Epithelial coxsackievirus adenovirus receptor promotes house dust mite-induced lung inflammation.

Airway inflammation and remodelling are important pathophysiologic features in asthma and other respiratory conditions. An intact epithelial cell layer is crucial to maintain lung homoeostasis, and this depends on intercellular adhesion, whilst damaged respiratory epithelium is the primary instigator of airway inflammation. The Coxsackievirus Adenovirus Receptor (CAR) is highly expressed in the epithelium where it modulates cell-cell adhesion stability and facilitates immune cell transepithelial migration. However, the contribution of CAR to lung inflammation remains unclear. Here we investigate the mechanistic contribution of CAR in mediating responses to the common aeroallergen, House Dust Mite (HDM). We demonstrate that administration of HDM in mice lacking CAR in the respiratory epithelium leads to loss of peri-bronchial inflammatory cell infiltration, fewer goblet-cells and decreased pro-inflammatory cytokine release. In vitro analysis in human lung epithelial cells confirms that loss of CAR leads to reduced HDM-dependent inflammatory cytokine release and neutrophil migration. Epithelial CAR depletion also promoted smooth muscle cell proliferation mediated by GSK3ß and TGF-ß, basal matrix production and airway hyperresponsiveness. Our data demonstrate that CAR coordinates lung inflammation through a dual function in leucocyte recruitment and tissue remodelling and may represent an important target for future therapeutic development in inflammatory lung diseases.

Towards a 21st-century roadmap for biomedical research and drug discovery: consensus report and recommendations.

Decades of costly failures in translating drug candidates from preclinical disease models to human therapeutic use warrant reconsideration of the priority placed on animal models in biomedical research. Following an international workshop attended by experts from academia, government institutions, research funding bodies, and the corporate and non-governmental organisation (NGO) sectors, in this consensus report, we analyse, as case studies, five disease areas with major unmet needs for new treatments. In view of the scientifically driven transition towards a human pathways-based paradigm in toxicology, a similar paradigm shift appears to be justified in biomedical research. There is a pressing need for an approach that strategically implements advanced, human biology-based models and tools to understand disease pathways at multiple biological scales. We present recommendations to help achieve this.

Leukotriene E4 is a full functional agonist for human cysteinyl leukotriene type 1 receptor-dependent gene expression.

Leukotriene E4 (LTE4) the most stable of the cysteinyl leukotrienes (cysLTs) binds poorly to classical type 1 (CysLT1) and 2 (CysLT2) receptors although it induces potent responses in human airways in vivo, such as bronchoconstriction, airway hyperresponsiveness and inflammatory cell influx suggesting the presence of a novel receptor that preferentially responds to LTE4. To identify such a receptor two human mast cell lines, LAD2 and LUVA, were selected that differentially responded to LTE4 when analysed by intracellular signalling and gene expression. Comparative transcriptome analysis and recombinant gene overexpression experiments revealed CysLT1 as a receptor responsible for potent LTE4-induced response in LAD2 but not in LUVA cells, an observation confirmed further by gene knockdown and selective inhibitors. Lentiviral overexpression of CysLT1 in LUVA cells augmented intracellular calcium signalling induced by LTE4 but did not restore full agonist responses at the gene expression level. Our data support a model where both an increased expression of Gαq-coupled CysLT1, and sustained intracellular calcium mobilisation and extracellular signal-regulated kinase (Erk) activation, are required for LTE4-mediated regulation of gene expression in human cells. Our study shows for the first time that CysLT1 expression is critically important for responsiveness to LTE4 within a human cell system.

Characterisation of P2Y(12) receptor responsiveness to cysteinyl leukotrienes.

Leukotriene E4 (LTE4), the most stable of the cysteinyl leukotrienes (cysLTs), binds poorly to classical type 1 and 2 cysLT receptors although in asthmatic individuals it may potently induce bronchial constriction, airway hyperresponsiveness and inflammatory cell influx to the lung. A recent study has suggested that the purinergic receptor P2Y12 is required for LTE4 mediated pulmonary inflammation in a mouse model of asthma and signals in response to cysLTs. The aim of the study was to characterise the responsiveness of human P2Y12 to cysteinyl leukotrienes. Models of human CysLT1, CysLT2 and P2Y12 overexpressed in HEK293, CHO cells and human platelets were used and responsiveness to different agonists was measured using intracellular calcium, cAMP and ß-arrestin recruitment assays. CysLTs induced concentration dependent calcium mobilisation in cells overexpressing CysLT1 and CysLT2 but failed to induce any calcium response in cells expressing P2Y12 or P2Y12+ Gα16. In contrast, selective P2Y12 agonists ADP and 2-MeS-ADP induced specific calcium flux in cells expressing P2Y12+ Gα16. Similarly, specific response to 2-MeS-ADP, but not to cysLTs was also observed in cells expressing P2Y12 when intracellular cAMP and ß-arrestin signalling were analysed. Platelets were used as a model of human primary cells expressing P2Y12 to analyse potential signalling and cell activation through P2Y12 receptor or receptor heterodimers but no specific LTE4 responses were observed. These results show that LTE4 as well as other cysLTs do not activate intracellular signalling acting through P2Y12 and suggest that another LTE4 specific receptor has yet to be identified.

Human T(H)2 cells respond to cysteinyl leukotrienes through selective expression of cysteinyl leukotriene receptor 1.

BACKGROUND: Allergic asthma is characterized by reversible airway obstruction and bronchial hyperresponsiveness associated with T(H)2 cell-mediated inflammation. Cysteinyl leukotrienes (CysLTs) are potent lipid mediators involved in bronchoconstriction, mucus secretion, and cell trafficking in asthmatic patients. Recent data have implicated CysLTs in the establishment and amplification of T(H)2 responses in murine models, although the precise mechanisms are unresolved. OBJECTIVES: Preliminary microarray studies suggested that human T(H)2 cells might selectively express cysteinyl leukotriene receptor 1 (CYSLTR1) mRNA. We sought to establish whether human T(H)2 cells are indeed a CysLT target cell type. METHODS: We examined the expression of CYSLTR1 using real-time PCR in human T(H)1 and T(H)2 cells. We functionally assessed cysteinyl leukotriene receptor 1 protein (CysLT(1)) expression using calcium flux, cyclic AMP, and chemotaxis assays. RESULTS: We show that human T(H)2 cells selectively express CYSLTR1 mRNA at high levels compared with T(H)1 cells after in vitro differentiation from naive precursors. Human T(H)2 cells are selectively responsive to CysLTs in a calcium flux assay when compared with T(H)1 cells with a rank order of potency similar to that described for CysLT(1) (leukotriene [LT] D(4) > LTC(4) > LTE(4)). We also show that LTD(4)-induced signaling in T(H)2 cells is mediated through CysLT(1) coupled to G(α)q and G(α)i proteins, and both pathways can be completely inhibited by selective CysLT(1) antagonists. LTD(4) is also found to possess potent chemotactic activity for T(H)2 cells at low nanomolar concentrations. CONCLUSIONS: These findings suggest a novel mechanism of action for CysLTs in the pathogenesis of asthma and provide a potential explanation for the anti-inflammatory effects of CysLT(1) antagonists.

Concentration-dependent noncysteinyl leukotriene type 1 receptor-mediated inhibitory activity of leukotriene receptor antagonists.

The use of cysteinyl leukotriene receptor antagonists (LTRAs) for asthma therapy has been associated with a significant degree of interpatient variability in response to treatment. Some of that variability may be attributable to noncysteinyl leukotriene type 1 receptor (CysLT(1))-mediated inhibitory mechanisms that have been demonstrated for this group of drugs. We used a model of CysLT(1) signaling in human monocytes to characterize CysLT(1)-dependent and -independent anti-inflammatory activity of two chemically different, clinically relevant LTRAs (montelukast and zafirlukast). Using receptor-desensitization experiments in monocytes and CysLT(1)-transfected HEK293 cells and IL-10- and CysLT(1) small interfering RNA-induced downregulation of CysLT(1) expression, we showed that reported CysLT(1) agonists leukotriene D(4) and UDP signal through calcium mobilization, acting on separate receptors, and that both pathways were inhibited by montelukast and zafirlukast. However, 3-log greater concentrations of LTRAs were required for the inhibition of UDP-induced signaling. In monocytes, UDP, but not leukotriene D(4), induced IL-8 production that was significantly inhibited by both drugs at micromolar concentrations. At low micromolar concentrations, both LTRAs also inhibited calcium ionophore-induced leukotriene (leukotriene B(4) and leukotriene C(4)) production, indicating 5-lipoxygenase inhibitory activities. We report herein that montelukast and zafirlukast, acting in a concentration-dependent manner, can inhibit non-CysLT(1)-mediated proinflammatory reactions, suggesting activities potentially relevant for interpatient variability in response to treatment. Higher doses of currently known LTRAs or new compounds derived from this class of drugs may represent a new strategy for finding more efficient therapy for bronchial asthma.

Leukotriene E4: perspective on the forgotten mediator.

Leukotriene (LT) E(4) mediates many of the principal features of bronchial asthma, such as bronchial constriction, hyperresponsiveness, eosinophilia, and increased vascular permeability. Furthermore, it is the most stable of the cysteinyl leukotrienes (CysLTs) and can be active at the site of release for a prolonged time after its synthesis. There might be several reasons why LTE(4) has been forgotten. LTE(4) demonstrated low affinity for CysLT(1) and CysLT(2) receptors in equilibrium competition assays. It was less potent than other CysLTs in functional assays, such as calcium flux, in cells transfected with CysLT(1) and CysLT(2). The introduction of CysLT(1) antagonists into clinical practice diverted interest into CysLT(1)-related mechanisms, which were mediated mainly by LTD(4). However, experiments with animal models and human studies have revealed that LTE(4) has unique characteristics that cannot be explained by the current knowledge of CysLT(1) and CysLT(2). These activities include its potency relative to other CysLTs to increase airway responsiveness to histamine, to enhance eosinophilic recruitment, and to increase vascular permeability. Asthmatic airways also demonstrate marked in vivo relative hyperresponsiveness to LTE(4), especially in patients with aspirin-sensitive respiratory disease. This has stimulated a search for additional LT receptors that would respond preferentially to LTE(4) stimulation.

IL-10 inhibits cysteinyl leukotriene-induced activation of human monocytes and monocyte-derived dendritic cells.

The immunoregulatory cytokine IL-10 plays an essential role in down-modulating adaptive and innate immune responses leading to chronic inflammatory diseases. In contrast, cysteinyl leukotrienes (cysLTs), important proinflammatory mediators of cell trafficking and innate immune responses, are thought to enhance immune reactions in the pathogenesis of diseases, such as bronchial asthma, atherosclerosis, and pulmonary fibrosis. The aim of this study was to determine the IL-10 regulatory role in cysLT-induced activation of human monocytes and monocyte-derived dendritic cells. Herein we show that cysLT-induced activation and chemotaxis of human monocytes and monocyte-derived immature dendritic cells (iDC) are inhibited by IL-10 pretreatment. IL-10 down-regulated cysLT type 1 and 2 receptors' mRNA in a time- and concentration-dependent fashion. cysLT-induced activation of monocytes and iDCs measured by intracellular calcium flux and immediate-early gene expression (FBJ murine osteosarcoma viral oncogen homolog B and early growth response-2) was potently decreased by IL-10 and by the cysLT antagonist MK571. Chemotaxis of monocytes and iDCs to increasing concentrations of leukotriene D(4) (LTD(4)) was also inhibited by IL-10. LTD(4) enhanced iDC migration in response to CCL5. IL-10 selectively inhibited LTD(4)-induced chemotaxis without affecting migration to CCL5. These data indicate that cysLT-induced activation of human monocytes and dendritic cells may be specifically inhibited by IL-10, suggesting a direct link between the 5-lipoxygenase proinflammatory pathway and IL-10 regulatory mechanisms. Antileukotriene therapies may reproduce some regulatory mechanisms played by IL-10 in inflammatory processes.

Leukotriene D(4) induces gene expression in human monocytes through cysteinyl leukotriene type I receptor.

BACKGROUND: Cysteinyl leukotrienes (CysLTs) are important mediators of innate immune responsiveness and chronic inflammatory diseases. CysLTs acting through CysLT receptors can influence the migration and activity of cells, such as eosinophils, monocytes, and dendritic cells. OBJECTIVE: We sought to determine the gene expression signature of human monocytes in response to CysLTs and to elucidate the signaling pathways involved in monocyte activation. METHODS: Gene expression was analyzed by using oligonucleotide microarrays. Responsiveness to CysLTs was assessed by using real-time PCR, calcium flux, kinase activation, and chemotaxis assays. RESULTS: CysLT type 1 receptor (CysLTR(1)) transcript 1 is predominantly expressed in human monocytes, and CysLTs signal through CysLTR(1) in these cells. Several immediate-early genes, including early growth response 2 and 3, FBJ murine osteosarcoma viral oncogene homolog B, activating transcription factor 3, and nuclear receptor subfamily 4 were significantly induced by leukotriene (LT) D(4). This effect was mediated by CysLTR(1) coupled to the G protein alpha inhibitory subunit, activation of phospholipase C, and inositol-1,4,5-triphosphate and store-operated calcium channels. LTD(4) induced p38 mitogen-activated protein kinase phosphorylation, a pathway also involved in the regulation of immediate-early gene expression in monocytes. LTD(4) stimulated monocyte chemotactic activity that was fully blocked by a selective CysLTR(1) inhibitor, MK571, and pertussis toxin, suggesting that CysLTR(1) coupled to the G protein alpha inhibitory subunit is a dominant functional pathway in human monocytes. CONCLUSION: Our data show that CysLTs acting through CysLTR(1) can significantly influence the activation and migration of human monocytes and that these effects can be fully inhibited by CysLTR(1) antagonists.

IFN-gamma induces cysteinyl leukotriene receptor 2 expression and enhances the responsiveness of human endothelial cells to cysteinyl leukotrienes.

Cysteinyl leukotrienes (cysLTs) are important mediators of cell trafficking and innate immune responses, involved in the pathogenesis of inflammatory processes, i.e., atherosclerosis, pulmonary fibrosis, and bronchial asthma. The aim of this study was to examine the regulation of cysLT signaling by IFN-gamma in human primary endothelial cells. IFN-gamma increased cysLT receptor 2 (CysLTR2) mRNA expression and CysLTR2-specific calcium signaling in endothelial cells. IFN-gamma signaled through Jak/STAT1, as both AG490, a Jak2 inhibitor, and expression of a STAT1 dominant-negative construct, significantly inhibited CysLTR2 mRNA expression in response to IFN-gamma. To determine mechanisms of IFN-gamma-induced CysLTR2 expression, the human CysLTR2 gene structure was characterized. The CysLTR2 gene has a TATA-less promoter, with multiple transcription start sites. It consists of six variably spliced exons. Eight different CysLTR2 transcripts were identified in endothelial and monocytic cells. Gene reporter assay showed potent basal promoter activity of a putative CysLTR2 promoter region. However, there were no significant changes in gene reporter and mRNA t(1/2) assays in response to IFN-gamma, suggesting transcriptional control of CysLTR2 mRNA up-regulation by IFN-gamma response motifs localized outside of the cloned CysLTR2 promoter region. Stimulation of endothelial cells by cysLTs induced mRNA and protein expression of early growth response genes 1, 2, and 3 and cycloxygenase-2. This response was mediated by CysLTR2 coupled to G(q/11), activation of phospholipase C, and inositol-1,4,5-triphosphate, and was enhanced further 2- to 5-fold by IFN-gamma stimulation. Thus, IFN-gamma induces CysLTR2 expression and enhances cysLT-induced inflammatory responses.

Functional characterization of human cysteinyl leukotriene 1 receptor gene structure.

The 5-lipoxygenase pathway has been strongly implicated in the pathogenesis of chronic inflammatory disorders, such as bronchial asthma and atherosclerosis. Cysteinyl leukotrienes (cysLTs), 5-lipoxygenase pathway products, are recognized now not only as important factors in asthmatic inflammation, but also as mediators of cell trafficking and innate immune responses. To study a role of cysLTs in inflammatory reactions we have characterized the gene structure of human cysteinyl leukotriene receptor type I (cysLT(1)R). The cysLT(1)R gene consists of 5 exons that are variably spliced and a single promoter region with multiple transcription start sites. Four different cysLT(1)R transcripts were identified. RT-PCR showed dominant and wide expression of the transcript I, containing exons 1, 4, and 5, with the strongest presence in blood leukocytes, spleen, thymus, lung, and heart. The expression of cysLT(1)R is functionally regulated at the transcriptional level by IL-4 through a STAT6 response element localized to the proximal cysLT(1)R promoter region. IL-4 stimulation increased cysLT(1)R mRNA (real-time PCR) and surface protein expression (flow cytometry) in a time-dependent fashion. CysLTs (LTD(4) and LTC(4)) induced an increased production of a potent monocyte chemoattractant CCL2 (MCP-1) in IL-4-primed THP-1 cells in a dose-dependent manner. This effect was effectively inhibited by the cysLT(1)R-selective antagonist MK571 in a dose-dependent manner and only partially by a nonselective cysLT(1)R/cysLT(2)R inhibitor BAY-u9773, implying a cysLT(1)R-mediated mechanism. Thus, cysLTs signaling through cysLT(1)R might contribute to inflammatory reactions by cooperating with IL-4 in enhanced CCL2 production in human monocytic cells.

[The role of genetic polymorphisms within tumor necrosis factor promoter gene in non-Hodgkin's lymphomas]. / Znaczenie genetycznych polimorfizmów promotora genu dla czynnika martwicy nowotworów w nieziarniczych chloniakach zlosliwych.

Elevated tumor necrosis factor (TNF) and its soluble receptors (p55 and p75) plasma levels in patients with non-Hodgkin's lymphoma (NHL) have been shown to correlate with various adverse prognostic factors and predict poor NHL outcome. In vitro studies demonstrated that TNF expression level could be influenced by TNF-376, -308, -238, -163 promoters' polymorphisms. To explore whether these polymorphisms confer the susceptibility to and influence NHL outcome, we genotyped the TNF-376, -308, -238, -163 polymorphisms in 204 NHL patients and 96 healthy controls. The frequency and distribution of polymorphic alleles were similar in both studied groups. TNF-308A was the only polymorphic allele related to elevated TNF, p55, p75 plasma levels (p = 0.009, p = 0.03, p = 0.007, respectively), lower complete remission rate (p = 0.01), higher progression (p = 0.06) and death (p = 0.01) incidences. TNF-308A was the sole allele of independent prognostic significance for shorter freedom from progression (FFP) and overall survival (OS) (p = 0.009 and p = 0.017, respectively). These data indicate that innate immunity as reflected by the genetic propensity of the host to regulate TNF expression influences clinical course and outcome of NHL.

[The role of HLA DRB1 genetic polymorphisms in non-Hodgkin's lymphomas]. / Znaczenie genetycznych polimorfizmów HLA DRB1 w nieziarniczych chloniakach zlosliwych.

The role of tumor necrosis factor (TNF)-308A polymorphic allele on non-Hodgkin's lymphoma (NHL) outcome was documented in the previous studies, although the role of the neighboring polymorphisms was unknown. The aim of the present study was to asses the frequencies and distributions of the HLA DRB1, TNF-308 and lymphotoxin alpha (LTalpha)+252 allelic polymorphisms in NHL patients and healthy controls and their influence on NHL outcome. The HLA DRB1, TNF-308 and LTalpha +252 allelic frequencies and distributions didn't differ significantly between patients and healthy controls, thus it is unlikely that polymorphisms within the above mentioned sites confer susceptibility for lymphoma occurrence. Among the polymorphic alleles HLA DRB1*03, TNF-308A and LTalpha +252A remaining in linkage disequilibrium, TNF-308A was the only allele associated with higher TNF and its p55 and p75 receptors' plasma levels (p = 0.009, p = 0.03, and p = 0.007), lower complete remission rates (p = .006), shorter freedom from progression (FFP) and overall survival (OS) (p = 0.009 and p = 0.017, respectively). Among the polymorphic HLA DRB1 alleles, null HLA DRB1*02 was the sole allele along with the TNF-308A that remained independent factors for shorter FFP (relative risk [RR] = 1.18, p < 0.02 and RR = 1.63, p < 0.0001, respectively) and OS (RR = 1.25, p < 0.0001 and RR = 1.51, p < 0.0001, respectively). Innate immunity reflected by inherited HLA DRB1 genes repertoire and genetic propensity of the host to regulate TNF production and/or other closely linked genes influences clinical course and outcome of NHL.

Cytosolic phospholipase A2 Group IValpha but not secreted phospholipase A2 Group IIA, V, or X induces interleukin-8 and cyclooxygenase-2 gene and protein expression through peroxisome proliferator-activated receptors gamma 1 and 2 in human lung cells.

It has been reported that interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) expression is regulated by peroxisome proliferator-activated receptor (PPAR)-gamma synthetic ligands. We have shown previously that cytosolic phospholipase A2 (cPLA2) is able to activate gene expression through PPAR-gamma response elements (Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H., and Wu, T. (2002) J. Biol. Chem. 277, 33153-33163). In this study we investigated the influence of cPLA2 and secreted phospholipase A2 (sPLA2) Group IIA, Group V, and Group X on IL-8 and COX-2 expression in human lung epithelial cells (A549 cells). We also studied the results of cPLA2 activation by epidermal growth factor (EGF) and calcium ionophore (A23187) on IL-8 and COX-2 reporter gene activity, mRNA level, and protein synthesis. cPLA2 overexpression and activation increased both IL-8 and COX-2 reporter gene activity. Overexpression and activation of Group IIA, Group V, or Group X sPLA2s did not increase IL-8 and COX-2 reporter gene activity. Methyl arachidonyl fluorophosphate, a cPLA2 inhibitor, inhibited the effect of A23187 and of EGF on both IL-8 and COX-2 reporter gene activity, steady state levels of IL-8 and COX-2 mRNA, and IL-8 and COX-2 protein expression. Small inhibitory RNAs directed against PPAR-gamma1 and -gamma2 blunted the effect of A23187 and of EGF on IL-8 and COX-2 protein expression. Moreover small inhibitory RNAs directed against cPLA2 decreased the effect of A23187 and EGF on IL-8 and COX-2 protein expression. These results demonstrate that cPLA2 has an influence on IL-8 and COX 2 gene and protein expression at least in part through PPAR-gamma.

Tumour necrosis factor-alpha polymorphism as one of the complex inherited factors in pemphigus.

The aim of our study was to analyse a significance of tumour necrosis factor (TNF)-alpha promoter gene polymorphisms in relation to the HLA-DR locus in genetic predisposition to pemphigus. TNF-alpha gene polymorphisms in position -238 and -308 were identified using a modified polymerase chain reaction-restriction fragment length polymorphism method in 53 patients with pemphigus (38 with pemphigus vulgaris, 15 with pemphigus foliaceus) and 87 healthy controls. The HLA-DRB1 locus was typed using the polymerase chain reaction SSO method in all the patients and 152 population controls. Carriers of the TNF-alpha polymorphic -308 A allele were found to be more frequent in the pemphigus foliaceus group in comparison with the control group (odds ratio (OR) = 8.12; p = 0.0005). A significant association between HLA-DRB1*04 (OR = 3.86; pcor = 0.0001) and DRB1*14 (OR = 8.4; pcor = 0.0001) and pemphigus vulgaris was found. In this group of patients a decreased frequency of HLA-DRB1*07 (OR = 0.08; pcor = 0.006) was also identified. We have shown for the first time a positive association of TNF-alpha polymorphism in position -308 with pemphigus foliaceus.

[Leukotrienes as inflammation mediators]. / Leukotrieny--mediatory zapalenia.

Leukotrienes are biologically active metabolites derived from arachidonic acid playing an important role in inflammatory responses. There are two main groups of leukotrienes: dihydroxyleukotrienes (LTB4) and cysteinyl-leukotrienes (LTC4, LTD4, LTE4). By activating specific G-protein coupled receptors, leukotrienes take part in immune responses, like activation and chemotaxis of leukocytes. Several studies have shown that leukotrienes may play a significant role in pathomechanisms of inflammatory diseases of human airways, skin, digestive tract and heart.

Comparison study for genotyping of a single-nucleotide polymorphism in the tumor necrosis factor promoter gene.

A variety of methods that address the detection of single-nucleotide polymorphism (SNP) have been used in molecular diagnostics. The allele-specific polymerase chain reaction (ASPCR) has been one of the most extensively studied, including its application in the tumor necrosis factor (TNF)(-308) genotyping. Many studies have demonstrated that the ASPCR sensitivity and specificity depends on various PCR parameters, with mismatches occurring to a degree of 4%. The purpose of our study was to evaluate a comparison of genotyping of the TNF(-308) using an ASPCR and automated sequencing (ASEQ). In a total of 204 DNA samples, their duplicate examination by the ASPCR and ASEQ revealed concordant results in 96.5% and mismatches in 3.5% genotypes. Depending on the target TNF(-308G/G), TNF(-308G/A) , TNF(-308A/A) sequences, this translated into decreased ASPCR sensitivity to a degree of 98.6%, 94.2%, 60.0%, specificity 94.7%, 97.4%, 100.0%, positive predictive values 97.9%, 92.5%, 100.0%, and negative predictive values 96.4%, 98.0%, 99.0%, respectively. Based on these results, we found ASEQ to be more accurate than ASPCR for the TNF(-308) genotyping. By eliminating the need of empirical determination of appropriate PCR conditions for each studied sequence, ASEQ provides a sensitive and reproducible quality-control benchmark for other SNP assays.

Human leukocyte antigens class II and tumor necrosis factor genetic polymorphisms are independent predictors of non-Hodgkin lymphoma outcome.

Tumor necrosis factor (TNF) production and non-Hodgkin lymphoma (NHL) outcome was found to be related to the TNF(-308) polymorphism. To explore whether this could be linked to neighboring polymorphisms, we genotyped the TNF(-376,-308,-238,-163), lymphotoxin alpha (LTalpha)(+252), and HLA DRB1 alleles in 204 patients with NHL and 120 controls. TNF(-308A) was the only allele associated with higher TNF and its p55 and p75 receptors' levels (P =.009, P =.03, and P =.007) and lower complete remission rates (P =.006). Freedom from progression (FFP) and overall survival (OS) were shorter in patients with TNF(-308A) (P =.009 and P =.02), null HLA DRB1*02 allele (P =.007 and P =.14), or both genetic markers (P =.004 and P =.005). Multivariate analysis incorporating International Prognostic Index (IPI) identified TNF(-308A) (P <.0001, relative risk [RR] = 1.63; P <.0001, RR = 1.51) and null HLA DRB1*02 alleles (P =.015, RR = 1.18; P <.0001, RR = 1.25) as independent factors for FFP and OS. These results indicate the existence of at least 2 inherited factors involved in NHL outcome.