROS) are elevated over significant periods. Nuclear factor erythroid 2-related factor (Nrf2) regulates the transcription of several gene products involved in the protective response to oxidative stress. The transcriptional regulatory and signaling relationships linking gene products involved in the response to oxidative stress are, currently, only partially resolved. Microarray data constitute RNA abundance measures representing gene expression patterns. In some cases, these patterns can identify the molecular interactions of gene products. They can be, in effect, proxies for protein-protein and protein-DNA interactions. Traditional techniques used for clustering coregulated genes on high-throughput gene arrays are rarely capable of distinguishing between direct transcriptional regulatory interactions and indirect ones. In this study, newly developed information-theoretic algorithms that employ the concept of mutual information were used: the Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNE), and Context Likelihood of Relatedness (CLR). These algorithms captured dependencies in the gene expression profiles of the mouse lung, allowing the regulatory effect of Nrf2 in response to oxidative stress to be determined more precisely. In addition, a characterization of promoter sequences of Nrf2 regulatory targets was conducted using a Support Vector Machine classification algorithm to corroborate ARACNE and CLR predictions. Inferred networks were analyzed, compared, and integrated using the Collective Analysis of Biological Interaction Networks (CABIN) plug-in of Cytoscape. Using the two network inference algorithms and one machine learning algorithm, a number of both previously known and novel targets of Nrf2 transcriptional activation were identified. Genes predicted as novel Nrf2 targets include Atf1, Srxn1, Prnp, Sod2, Als2, Nfkbib, and Ppp1r15b. Furthermore, microarray and quantitative RT-PCR experiments following cigarette-smoke-induced oxidative stress in Nrf2(+/+) and Nrf2(-/-) mouse lung affirmed many of the predictions made. Several new potential feed-forward regulatory loops involving Nrf2, Nqo1, Srxn1, Prdx1, Als2, Atf1, Sod1, and Park7 were predicted. This work shows the promise of network inference algorithms operating on high-throughput gene expression data in identifying transcriptional regulatory and other signaling relationships implicated in mammalian disease.

Soluble ICAM-1 (sICAM-1) is an endothelium-derived inflammatory marker that has been associated with diverse conditions such as myocardial infarction, diabetes, stroke, and malaria. Despite evidence for a heritable component to sICAM-1 levels, few genetic loci have been identified so far. To comprehensively address this issue, we performed a genome-wide association analysis of sICAM-1 concentration in 22,435 apparently healthy women from the Women's Genome Health Study. While our results confirm the previously reported associations at the ABO and ICAM1 loci, four novel associations were identified in the vicinity of NFKBIK (rs3136642, P = 5.4 × 10(-9)), PNPLA3 (rs738409, P = 5.8 × 10(-9)), RELA (rs1049728, P = 2.7 × 10(-16)), and SH2B3 (rs3184504, P = 2.9 × 10(-17)). Two loci, NFKBIB and RELA, are involved in NFKB signaling pathway; PNPLA3 is known for its association with fatty liver disease; and SH3B2 has been associated with a multitude of traits and disease including myocardial infarction. These associations provide insights into the genetic regulation of sICAM-1 levels and implicate these loci in the regulation of endothelial function.

BACKGROUND

Epigenetic mechanisms may be important in the progression of chronic kidney disease (CKD).

METHODS

We studied the genome-wide DNA methylation pattern associated with rapid loss of kidney function using the Infinium HumanMethylation 450 K BeadChip in 40 Chronic Renal Insufficiency (CRIC) study participants (n = 3939) with the highest and lowest rates of decline in estimated glomerular filtration rate.

RESULTS

The mean eGFR slope was 2.2 (1.4) and -5.1 (1.2) mL/min/1.73 m(2) in the stable kidney function group and the rapid progression group, respectively. CpG islands in NPHP4, IQSEC1 and TCF3 were hypermethylated to a larger extent in subjects with stable kidney function (P-values of 7.8E-05 to 9.5E-05). These genes are involved in pathways known to promote the epithelial to mesenchymal transition and renal fibrosis. Other CKD-related genes that were differentially methylated are NOS3, NFKBIL2, CLU, NFKBIB, TGFB3 and TGFBI, which are involved in oxidative stress and inflammatory pathways (P-values of 4.5E-03 to 0.046). Pathway analysis using Ingenuity Pathway Analysis showed that gene networks related to cell signaling, carbohydrate metabolism and human behavior are epigenetically regulated in CKD.

CONCLUSIONS

Epigenetic modifications may be important in determining the rate of loss of kidney function in patients with established CKD.

BACKGROUND

Increasing evidence supports a key role for the transcription factor nuclear factor (NF)-kappaB in the host response to pneumococcal infection. Control of NF-kappaB activity is achieved through interactions with the IkappaB family of inhibitors, encoded by the genes NFKBIA, NFKBIB, and NFKBIE. Rare NFKBIA mutations cause immunodeficiency with severe bacterial infection, raising the possibility that common IkappaB gene polymorphisms confer susceptibility to common bacterial disease.

OBJECTIVE

To determine whether polymorphisms in NFKBIA, NFKBIB, and NFKBIE associate with susceptibility to invasive pneumococcal disease (IPD) and thoracic empyema.

METHODS

We studied the frequencies of 62 single-nucleotide polymorphisms (SNPs) across NFKBIA, NFKBIB, and NFKBIE in individuals with IPD and control subjects (n=1,060). Significantly associated SNPs were then studied in a group of individuals with thoracic empyema and a second control group (n=632).

RESULTS

Two SNPs in the NFKBIA promoter region were associated with protection from IPD in both the initial study group and the pneumococcal empyema subgroup. Significant protection from IPD was observed for carriage of mutant alleles at these two loci on combining the groups (SNP rs3138053: Mantel-Haenszel 2x2 chi2=13.030, p=0.0003; odds ratio [OR], 0.60; 95% confidence interval [CI], 0.45-0.79; rs2233406: Mantel-Haenszel 2x2 chi2=18.927, p=0.00001; OR, 0.55; 95% CI, 0.42-0.72). An NFKBIE SNP associated with susceptibility to IPD but not pneumococcal empyema. None of the NFKBIB SNPs associated with IPD susceptibility.

CONCLUSIONS

NFKBIA polymorphisms associate with susceptibility to IPD. Genetic variation in an inhibitor of NF-kappaB therefore not only causes a very rare immunodeficiency state but may also influence the development of common infectious disease.

T-box 3 (Tbx3) is a member of the T-box family of genes. Mutations that result in the haploinsufficiency of TBX3 cause ulnar mammary syndrome in humans characterized by mammary gland hypoplasia as well as other congenital defects. In mice, homozygous mutations are embryonic lethal, suggesting that Tbx3 is essential for embryo development. Studies in mice have shown that Tbx3 is essential in the maintenance of mouse embryonic stem cell (ESC) self-renewal and in their differentiation into extraembryonic endoderm (ExEn). The role TBX3 plays in regulating human ESCs (hESCs) has not been explored. Since mouse and hESCs are known to represent distinct pluripotent states, it is important to address the role of TBX3 in hESC self-renewal and differentiation. Using overexpression and knockdown strategies, we found that TBX3 overexpression promotes hESC proliferation possibly by repressing the expression of both NFκBIB and p14(ARF) , known cell cycle regulators. During differentiation, TBX3 knockdown resulted in decreased neural rosette formation and in decreased expression of neuroepithelial and neuroectoderm markers (PAX6, LHX2, FOXG1, and RAX). Taken together, our data suggest a role for TBX3 in hESC proliferation and reveal an unrecognized novel role of TBX3 in promoting neuroepithelial differentiation. Our results suggest that TBX3 plays distinct roles in regulating self-renewal and differentiation in both hESCs and mouse ESCs.

BACKGROUND

The T-box transcription factor TBX3 is necessary for early embryonic development and for the normal development of the mammary gland. Homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. In humans, mutations that result in the haploinsufficiency of TBX3 causes Ulnar Mammary Syndrome (UMS) characterized by mammary gland hypoplasia as well as other congenital defects. In addition to its role in mammary gland development, various studies have also supported a role for Tbx3 in breast cancer development. TBX3 is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. Previous studies have suggested that TBX3 contributes to cancer development by its ability to bypass senescence by repressing the expression of p14(ARF)-tumor suppressor. Although many studies have shown that a dysregulation of TBX3 expression may contribute to cancer progression, no direct evidence shows TBX3 causes breast cancer.

RESULTS

In this study, we created doxycycline inducible double transgenic mice (MMTV-rtTA;tet-myc-TBX3-IRES-Luciferase) to test whether TBX3 over-expression can induce tumor formation within the mammary gland. Although over-expression of TBX3, alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. We also show that TBX3 directly binds to and represses NFκBIB, an inhibitor of the NF-κB pathway known to play a role in regulating cell proliferation. Lastly, we also show that the over-expression of TBX3 is associated with an increase in mammary stem-like cells.

CONCLUSIONS

Overall, our data suggests that over-expression of TBX3 may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the NF-κB pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation.

Despite shared environmental exposures, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease are usually studied in isolation, and the presence of shared molecular mechanisms is unknown.

We applied an integrative genomic approach to identify convergent transcriptomic pathways in emphysema and IPF.

We defined the transcriptional repertoire of chronic obstructive pulmonary disease, IPF, or normal histology lungs using RNA-seq (n = 87).

Genes increased in both emphysema and IPF relative to control were enriched for the p53/hypoxia pathway, a finding confirmed in an independent cohort using both gene expression arrays and the nCounter Analysis System (n = 193). Immunohistochemistry confirmed overexpression of HIF1A, MDM2, and NFKBIB members of this pathway in tissues from patients with emphysema or IPF. Using reads aligned across splice junctions, we determined that alternative splicing of p53/hypoxia pathway-associated molecules NUMB and PDGFA occurred more frequently in IPF or emphysema compared with control and validated these findings by quantitative polymerase chain reaction and the nCounter Analysis System on an independent sample set (n = 193). Finally, by integrating parallel microRNA and mRNA-Seq data on the same samples, we identified MIR96 as a key novel regulatory hub in the p53/hypoxia gene-expression network and confirmed that modulation of MIR96 in vitro recapitulates the disease-associated gene-expression network.

Our results suggest convergent transcriptional regulatory hubs in diseases as varied phenotypically as chronic obstructive pulmonary disease and IPF and suggest that these hubs may represent shared key responses of the lung to environmental stresses.

Genome-wide association studies (GWAS) and candidate gene studies have identified a number of risk loci associated with the smoking-related disease COPD, a disorder that originates in the airway epithelium. Since airway basal cell (BC) stem/progenitor cells exhibit the earliest abnormalities associated with smoking (hyperplasia, squamous metaplasia), we hypothesized that smoker BC have a dysregulated transcriptome, enriched, in part, at known GWAS/candidate gene loci. Massive parallel RNA sequencing was used to compare the transcriptome of BC purified from the airway epithelium of healthy nonsmokers (n = 10) and healthy smokers (n = 7). The chromosomal location of the differentially expressed genes was compared to loci identified by GWAS to confer risk for COPD. Smoker BC have 676 genes differentially expressed compared to nonsmoker BC, dominated by smoking up-regulation. Strikingly, 166 (25%) of these genes are located on chromosome 19, with 13 localized to 19q13.2 (p<10⁻⁴ compared to chance), including 4 genes (NFKBIB, LTBP4, EGLN2 and TGFB1) associated with risk for COPD. These observations provide the first direct connection between known genetic risks for smoking-related lung disease and airway BC, the population of lung cells that undergo the earliest changes associated with smoking.

BACKGROUND

The innate immune system is essential for host survival because of its ability to recognize invading pathogens and mount defensive responses.

OBJECTIVE

We sought to identify genetic associations of innate immunity genes with atopy and asthma and interactions with early viral infections (first 12 months of life) in a high-risk birth cohort.

METHODS

Three Canadian family-based studies and 1 Australian population-based case-control study (n = 5565) were used to investigate associations of 321 single nucleotide polymorphisms (SNPs) in 26 innate immunity genes with atopy, asthma, atopic asthma, and airway hyperresponsiveness. Interactions between innate immunity genes and early viral exposure to 3 common viruses (parainfluenza, respiratory syncytial virus, and picornavirus) were examined in the Canadian Asthma Primary Prevention Study by using both an affected-only family-based transmission disequilibrium test and case-control methods.

RESULTS

In a joint analysis of all 4 cohorts, IL-1 receptor 2 (IL1R2) and Toll-like receptor 1 (TLR1) SNPs were associated with atopy after correction for multiple comparisons. In addition, an NFKBIA SNP was associated with atopic asthma. Six SNPs (rs1519309 [TLR3], rs740044 [ILIR2], rs4543123 [TLR1], rs5741812 [LBP], rs917998 [IL18RAP], and rs3136641 [NFKBIB]) were significant (P < .05, confirmed with 30,000 permutations) in both the combined analysis of main genetic effects and SNP-virus interaction analyses in both case-control and family-based methods. The TLR1 variant (rs4543123) was associated with both multiple viruses (respiratory syncytial virus and parainfluenza virus) and multiple phenotypes.

CONCLUSIONS

We have identified novel susceptibility genes for asthma and related traits and interactions between these genes and early-life viral infections.

Drug resistance of cancer cells can be regulated by the dysregulated miRNAs, and sustained NFκB activation also plays an important role in tumor resistance to chemotherapy. Here, we sought to investigate whether there was a correlation between miR-20a and the NFκB pathway to clarify the effects that miR-20a exerted on gastric cancer (GC) chemoresistance. We found that miR-20a was significantly upregulated in GC plasma and tissue samples. In addition, it was upregulated in GC plasma and tissues from patients with cisplatin-resistant gastric cancer cell line SGC7901/cisplatin (DDP). And the upregulation of miR-20a was concurrent with the downregulation of NFKBIB (also known as IκBβ) as well as upregulation of p65, livin, and survivin. The luciferase activity suggested that NFKBIB was the direct target gene of miR-20a. Transfection of miR-20a inhibitor could increase NFKBIB level; downregulate the expression of p65, livin, and survivin; and lead to a higher proportion of apoptotic cells in SGC7901/DDP cells. Conversely, ectopic expression of miR-20a dramatically decreased the expression of NFKBIB; increased the expression of p65, livin, and survivin; and resulted in a decrease in the apoptosis induced by DDP in SGC7901 cells. Taken together, our findings suggested that miR-20a could promote activation of the NFκB pathway and downstream targets livin and survivin by targeting NFKBIB, which potentially contributed to GC chemoresistance.

Lack of reproducibility of findings has been a criticism of genetic association studies on complex diseases, such as chronic obstructive pulmonary disease (COPD). We selected 257 polymorphisms of 16 genes with reported or potential relationships to COPD and genotyped these variants in a case-control study that included 953 COPD cases and 956 control subjects. We explored the association of these polymorphisms to three COPD phenotypes: a COPD binary phenotype and two quantitative traits (post-bronchodilator forced expiratory volume in 1 s (FEV₁) % predicted and FEV₁/forced vital capacity (FVC)). The polymorphisms significantly associated to these phenotypes in this first study were tested in a second, family-based study that included 635 pedigrees with 1,910 individuals. Significant associations to the binary COPD phenotype in both populations were seen for STAT1 (rs13010343) and NFKBIB/SIRT2 (rs2241704) (p<0.05). Single-nucleotide polymorphisms rs17467825 and rs1155563 of the GC gene were significantly associated with FEV₁ % predicted and FEV₁/FVC, respectively, in both populations (p<0.05). This study has replicated associations to COPD phenotypes in the STAT1, NFKBIB/SIRT2 and GC genes in two independent populations, the associations of the former two genes representing novel findings.

Establishment and maintenance of pregnancy in the pig involves intricate communication between the developing conceptuses and the maternal endometrium. This process occurs during trophoblast elongation which is spaciotemporally associated with conceptus synthesis and release of IL1B concomitant with pregnancy-specific endometrial up-regulation of IL-1 receptors, providing the potential for activation of the transcription factor, NFKB. The objective of the current investigation was to determine changes in expression and cellular localization of NFKB and associated factors during the estrous cycle and early pregnancy in the pig. In situ hybridization was used to localize changes in PGR, ESR1, and TNFRSF11A during the peri-implantation period. Quantitative RT-PCR was utilized to demonstrate gene expression changes for NFKB1, RELA, TNFRSF11A, TLR4, NFKBIA and NFKBIB. Transcription factor ELISA demonstrated an overall increase in RELA during the peri-implantation period in both cyclic and pregnant gilts. While the presence of TNFSF11A and TLR4 were both detected, TLR4 expression changes were temporally associated with NFKB expression and activation. Collectively, these data demonstrate that NFKB activation may occur during the period of uterine receptivity in both the cyclic and pregnant endometrium.

1. Vascular inflammation plays a critical role in atherogenesis. Previously, we showed that baboon arterial endothelial cells (BAEC) were hyporesponsive to lipopolysaccharide (LPS) compared with human arterial endothelial cells (HAEC). 2. In the present study, we investigated mechanisms underlying differential responses between HAEC and BAEC to tumour necrosis factor (TNF)-alpha and LPS. 3. Both HAEC and BAEC responded similarly to TNF-alpha. However, BAEC showed retarded responses to LPS in expression of E-selectin, intercellular adhesion molecule-1, monocyte chemotactic protein-1 and interleukin-8 (P < 0.05). These changes were confirmed at the mRNA level. Tumour necrosis factor-alpha activated nuclear factor-kappaB members such as p50, p52, p65, c-rel and RelB in both HAEC and BAEC. In contrast, LPS activated p50 and p65 only in HAEC. Using microarray assays, we found that TNF receptor-associated factor 2 (TRAF-2), TNF receptor superfamily, member 1A-associated via death domain (TRADD) and nuclear factors such as nuclear factor of kappa in B-cells inhibitor, alpha (NFKBIA) and nuclear factor of kappa in B-cells inhibitor, beta (NFKBIB) were upregulated by LPS only in HAEC. Although the baseline expression of Toll-like receptor (TLR) 4 was low in both HAEC and BAEC, TNF-alpha activated TLR4 expression in both cell types. Although LPS increased TLR4 expression only in HAEC, human and baboon peripheral blood mononuclear cells exhibited similar TLR4 expression and response to LPS. Transfecting BAEC with TLR4/myeloid differentiation protein-2 overexpression vector conferred BAEC responsiveness to LPS. 4. The findings of the present study indicate that an altered TLR4 system may be responsible for the resistance of baboon endothelial cells to LPS. Given the importance of TLR4 in human immune responses and vascular diseases, the natural resistance of baboons to LPS/TLR4-initiated inflammation could make the baboon a valuable animal model in which to study how inflammation affects atherogenesis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects ~1% of the Caucasian population. Over the last decades, the availability of biological drugs targeting the proinflammatory cytokine tumour necrosis factor α, anti-TNF drugs, has improved the treatment of patients with RA. However, one-third of the patients do not respond to the treatment. We wanted to evaluate the status of pharmacogenomics of anti-TNF treatment. We performed a PubMed literature search and all studies reporting original data on associations between genetic variants and anti-TNF treatment response in RA patients were included and results evaluated by meta-analysis. In total, 25 single nucleotide polymorphisms were found to be associated with anti-TNF treatment response in RA (19 from genome-wide association studies and 6 from the meta-analyses), and these map to genes involved in T cell function, NFκB and TNF signalling pathways (including CTCN5, TEC, PTPRC, FCGR2A, NFKBIB, FCGR2A, IRAK3). Explorative prediction analyses found that biomarkers for clinical treatment selection are not yet available.

BACKGROUND

The nuclear factor-kappaB (NF-kappaB) family is a set of transcription factors with key roles in the induction of the inflammatory response and may be the link between inflammation and cancer development. This pathway has been shown to influence ovarian epithelial tissue repair. Inhibitors of kappaB (IkappaB) prevent NF-kappaB activation by sequestering NF-kappaB proteins in the cytoplasm until IkappaB proteins are phosphorylated and degraded.

METHODS

We used a case-control study to evaluate the association between single nucleotide polymorphisms (SNPs) in NFKBIA and NFKBIB (the genes encoding IkappaBalpha and IkappaBbeta, respectively) and risk of epithelial ovarian cancer. We queried 19 tagSNPs and putative-functional SNPs among 930 epithelial ovarian cancer cases and 1,037 controls from two studies.

RESULTS

The minor allele for one synonymous SNP in NFKBIA, rs1957106, was associated with decreased risk (p = 0.03).

CONCLUSIONS

Considering the number of single-SNP tests performed and null gene-level results, we conclude that NFKBIA and NFKBIB are not likely to harbor ovarian cancer risk alleles. Due to its biological significance in ovarian cancer, additional genes encoding NF-kappaB subunits, activating and inhibiting molecules, and signaling molecules warrant interrogation.

OBJECTIVE

To examine genetic association between rheumatoid arthritis (RA) and known polymorphisms in core genes of the nuclear factor (NF)kappaB pathway, the major intracellular pathway in RA pathogenesis.

METHODS

Discovery and replication sample sets of Spanish patients with RA and controls were studied. A total of 181 single nucleotide polymorphisms (SNPs) uniformly spaced along the genomic sequences of 17 core genes of the NFkappaB pathway (REL, RELA, RELB, NFKB1, NFKB2, NFKBIA, NFKBIB, NFKBIE, IKBKA, IKBKB, IKBKE, IKBKAP, KBRAS1, KBRAS2, MAP3K1, MAP3K14, TAX1BP1) were studied by mass spectrometry analysis complemented with 5'-nuclease fluorescence assays in the discovery set, 458 patients with RA and 657 controls. SNPs showing nominal significant differences were further investigated in the replication set of 1189 patients with RA and 1092 controls.

RESULTS

No clear reproducible association was found, although 12 SNPs in IKBKB, IKBKE and REL genes showed significant association in the discovery set. Interestingly, two of the SNPs in the IKBKE gene, weakly associated in the discovery phase, showed a trend to significant association in the replication phase. Pooling both sample sets together, the association with these two SNPs was significant.

CONCLUSIONS

We did not find any major effect among the explored members of the NFkappaB pathway in RA susceptibility. However, it is possible that variation in the IKBKE gene could have a small effect that requires replication in additional studies.

Multiple mediators of septic shock are regulated by the transcription factor nuclear factor κB (NFκB). However, complete NFκB inhibition can exacerbate disease, necessitating evaluation of targeted strategies to attenuate the pro-inflammatory response. Here, we demonstrate that in murine macrophages, low-dose NFκB inhibitors specifically attenuates lipopolysaccharide (LPS)-induced IκBβ degradation and the expression of a select subset of target genes (encoding IL1β, IL6, IL12β). Gain- and loss-of-function experiments demonstrate the necessary and sufficient role of inhibitor of NFκB family member IκBβ (also known as NFKBIB) in the expression of these genes. Furthermore, both fibroblasts and macrophages isolated from IκBβ overexpressing mice demonstrate attenuated LPS-induced IκBβ-NFκB signaling and IL1β, IL6 and IL12β expression. Further confirming the role of IκBβ and its NFκB subunit binding partner cRel in LPS-induced gene expression, pre-treatment of wild-type mouse embryonic fibroblasts with a cell-permeable peptide containing the cRel nuclear localization sequence attenuated IL6 expression. We prove that LPS-induced IκBβ-NFκB signaling can be selectively modulated to attenuate the expression of select pro-inflammatory target genes, thus providing therapeutic insights for patients exposed to systemic inflammatory stress.

The Chromosome 19 Consortium, a part of the Chromosome-Centric Human Proteome Project (C-HPP, http://www.C-HPP.org ), is tasked with the understanding chromosome 19 functions at the gene and protein levels, as well as their roles in lung oncogenesis. Comparative genomic hybridization (CGH) studies revealed chromosome aberration in lung cancer subtypes, including ADC, SCC, LCC, and SCLC. The most common abnormality is 19p loss and 19q gain. Sixty-four aberrant genes identified in previous genomic studies and their encoded protein functions were further validated in the neXtProt database ( http://www.nextprot.org/ ). Among those, the loss of tumor suppressor genes STK11, MUM1, KISS1R (19p13.3), and BRG1 (19p13.13) is associated with lung oncogenesis or remote metastasis. Gene aberrations include translocation t(15, 19) (q13, p13.1) fusion oncogene BRD4-NUT, DNA repair genes (ERCC1, ERCC2, XRCC1), TGFβ1 pathway activation genes (TGFB1, LTBP4), Dyrk1B, and potential oncogenesis protector genes such as NFkB pathway inhibition genes (NFKBIB, PPP1R13L) and EGLN2. In conclusion, neXtProt is an effective resource for the validation of gene aberrations identified in genomic studies. It promises to enhance our understanding of lung cancer oncogenesis.

BACKGROUND

Wheezing during early life is a very common disorder, but the reasons underlying the different wheezing phenotypes are still unclear. The aims of this study were to analyse the potential correlations between the risk of developing recurrent wheezing and the presence of specific polymorphisms of some genes regulating immune system function, and to study the relative importance of the associations of different viruses and genetic polymorphisms in causing recurrent episodes.

METHODS

The study involved 119 otherwise healthy infants admitted to hospital for a first episode of wheezing (74 of whom subsequently experienced recurrent episodes) and 119 age- and sex-matched subjects without any history of respiratory problem randomly selected from those attending our outpatient clinic during the study period. All of the study subjects were followed up for two years, and 47 single nucleotide polymorphisms (SNPs) in 33 candidate genes were genotyped on whole blood using an ABI PRISM 7900 HT Fast Real-time instrument.

RESULTS

IL8-rs4073AT, VEGFA-rs833058CT, MBL2-rs1800450CT and IKBKB-rs3747811AT were associated with a significantly increased risk of developing wheezing (p = 0.02, p = 0.03, p = 0.05 and p = 0.0018), whereas CTLA4-rs3087243AG and NFKBIB-rs3136641TT were associated with a significantly reduced risk (p = 0.05 and p = 0.04). IL8-rs4073AT, VEGFA-rs2146323AA and NFKBIA-rs2233419AG were associated with a significantly increased risk of developing recurrent wheezing (p = 0.04, p = 0.04 and p = 0.03), whereas TLR3-rs3775291TC was associated with a significantly reduced risk (p = 0.03). Interestingly, the study of gene-environment interactions showed that rhinovirus was significantly associated with recurrent wheezing in the presence of IL4Ra-rs1801275GG and G (odds ratio [OR] 6.03, 95% confidence interval [CI]: 1.21-30.10, p = 0.03) and MAP3K1-rs702689AA (OR 4.09, 95% CI: 1.14-14.61, p = 0.03).

CONCLUSIONS

This study shows a clear relationship between the risk of wheezing and polymorphisms of some genes involved in the immune response. Although further studies are needed to confirm the results, these findings may be useful for the early identification of children at the highest risk of developing recurrent episodes and possibly subsequent asthma.

Parasite recognition by Toll-like receptors (TLRs) contributes to macrophage activation and subsequent control of Leishmania infection through the coordinated production of pro-inflammatory and microbicidal effector molecules. The modulation of microRNA (miRNA) expression by Leishmania infection potentially mediates the post-transcriptional regulation of the expression of genes involved in leishmanicidal activity. Here, the contribution of TLR signaling to the miRNA profile and gene expression was evaluated in Leishmania amazonensis-infected murine macrophages. The infectivity of L. amazonensis was higher in murine bone marrow-derived macrophages from mice knockout for myeloid differentiation factor 88 (MyD88^-/-), TLR2 (TLR2^-/-), or TLR4 (TLR4^-/-) than wild type C57BL/6 (WT). L. amazonensis infection of WT macrophages modulated the expression of 32% of the miRNAs analyzed, while 50% were upregulated. The absence of MyD88, TLR2, and TLR4 altered the percentage of miRNAs modulated during L. amazonensis infection, including the downregulation of let-7e expression. Moreover, the absence of signals mediated by MyD88, TLR2, or TLR4 reduced nitric oxide synthase 2 (Nos2) mRNA expression during infection. Indeed, the inhibition of let-7e increased levels of the NosL. amazonensis-infected macrophages (4-24 h). The absence of TLR2 and inhibition of let-7e increased the expression of the arginase 1 (ArgCatCatTlrTlrLyTirap, Traf 6, TicamTollip, CaspMapkMapkNfkbib, NfkbilPpara, Mapk8ip3, HspdUbe2n, as well as immunomodulators, such as PtgsCoxCsf 2, Csf 3, IfnbIlra, and IlrL. amazonensis infection alters the TLR signaling pathways by modulating the expression of miRNAs in macrophages to subvert the host immune responses.

The aim of the present research was to investigate the regulation of gene expression in ovine blood leukocytes during ACTH-induced cortisol release and the effect of dietary administration of botanicals to counteract the evoked response in polymorphonucleate cells (PMNCs). Thirty-six homogeneous Sarda sheep (age, 18±4.1 mo; BW, 38.7±1.3 kg) were allotted to six groups of six sheep each. One group was used as a negative control (Saline) and five groups were treated, every 12 h for 48 h, with 0.5 mL of ACTH agonist (250 μg/mL of tetracosactrin). Before ACTH treatment, four of the five ACTH-treated groups were separated and fed for 22 d with a basal diet supplemented with extracts from Echinacea angustifolia roots (PO+ACTH), Echinacea angustifolia flowers (EA+ACTH), Andrographis paniculata (AP+ACTH), and the bark of Larix decidua milled (LB+ACTH). Control groups (Saline and ACTH) were fed with the same basal diet without botanicals. Total RNA was extracted from blood samples collected before (T0) and after 3 h (T3) and 51 h (T51) from the first ACTH injection, and transcriptome analysis was performed using a custom oligoarray, designed from 12,194 Ovis aries UniGenes on a CombiMatrix platform. At T3, treatment with ACTH caused down-regulation of transcripts (P<0.001) involved in "response to stress" (GADD45A, GADD45B, WRNIP1, and XRCC6) and in "innate immune response" (MAPK3 and NFkBIB). At T51, treatment with ACTH caused down-regulation (P<0.001) of genes involved in "immune response" (IFNG and IL2) and up-regulation (P<0.001) of NF-κB1 and TP53. Each botanical produced a different (P<0.001) molecular signature for these genes at T3 and T51. The most active botanical in modulating transcriptome modifications in PMNCs after ACTH-induced cortisol release was Larix decidua Mill bark followed by Polinacea roots. These botanicals can be viewed as promising feed supplements in ruminants to cope with conditions associated with increased concentrations of plasma cortisol.

Tripterygium hypoglaucum (levl.) Hutch (Celastraceae) (THH) root is a Chinese medicinal herb commonly used for treating autoimmune diseases. In the present study, alkaloids of THH were prepared and their cytotoxicity against the HL-60 cell was investigated. THH-induced apoptosis was observed using flow cytometry, confocal fluorescence microscope, and DNA laddering and caspase assays. The molecular mechanism involved in the induction of HL-60 cell apoptosis by THH alkaloids was examined using cDNA microarrays containing 3000 human genes derived from a leukocyte cDNA library. Sixteen genes were identified to be differentially expressed in HL-60 cells upon THH treatment. Several genes related to the NF-kappaB signaling pathway and cell apoptosis (such as NFKBIB, PRG1 and B2M) were up-regulated. In addition, c-myc binding protein and apoptosis-related cysteine proteases caspase-3 and caspase-8 were also regulated. The changes in c-Myc RNA expression and c-myc protein level were further confirmed by RT-PCR and Western blot analysis. The results demonstrated that THH alkaloids induced apoptosis of HL-60 cells though c-myc and NF-kappaB signaling pathways.

Activating MYD88 mutations are present in 95% of Waldenström macroglobulinemia (WM) patients, and trigger NF-κB through BTK and IRAK. The BTK inhibitor ibrutinib is active in MYD88-mutated (MYD88 MUT ) WM patients, but shows lower activity in MYD88 wild-type (MYD88 WT ) disease. MYD88 WT patients also show shorter overall survival, and increased risk of disease transformation in some series. The genomic basis for these findings remains to be clarified. We performed whole exome and transcriptome sequencing of sorted tumor samples from 18 MYD88 WT patients and compared findings with WM patients with MYD88 MUT disease. We identified somatic mutations predicted to activate NF-κB (TBL1XR1, PTPN13, MALT1, BCL10, NFKB2, NFKBIB, NFKBIZ, and UDRL1F), impart epigenomic dysregulation (KMT2D, KMT2C, and KDM6A), or impair DNA damage repair (TP53, ATM, and TRRAP). Predicted NF-κB activating mutations were downstream of BTK and IRAK, and many overlapped with somatic mutations found in diffuse large B-cell lymphoma. A distinctive transcriptional profile in MYD88 WT WM was identified, although most differentially expressed genes overlapped with MYD88 MUT WM consistent with the many clinical and morphological characteristics that are shared by these WM subgroups. Overall survival was adversely affected by mutations in DNA damage response in MYD88 WT WM patients. The findings depict genomic and transcriptional events associated with MYD88 WT WM and provide mechanistic insights for disease transformation, decreased ibrutinib activity, and novel drug approaches for this population.