TNF genetic variants and the combined effect between TNF gene and cigarette smoking in the development of gastric cancer in the Korean population.The aim of this study was to investigate the role of TNF gene, TNF-α-238 G/A, -308 G/A, -857 C/T, -863 C/A, -1031 T/C, and TNF-β 252 A/G were genotyped. The ORs (95% CIs) were calculated using unconditional logistic regression model to detect each SNP and haplotype-pair effects for gastric cancer. The combined effects between the TNF gene and smoking on gastric cancer risk were also evaluated. Multi dimensionality reduction (MDR) analyses were performed to explore the potential TNF gene-gene interactions.We selected 84 incident gastric cancer cases and 336 matched controls nested within the Korean Multi-Center Cancer Cohort. Six SNPs on the TNF-α-857 C/T containing the T allele was significantly associated with an increased risk of gastric cancer and a linear trend effect was observed in the additive model . All haplotype-pairs that contained TCT or CCC of TNF-α-1031 T/C, TNF-α-863 C/A, and TNF-α-857 C/T were associated with a significantly higher risk for gastric cancer only among smokers. In the MDR analysis, regardless of smoking status, TNF-α-857 C/T was included in the first list of SNPs with a significant main effect.TNF-α-857 C/T polymorphism may play an independent role in gastric carcinogenesis and the risk for gastric cancer by TNF genetic effect is pronounced by cigarette smoking. H. pylori) which is labeled a group I human gastric cancer carcinogen by the International Agency for Research on Cancer [H. pylori infection does not always result in a high incidence of gastric cancer. Only a small portion of H. pylori infected persons have gastric cancer, thereby suggesting that other susceptible factors such as genetic variants or environmental differences must additionally be involved in gastric carcinogenesis.Gastric cancer is a multi-factorial disease that requires the study of environmental, genetics, and host-related factors in order to understand its pathology. The strongest risk factor for gastric cancer is Helicobacter pylori  expressed by TNF-α and TNF-β genes [TNF and gastric cancer [TNF can modify the risk of gastric cancer [TNF as a gastric carcinogen is still controversial.Genetic variants of inflammation-related cytokines are a potential risk factor because -β genes -6. Many c cancer -15. Althc cancer -11, the TNF-α and Interleukin-1B may change normal mucosa to H. pylori infected mucosa that may be a first step towards gastric carcinogenesis [In terms of environmental factors, cigarette smoking has been suggested to play a crucial role in increasing the risk of gastric cancer. Previous epidemiologic studies indicated that cigarette smoking was an independent risk factor for gastric cancer development -21. Moreogenesis .TNF cytokines and cigarette smoking may play a role in gastric carcinogenesis. They seem to play independent or synergetic roles in gastric cancer but the mechanism is still unclear. Thus, we hypothesized that genetic variants of TNF underlies the association with gastric cancer risk and/or their combined effect with cigarette smoking may modify the risk of gastric cancer. The aim of this study was to assess the role of TNF gene variants and the combined effect between TNF genes and cigarette smoking in the development of gastric cancer in the Korean population.Genetic variants of In this population-based nested case-control study, we selected subjects from the Korean Multi-Center Cancer Cohort (KMCC), a prospective cohort of participants  recruited from four urban and rural areas in Korea  . The parTNF-α gene was genotyped for five single nucleotide polymorphisms (SNPs) that were -238 G/A(rs361525), -308 G/A(rs1800629), -857 C/T(rs1799724), -863 C/A(rs1800630), and -1031 T/C(rs1799964). These five polymorphisms were located in the promoter region of TNF-α gene. The TNF-β gene that is synonymous with Lymphotoxin-α (LTA) was genotyped for 252 A/G (rs909253) SNP. Genotyping was performed using SNaPshot method. Ten samples were randomly selected and genotyped for each SNP to check for reliability. All assays were 100% concordant. More than 95% of the total subjects were successfully genotyped for all SNPs.H. pylori and seropositivity of CagA/VacA by Helico Blot 2.1™  according to the manufacturer's protocol. [H. pylori, CagA and VacA of Helico Blot 2.1™ were very high  [Serum was evaluated to identify IgG antibodies specific for rotocol. ,25. The ctively) .H. pylori seropositivity, alcohol consumption, and cigarette smoking status for gastric cancer risk. To evaluate significant covariates, we reviewed the literature and conducted logistic regression analysis; smoking status, H. pylori infection, and CagA seropositivity were selected. Because of the significant association between age/sex and smoking status, we also included age and sex as major covariates. In the analysis, all five covariates were selected adjusted variables, and smoking status was also selected as a stratified variable.Unconditional logistic regression model was used to calculate the odds ratios (ORs) and 95% confidence intervals (95% CIs) of selected characteristics such as follow time (years), H. pylori infection, and CagA seropositivity. Also, in order to correct for multiple comparisons, false discovery rate (FDR) that controls the expected proportion of incorrectly rejected null hypotheses (type I errors) was computed [The Hardy-Weinberg equilibrium assessed allele frequencies using the chi-square test. Both single SNPs and haplotype analysis were performed. To detect each SNP effect for gastric cancer, three genetic models, dominant, co-dominant and additive model, were used. Tests for significance were computed after adjusting for age, sex, smoking status, computed .2 values were calculated with 95% CIs when the genotype frequencies were in Hardy-Weinberg equilibrium [TNF-α SNPs -857, -863, and -1031; 2) three TNF-α SNPs in block 1 and TNF-β 252; and 3) all six SNPs on TNF-α and TNF-β were generated by the Haploview software using the method suggested by Gabriel et al [To evaluate putative haplotype blocks, linkage disequilibrium (LD) between loci expressing genetic variation was analyzed using Haploview 4.0. Moreover, pairwise Lewontin's D' and rilibrium . Three hel et al   of the best 1-, 2-, 3-, and 4- factor models .Multi dimensionality reduction (MDR) analyses were performed to explore the potential of genes ,31. MDR of genes -32. TNF The study protocols for the KMCC study and the present nested case-control study were approved by the institutional review boards of Seoul National University Hospital and the National Cancer Center of Korea .H. pylori infection, CagA/VacA seropositivity, and alcohol consumption. All smoking related factors were associated with an increased risk for gastric cancer. Current smokers had a significantly higher risk for gastric cancer compared to never smokers . Moreover, a dose-response relationship between smoking intensity and gastric cancer risk was found. Smoking more cigarettes per day in addition to longer smoking duration showed a significantly higher risk compared to never smokers . 41 or more pack-years of smoking was related to a statistically increased risk for gastric cancer .Table TNF polymorphisms among cases and controls and the ORs (95% CIs) for gastric cancer risk in relation to TNF genetic polymorphisms. All genotype frequencies of each SNP did not deviate from the Hardy-Weinberg equilibrium (p > 0.1). In single SNPs analysis, TNF-α-857 C/T containing the T allele was significantly associated with an increased risk of gastric cancer. CT genotype of TNF-α-857 showed a 1.7 fold increased risk for gastric cancer in the co-dominant and dominant models that were statistically significant . However the TT genotype of TNF-α-857 showed an insignificantly increased risk in the co-dominant model . In the additive model which showed a linear trend effect, the CT genotype of TNF-α-857 showed a 1.6 fold increased risk for gastric cancer (95% CI 1.0–2.5) and the TT genotype of TNF-α-857 had a 2.6 fold increased risk (95% CI 1.0–6.4). Both increase risks for the T allele in the additive model were statistically significant. Under multiple comparison tests by FDR, all p-values of TNF SNPs in each additive model were greater than 0.2.Table TNF gene and gastric cancer risk according to smoking status. In block 1 that is composed of TNF-α-1031 T/C, TNF-α-863 C/A and TNF-α-857 C/T (D' = 1.0 and r2 = 0.037), compared to TCC-TCC without a variant allele, TCC-TCT showed a significantly increased risk for gastric cancer among total subject and smokers  but was not associated with gastric cancer risk among non-smokers . TCC-CCC was associated with an increased risk only among smokers . Similar results were represented in block 2 that is composed of the three TNF-α SNPs in block 1 and TNF-β 252 A/G (D' = 1.0 and r2 = 0.184), and block 3 that is composed of all six TNF SNPs (D' = 1.0 and r2 = 0.068). Compared to the most frequent haplotype-pairs GTCC-GTCC in block 2, ATCT-XXXX or ACCC-XXXX that contained TCT or CCC of TNF-α-1031 T/C, TNF-α-863 C/A and TNF-α-857 C/T showed a significantly higher risk among smokers but not among non-smokers . Particularly, among non-smokers, other haplotype-pairs which did not contain ATCT or ACCC were associated with a significantly reduced risk for gastric cancer compared to the reference haplotype-pairs . Similarly for block 1 and 2, ATCTGG or ACCCXX haplotype-pairs that contained TCT or CCC of TNF-α-1031 T/C, TNF-α-863 C/A and TNF-α-857 C/T were also associated with a significantly higher risk for gastric cancer only among smokers .Table TNF SNPs among total subjects included TNF-α-1031 T/C, TNF-α-863 C/A, TNF-α-857 C/T and TNF-α-308. This model had maximum TA of 0.602 and maximum CVC of 9/10 (p = 0.0015). The global OR adjusted for the age, sex, smoking status, H. pylori infection, and CagA seropositivity was 2.2 (95% CI 1.3–3.6). Regardless of smoking status, TNF-α-857 C/T was included in the first list of SNPs with a significant main effect. TNF-α-857 model among total subjects had TA of 0.554 and CVC of 9/10 but was not significant at the level of 0.0799. Similar results were shown among non-smokers and smokers. Among non-smokers, the model included only TNF-α-857 and had a maximum CVC of 9/10 and a higher prediction for gastric cancer risk of  though showing minimum TA of 0.603 (p = 0.0636). In contrast, although the model included TNF-β 252 A/G, TNF-α-1031 T/C, TNF-α-857 C/T, and TNF-α-308 G/A had a higher CVC of 8/10 and maximum TA of 0.697 (p = 0.0009), it had a lower predictability for gastric cancer risk since the adjusted global OR by this model was not significant . The best model of smokers was selected with TNF-α-1031 T/C, TNF-α-863 C/A and TNF-α-857 C/T. This model had a maximum CVC of 10/10, TA of 0.615 (p = 0.0023) and adjusted global OR of 2.0 (95% CI 1.2–3.3). Although the model included TNF-α-1031 T/C, TNF-α-863 C/A, TNF-α-857 C/T, and TNF-α-308 G/A had maximum TA with the lowest p value, its CVC was relatively low (7/10).Table TNF-α-857 C/T genetic variant containing the T allele was associated with a significantly increased risk for gastric cancer. SNP-SNP interaction in the TNF gene including TNF-α-857 C/T or TNF-α-1031 T/C genetic variant was associated with a risk for gastric cancer among smokers but not among non-smokers. Moreover this effect of the TNF-α-857 C/T genetic variant was ascertained in both haplotype and MDR analysis.In our study, TNF gene polymorphisms modified the risk of gastric cancer [TNF-α polymorphisms and risk of gastric cancer [TNF-α-308 polymorphism had a significantly increased risk for gastric cancer but TNF-α-857 polymorphism only showed a marginally significant risk due to the small number of studies. In contrast to the meta-analysis, our results indicated that TNF-α-857 polymorphism was associated with a significantly increased risk for gastric cancer but other SNPs including TNF-α-308 did not show a significant association. Our findings about the single SNP effect of TNF-α-857 polymorphism can help clarify results of the previous meta-analyses. Inconsistency in TNF-α-308 polymorphism may be due to an ethnic difference since TNF-α-308 AA polymorphism is rare in the East Asian population. Also, we were not able to clearly evaluate TNF-α-308 AA polymorphism and gastric cancer because none of our gastric cancer cases had the TNF-α-308 AA polymorphism.Previous studies reported that c cancer ,8-11, whc cancer ,7,12-15.c cancer ,34. AccoTNF SNPs. Individual haplotype-pairs including TNF-α-1031, -863 and -857 were consistently associated with a significantly increased risk of gastric cancer. Compared to the single SNP effect of the TNF-α-857 polymorphism, a greater odds ratio for haplotype-pairs including TNF-α-857 indicated that a synergistic interaction among TNF SNPs was more strongly associated with gastric cancer development. These results were nearly replicated in the MDR analysis. Our consistent findings from the different statistical methodologies are quite meaningful.In haplotype-pairs and MDR analysis, we observed a genetic combined effect among TNF cytokine may interact with cigarette smoking to promote gastric cancer development. Biologically, cigarette smoking can activate systemic inflammations [TNF-α through changes in inflammatory markers or cytokine level in animal models [TNF gene in the etiology of gastric cancer [TNF-α were increased and higher TNF-α level was associated with such diseases in smokers compared to non-smokers [TNF-α levels were presented among smokers, especially subjects with the 857 T allele and rare haplotype of the TNF-α promoter [TNF-α-857 polymorphism was especially susceptible to the hazards of smoking and the TNF cytokine was strongly affected by cigarette smoking.mmations  and augml models -38. Althc cancer -42. Some-smokers -42. In opromoter . SimilarTNF gene was definitely expressed in our haplotype-pairs and MDR analysis. Haplotype-pairs formed with TNF-α-1031, -863 and -857 were associated with a significantly greater increased risk for gastric cancer only among smokers but not among non-smokers. Both single SNP and haplotype-pair effect of TNF-α-857 T allele were related with an increased risk among smokers. Haplotype-pairs including TNF-α-1031 and -863 genetic variation that did not show significance for single SNP effect were also associated with a significantly increased risk of gastric cancer among smokers only. In contrast to TNF-α-857, TNF-α-1031 and -863 may interact mutually based on haplotype specificity and not allele specificity, and this effect may be strongly affected by smoking. If only allele specificity by high-producing or mutant alleles plays a role as risk factors for gastric cancer, haplotype-pairs composed of a greater number of high-producing or mutant alleles may have an even greater risk. Based on this hypothesis, CAT haplotype should be the most powerful risk factor for gastric cancer development and CAC haplotype composed of two high-producing alleles should show a greater risk than CCC or TCT haplotype that includes only one high-producing allele. However, we were not able to fully test this hypothesis because only four haplotypes, TCC, TCT, CCC and CAC, were observed in our population. Considering our results, independent allele specificity of TNF-α-857 T and mutual haplotype specificity of TNF-α-1031 and -863 may be more important risk factors rather than the total number of high-producing alleles, especially for smokers. Moreover, these results were reproduced in all haploblocks regardless of different SNP combinations and were nearly replicated in the MDR analysis. This supports our conclusion that the interaction between the TNF gene and smoking may play a crucial role in the etiology of gastric cancer.The combined effect between smoking and TNF gene and cigarette smoking on gastric cancer development, our study had several limitations. First, because of a small number of gastric cancer cases and small sample size, we did not have sufficient statistical power and were not able to stratify on different factors. Second, although we had information on cardiac or non-cardiac cancer of gastric cancer cases, because of our small number of cases, we were not able to observe a difference for gastric cancer risk according to cancer type. Moreover, we did not collect information on cancer histology. Finally, a part of SNPs which is related to signal pathway (NF-κB) from H. pylori to cytokine gene expression were genotyped. Other cytokines, such as interleukins and GM-CSF, which are involved in NF-κB pathway, were not considered for analysis so we examined only a small portion of signal pathway for gastric carcinogenesis.Although, to our knowledge, this is the first study to report an interaction between the In spite of these limitations, our study had several strengths. This is a population-based, nested case-control study that is free of biases that are common in retrospective studies. Additionally, we matched cases and controls according to basic confounders, such as age and sex, and significant confounding factors selected in the full model. Furthermore, we used various approaches to detect the potential association between genetic and environmental factors on gastric cancer, and derived consistent results through different approaches. Finally, the minor allele frequencies (MAFs) of all cytokine genes analyzed in our study showed very similar results in the Korean, Chinese, and Japanese Hapmap projects ,44 and tTNF-α-857 C/T polymorphism may play an independent role in gastric carcinogenesis and the gene-gene interaction of TNF also affects gastric cancer development. The combined effect between TNF gene and cigarette smoking can be a major risk factor for gastric cancer. Tailored smoking cessation programs should be targeted for smokers with TNF genetic variants. Additional studies with a greater number of cases and information about gastric cancer type and various genes will allow us to conduct stratified analysis to obtain more detailed results that will further clarify the role of genetic and environment factors on gastric cancer.This study demonstrates that The authors declare that they have no competing interests.All the authors participated in the design, conduct, and analysis of the study, and approved the final version of this paper.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/9/238/prepubTNF gene: A) only three TNF-α SNPs -857, -863, and -1031; B) three TNF-α SNPs in block 1 and TNF-β 252; and C) all six SNPs on TNF-α and TNF-βLD blocks of . These LD blocks are generated by the Haploview software using the method suggested by Gabriel et al.Click here for fileTNF genetic variants among A) total subjects, B) smokers, and C) non-smokers modeled by MDR analysisDendrograms for . These dendrograms show high-order combination of TNF genes and indicate TNF gene-gene interaction.Click here for file


The role of tumor necrosis factor (TNF) as an immune mediator has long been appreciated but its function in the brain is still unclear. TNF receptor 1 (TNFR1) is expressed in most cell types, and can be activated by binding of either soluble TNF (solTNF) or transmembrane TNF (tmTNF), with a preference for solTNF; whereas TNFR2 is expressed primarily by microglia and endothelial cells and is preferentially activated by tmTNF. Elevation of solTNF is a hallmark of acute and chronic neuroinflammation as well as a number of neurodegenerative conditions including ischemic stroke, Alzheimer's (AD), Parkinson's (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). The presence of this potent inflammatory factor at sites of injury implicates it as a mediator of neuronal damage and disease pathogenesis, making TNF an attractive target for therapeutic development to treat acute and chronic neurodegenerative conditions. However, new and old observations from animal models and clinical trials reviewed here suggest solTNF and tmTNF exert different functions under normal and pathological conditions in the CNS. A potential role for TNF in synaptic scaling and hippocampal neurogenesis demonstrated by recent studies suggest additional in-depth mechanistic studies are warranted to delineate the distinct functions of the two TNF ligands in different parts of the brain prior to large-scale development of anti-TNF therapies in the CNS. If inactivation of TNF-dependent inflammation in the brain is warranted by additional pre-clinical studies, selective targeting of TNFR1-mediated signaling while sparing TNFR2 activation may lessen adverse effects of anti-TNF therapies in the CNS. The potent pro-inflammatory cytokine Tumor necrosis factor (TNF) is a member of the TNF superfamily of ligands, many of which promote inflammatory signaling -3. TNF iTNF receptor 1  and TNF receptor 2  are membrane glycoprotein receptors that specifically bind TNF and homotrimers of lymphotoxin A, but the two receptors differ in their expression profiles, ligand affinity, cytoplasmic tail structure, and downstream signaling pathway activation (reviewed in ). SignalTNF signaling through TNFR1 and TNFR2 can elicit a variety of cellular responses depending on many factors including the metabolic state of the cell and the adaptor proteins present in the cell. These differences then influence the activation of a number of intracellular signaling pathways including nuclear factor kappa-B (NF-κB), p38, c-jun N-terminal kinase (JNK), and the ceramide/sphingomyelinase signaling pathway, resulting in a number of responses including inflammation, proliferation, cell migration, apoptosis, and necrosis -20.SolTNF signaling is thought to elicit its biological effects primarily through TNFR1 activation. TNFR1 contains a cytoplasmic death domain (DD) characteristic of many members of the TNF superfamily that permits the assembly of the TNFR1 signaling complex through the dissociation of silencer of death domains (SODD) and subsequent binding of TNF receptor associated death domain (TRADD) ,22. BindIn addition to TNFR1-induced activation of stress-induced kinase signaling in contributing to apoptotic signaling, TNFR1 can be internalized after TNF binding and this leads to dissociation of the TRADD/TRAF2/RIP complex and association of Fas-associated DD (FADD), recruitment of pro-caspase 8, and formation of the death-inducing signaling complex (DISC), triggering activation of the executioner caspases through the extrinsic apoptosis pathway ,35. CaspTNFR2 is restricted to expression in endothelial, hematopoetic, and some neuronal populations and displays preferential binding to tmTNF, both of these characteristic are likely to result in fewer biological effects compared to those mediated by TNFR1-dependent signaling . Signaliin vivo significance of these reverse signaling pathways remains to be established.Upon receptor binding, tmTNF has been demonstrated to initiate intracellular signaling in the tmTNF-expressing cell ,45. ThisA receptor cell surface expression, suggesting that TNF may control synaptic strength at excitatory synapses by increasing excitatory synaptic transmission and reducing inhibitory transmission [TNF signaling has been shown to have several important functions within the CNS  includinsmission ,59,60. Tsmission ,59. In asmission ,59. SynaThe function of TNF signaling through TNFR1 and TNFR2 in the hippocampus and cortex has not been fully elucidated. On the one hand, TNF appears to regulate hippocampal neuronal development as TNF-deficient mice display accelerated maturation of the dentate gyrus region and smaller dendritic trees in the hippocampus . In contGenetic manipulation of TNF or TNF receptors in mouse models of disease have provided valuable insight into the biological roles of TNF in the CNS . ElevateA number of endogenous mechanisms limit TNF activity during inflammatory responses as part of resolution of those responses (reviewed in ). Identibiologics are available or currently under development  mechanism of action has emerged and was developed using structure-based computational design leading to the generation of TNF variants referred to as DN-TNFs which are able to exchange with native soluble TNF monomers to form heterotrimers Figure  with draA small molecule TNF inhibitor that has been described is composed of trifluoromethylphenyl indole and dimethyl chromone moieties linked by a dimethylamine spacer . This smLess specific inhibitors of TNF include minocycline and thalidomide which have been used in the treatment of inflammatory conditions. Minocycline, a broad-spectrum tetracycline antibiotic, has been shown to have both bacteriostatic and anti-inflammatory actions . Minocycin vivo [Another method by which inhibition of soluble TNF production can be achieved is by inhibition of TACE, the protease that cleaves and converts tmTNF into its circulating form. TACE, a member of the ADAM  family of matrix metalloproteases, is the primary enzyme responsible for TNF shedding in vivo . Unlike in vivo ). Develoin vivo . Other Tin vivo ).Evidence that implicates TNF in the underlying pathology of multiple sclerosis (MS) includes: the observation that at autopsy MS patients have elevated TNF levels at the site of active MS lesions ; reportsThere is conflicting evidence concerning the role of TNF in ischemia (stroke) models (reviewed in ). For exin vivo administration of a neurotoxic dopamine analog, 6-hydroxydopamine (6-OHDA) [in vitro primary cultures [in vivo [As in many other neurodegenerative diseases, TNF and solTNFR1 levels are elevated in cerebrospinal fluid and tissues of PD patients as well as in postmortem PD brains with the highest TNF levels present in areas that have the greatest loss of dopaminergic neurons -81. Neve(6-OHDA) , the mit(6-OHDA) -146, and(6-OHDA) . In MPTP(6-OHDA) . Multiplcultures -151 and [in vivo ,153. The[in vivo . Importa[in vivo . A secon[in vivo ,157. Las[in vivo . In summGenetic approaches to investigate the role of TNF signaling in the nigrostriatal pathway have involved use of mice deficient in TNF and TNFRs but have yielded conflicting results. In some studies TNFR1 or TNFR2 single-knockout mice or double receptor deficient mice are not protected from MPTP-induced striatal terminal damage or nigral dopaminergic neuron loss ,158; whein vivo degeneration of DA neurons in both the 6-OHDA oxidative neurotoxin and chronic LPS models of PD has been demonstrated by the ability of dominant-negative (DN) TNF inhibitors or a lentivirus encoding DN-TNF to attenuate nigral DA neuron loss when delivered intranigrally [Use of non-specific TNF inhibitors including thalidomide, a potent anti-inflammatory and sedative, and minocycline, a tetracycline antibiotic that inhibits TNF synthesis, has also been investigated in MPTP intoxication paradigms in mice and in intranigral LPS rat models of PD with protective outcomes in some ,160 but nigrally ,163. In The first indication of a contribution for TNF signaling in AD was the presence of TNF at amyloidogenic plaques in post-mortem analysis of AD brains . In pre-Swe, tauP301L, and PS1M146V) which lead to accumulation of intraneuronal amyloid immunoreactivity in regions that include entorhinal cortex, elevated TNF mRNA levels were reported in the same regions and correlated with the onset of cognitive deficits in these mice [Swe, tauP301L, and PS1M146V) by either hippocampal infusion of DN-TNF inhibitors or intracerebroventricular injection of a lentivirus encoding DN-TNF reduced inflammation-induced accumulation of C-terminal APP fragments in the hippocampus, cortex, and amygdale. In triple (3xTgAD) transgenic mice lacking TNFR1, exposure to chronic systemic inflammation did not result in intraneuronal accumulation of amyloid immunoreactivity, suggesting that solTNF/TNFR1 signaling may be an important regulator of APP processing and that pathological elevation of TNF may contribute to pre-plaque pathology and acceleration of cognitive deficits . In support of this idea, the contribution of TNFR1 signaling to AD pathogenesis was recently shown in APP23 transgenic mice which overexpress APPKM670/671NL. In these transgenic mice, TNFR1 deletion reduces Aß pathology, microglia activation, BACE1 activity, neuron loss, and memory deficits compared to transgenic APP23 mice expressing normal levels of TNFR1 [Other evidence of increased inflammation and elevated TNF levels in AD pathology includes the dysregulation of TNF levels and other pro-inflammatory cytokines in AD patients and in transgenic mouse models of AD. Elevated TNF levels appear to correlate with disease progression as higher serum levels of TNF, as well as an increase in the TNF/IL-1β ratio, are present in patients with severe AD compared to individuals with mild-to-moderate disease . Howeverese mice ,175. Inhof TNFR1 . Althougof TNFR1 . These oMycobacterium and Listeria). Given the growing evidence that TNFR2 may antagonize TNFR1 signaling, a second strategy, which may bestow beneficial effects and allow protection of neuronal populations highly sensitive to solTNF/TNFR1-mediated toxicity, may be to increase TNFR2 expression levels through gene therapy. Lastly, it is important to note that while brain-permeant small molecule inhibitors of TNF, including the immunomodulatory drugs thalidomide and minocycline, may be attractive for CNS applications, their lack of selectivity for solTNF over tmTNF and their inhibitory effects on other inflammatory mediators may contribute to undesirable effects as TNF signaling and other inflammatory cascades would be suppressed globally in the CNS [TNF exerts pleiotropic effects in the CNS and its role in normal brain functions, in particular synaptic scaling, has yet to be fully elucidated. While it is clear that solTNF is toxic to dopaminergic neurons and compromises their survival during chronic inflammatory stimuli in the ventral midbrain, tmTNF and TNFR2 appear to have important roles in myelination and may contribute to hippocampal neurogenesis after ischemic injury. These observations strongly suggest that one potential approach to lessen adverse effects of anti-TNF therapies in the CNS may be to selectively target TNFR1 signaling with localized delivery of inhibitors which spare TNFR2-mediated signaling. As solTNF signals preferentially through TNFR1, selective inhibition of solTNF signaling may be advantageous. This approach may allow efficient neutralization of solTNF in the desired region (i.e. substantia nigra) without eliciting collateral damage to TNFR2/tmTNF-dependent processes in regions of the brain where tmTNF has important homeostatic functions. An additional benefit to selective targeting of solTNF would be sparing tmTNF-dependent functions in the immune system, including proper development and maintenance of immune cell populations as well as self-tolerance and resistance to infectious agents (such as  the CNS .The authors declare that they have no competing interests.The manuscript was written by MKM as part of her doctoral thesis. MGT provided historical perspectives and editorial assistance.


The lytic process was inhibited by mAb IV.3, specific for the Fcγ receptor (FcγR) type II. The mAb 3G8, which reacts with FcγR type III, was ineffective. Moreover, the lysis was inhibited by the anti-CD18 mAb MEM-48. These data suggest that FMLP/Lym-1 as well as TNF-α/Lym-1 cytolytic systems strictly require FcγRII and CD18 integrins. As the lysis induced by TNF-α/Lym-1 was prevented by pertussis toxin (PT), PT-sensitive G-proteins are likely to intervene in post-FcγRII signal transduction. Both the FMLP- and the TNF-α-dependent systems were also found to be equally susceptible to inhibition by various inhibitors of kinases -2-methylpiperazine and wortmannin). On the contrary, an inhibitor of protein kinase C  was effective only in the FMLP/Lym-1 cytolytic system. Therefore, it appears that signals delivered by FMLP or TNF-α, BIM-sensitive and insensitive respectively, converge and synergize with those from G-protein-coupled FcγRII and, probably, CD18-integrins to promote the expression of the neutrophil cytolytic potential. © 1999 Cancer Research CampaignHuman neutrophils, incubated with Cr


To further elucidate the potential immuno-modulatory functions of flavanol-rich cocoa, the present investigation examined whether isolated CFP fractions (monomers through decamers) influence the secretion of tumor necrosis factor-α (TNF-α) from resting and phytohemagluttinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC). We used an in vitro culture system where PBMC from 14 healthy subjects were introduced to individual CFP fractions for 72 h prior to measuring the levels of TNF-α released. The intermediate-sized CFP fractions (tetramers through octamers) were the most active on resting cells, causing a 3–4 fold increase in TNF-α relative to media baseline. The monomers and dimers were the least stimulatory of the fractions tested, displaying a 42 and 31% increase, respectively, over media control, whereas the trimers, nonamers and decamers showed an intermediate stimulation of this cytokine. In the presence of PHA, the intermediate-sized CFP fractions again were the most active, enhancing TNF-α secretion in the range of 48–128% relative to the PHA control. The monomers and dimers were slightly inhibitory , while trimers, nonamers and decamers stimulated moderate increases in TNF-α levels . The above results lend support to the concept that CFP can be immunomodulatory. The stimulation of TNF-α secretion may contribute to the putative beneficial effects of dietary flavanoids against microbial infection and tumorigenesis.Epidemiological reports have suggested that the consumption of foods rich in flavonoids is associated with a lower incidence of certain degenerative diseases, including cardiovascular disease. Flavanols and their related oligomers, the procyanidins CFP, isolated from cocoa can modulate the production and level of several signaling molecules associated with immune function and inflammation


TNF and PGE2 were found in both cases while IL-1 alpha was not detected. Both TNF and PGE2 levels were higher in the medium of the MdM infected with K biovars. TNF reached maximum levels 24 h postinfection, and then declined, while PGE2 levels increased continuously during the infection time up to 96 h post-infection. Addition of dexamethasone inhibited production of TNF and PGE2. Inhibition of PGE2 production by indomethacin resulted in increased production of TNF, while addition of PGE2 caused partial inhibition of TNF production from infected MdM.In this study levels of prostaglandin E


HIV-1 escape from surveillance by cytotoxic T lymphocytes (CTL) is thought to cause at least transient weakening of immune control. However, the CTL response is highly adaptable and the long-term consequences of viral escape are not fully understood. The objective of this study was to address the question “to what extent does HIV-1 escape from CTL contribute to HLA-associated AIDS progression?” We combined an analysis of 21 escape events in longitudinally-studied HIV-1 infected people with a population-level analysis of the functional CTL response in 150 subjects (by IFNg ELISpot) and an analysis of the HIV-1 sequence database to quantify the contribution of escape to the HLA-associated rate of AIDS progression. We found that CTL responses restricted by protective HLA class I alleles, which are associated with slow progression to AIDS, recognised epitopes where escape variants had a weak evolutionary selective advantage (P = 0.008) and occurred infrequently (P = 0.017). Epitopes presented by protective HLA class I alleles were more likely to elicit a CTL response (P = 0.001) and less likely to contain sequence variation (P = 0.006). A third of between-individual variation in HLA-associated disease risk was predicted by the selective advantage of escape variants: a doubling in the evolutionary selective advantage was associated with a decrease in the AIDS-free period of 1.2 yrs. These results contribute to our understanding of what makes a CTL response protective and why some individuals progress to AIDS more rapidly than others. HIV-1-infected individuals mount a large, sustained HIV-1-specific antibody and CD8+ cytotoxic T lymphocyte (CTL) response. Despite this large immune response, untreated individuals progress to AIDS in all but a small minority of cases. There are a number of hypotheses to explain why the immune response ultimately fails to control HIV-1 infection including Nef-induced downregulation of MHC class I, CD8+ cell exhaustion, and destruction of HIV-1-specific CD4+ cells There is clear evidence that HIV-1 variant strains that escape CTL surveillance  evolve during the course of natural infection The aim of this study was to quantify the contribution of viral escape from CTL to the HLA-associated rate of progression to AIDS.This study involved the analysis of three datasets: a quantification of all detailed longitudinal escape events reported in the literature , analysis of the functional CTL response in 150 HIV-1 infected individuals and analysis of sequence variation in clade B HIV-1 sequences from the Los Alamos National Laboratory (LANL) Sequence Repository.Different HLA class I alleles are significantly associated with different rates of progression to AIDS An escape variant grows out and replaces the wild type strain because it grows faster than the wild type i.e. it has an evolutionary selective advantage compared with the wild type. The magnitude of the selective advantage will depend on the strength of the CTL response evaded offset by any fitness costs associated with the escape mutations As a further test, we compared the selective advantage of escape variants in epitopes bound by alleles associated with significant differences in progression to AIDS. We found that variants escaping CTL responses restricted by the class I alleles B*27 and B*51, which are significantly associated with a delay in the onset of AIDS-defining illnesses We have previously shown that viral escape is significantly more rapid (higher selective advantage) in primary infection than in chronic infection Alternatively, analysing the same dataset by epitope location, we found that escape variants in epitopes located in Gag had a significantly lower selective advantage than escape variants in other proteins  was associated with a decrease in the AIDS-free period of about 1.2 years  of the variation in HLA-associated rate of progression in our subject group could be explained by variation in the selective advantage. We found that an increase in the selective advantage from 0.028 day.2 years .Although variants with a weak selective advantage do replace the wild type more slowly than variants with a strong selective advantage the time difference is not large and it is hard to reconcile with the considerable protective effect seen. That is, it is difficult to understand why a decrease in the time to fixation of only 160 days could cause a decrease in the AIDS-free period of about 1.2 years. One possible explanation is that escape variants with a weak selective advantage as well as having a longer fixation time from first appearance will also grow out less frequently. In the absence of a genetic barrier, population genetics theory If the above hypothesis is correct then we would predict that epitopes at which variants have a large selective advantage should have experienced more escape and therefore be less likely to elicit CTL responses in the HIV-1-infected population as a whole. We tested this prediction by investigating the association between the selective advantage of variants in the 21 escape events studied and the functional CTL response to that epitope. The functional CTL response was quantified by estimating the proportion of individuals  who had a detectable CTL response to the epitope of interest. This was done using a comprehensive cross-sectional IFNg ELISpot database in which CD8+ T cells from 150 HIV-1-infected individuals were screened for responses against 410 overlapping clade B consensus peptides spanning the entire expressed viral genome We found a significant negative correlation  between As expected- given the positive association between the selective advantage and the relative hazard, and the negative association between the selective advantage and the presence of a CTL response- we found a significant negative association between the presence of a CTL response and the relative hazard . We have replicated this significant negative association between the presence of a CTL response and relative hazard in an independent dataset of 84 HIV-1-infected subjects (P = 0.0007 www.hiv.lanl.gov). Env sequences were not analysed as Envelope is thought to be under dominant selection by the humoral response. We found a positive correlation between the variability at an epitope (at which escape had been described) and the relative hazard of the presenting allele, but this was not statistically significant (P = 0.06 weighted linear regression two-tailed). However, because sequence is analysed regardless of subject HLA genotype, the high density of CTL epitopes in many regions of the genome means that overlap between CTL epitopes presented by susceptibility alleles and epitopes presented by protective alleles can occur introducing noise into the dataset. To reduce noise and to see if our hypothesis was applicable to a greater range of CTL epitopes (not just the 21 at which CTL escape had been described) we calculated the Shannon entropy of all confirmed CTL epitopes from the LANL database defined to within 11 amino acids that did not overlap with other epitopes presented by alleles with a different relative hazard. We found a significant positive correlation between the Shannon entropy of the CTL epitope and the relative hazard of the presenting allele  sequences for every viral protein and HLA allele necessitating the use of public databases in which the HLA type of the individual is rarely recorded. We therefore quantified epitope sequence variation in all individuals regardless of HLA type. The average Shannon entropy, a measure of amino acid variation The interpretation that much of the amino acid variation in CTL epitopes is induced by the selective force of the CTL response is supported by the observation that the Shannon entropy was significantly higher at the known anchor residues in vivo evolutionary selective advantage of viral variants that escape the CTL response and related these estimates to the degree of protection conferred by the targeting CTL response. This analysis suggested that viral escape from the CTL response is a significant determinant of the HLA-associated rate of AIDS progression. We corroborated this work by analysing an IFNg ELISpot and an HIV-1 sequence database using two independent methods. The results were consistent with the conclusions that (1) viral escape is a significant determinant of the HLA-associated rate of AIDS progression and (2) protective CTL responses target epitopes where escape variants have a weak selective advantage and escape is thus less frequent  of cases of detailed longitudinal escape in the literature. However, both associations utilising these 21 events (selective advantage-relative hazard and selective advantage-frequency of CTL targeting) were significant  and the latter is also predicted by classical population genetics theory. Furthermore, the association between frequency of escape and relative hazard predicted by our hypothesis, as well as being confirmed in a large (N = 150) cohort (P = 0.001), was also confirmed using an alternative method in two large independent cohorts  using two surrogate markers of the frequency of escape. All the results are consistent with each other and support the hypothesis . We hopeThese results contribute to our understanding of why HIV-1-infected individuals differ in their rate of progression to disease and of what constitutes a protective CTL response and thus have direct implications for vaccine design. Most importantly they suggest that viral escape from CTL is a determinant of the rate of HLA-associated progression to AIDS.r and cells productively infected with the escape variant grow at a rate k+r, then the selective advantage of the escape variant is k and the dynamics of the two cell populations will be given byp(t) is the proportion of viral sequences that have escape mutations in the epitope of interest at time t then solving (1) we haveg = y(0)/x(0).The selective advantage of an escape variant compared with the wild type was estimated from longitudinal viral sequence data showing the replacement of the wild type by the escape variant k estimated. A selective advantage of k per day is roughly equivalent to a time to fixation  of about 9/k i.e. a variant with a selective advantage of 0.01 day−1 takes approximately 900 days to replace the wild type population.This model has been derived in population genetic theory to describe the fixation trajectory of an advantageous mutation in a large haploid population To quantify the selective advantage of HIV-1 escape variants it was necessary to have longitudinal data with two or more time points where the frequency of the escape variant was neither zero nor one. There are twenty-three such data sets in the literature. For two of these datasets the presenting HLA class I allele was unknown and so these two datasets were excluded from further analysis. Information on the escape events studied is given in k) was estimated using the relationship between the selective advantage and relative hazard  and the time since seroconversion  were removed from the list of epitopes and all the calculations were repeated.nd and C-terminus positions were the anchor residues for every allele and the conclusions were unaltered). Non-anchor residues were represented by the 1st and 6th position .Anchor residues for each HLA allele studied were determined from When we widened the analysis beyond the 21 escape events at defined CTL epitopes in the literature, epitopes were taken from the LANL HIV immunology database. All epitopes that were confirmed (appeared twice or more in the database), for which the presenting allele was known  and determined to within 11 amino acids were selected. All replicates were removed . We wished to calculate the Shannon entropy for each CTL epitope to investigate the relationship between the relative hazard of the presenting allele and the amount of sequence variation. Ideally we would have estimated the entropy of say an A2-presented epitope only in A2+ subjects. However, since the vast majority of the HIV-1 sequences used for our entropy calculations were from subjects of unknown HLA type this was not possible. Instead, to reduce the noise that looking at say A2-presented epitopes in both A2+ and A2- subjects will introduce, we removed all epitopes that were presented by infrequent alleles (frequency<5% in the Caucasian population) and further removed all epitopes that overlapped by more than 1/3 with another epitope where the relative hazard of the presenting allele differed by more than 10%. This left a total of 38 epitopes . For the3, a mutation rate of 3×10−5 per nucleotide per generation and a generation time of 1 day. 3 possible nucleotide mutations were considered, one of which had a selective advantage compared to the wild type, the other two had the same fitness as the wild type. Changes to these parameters will not alter the positive association between the selective advantage of an escape variant and its frequency of escape.The illustrative simulation  was implAll statistical tests used were non-parametric except when we needed to perform a weighted test. In the latter case linear regression with the data weighted either by the number of subjects in the group or by the number of sequences contributing to the entropy calculation was used. Whenever weighted regression was used we additionally checked that the non-parametric Spearman rank test yielded the same conclusion. All tests were suitable for the sample size. All P-values reported are two-tailed.


Spontaneous bacterial peritonitis (SBP) is a common clinical disease and one of the most severe complications of acute liver failure (ALF). Although the mechanism responsible for SBP is unclear, cytokines play an important role. The aim of this study was to investigate the effects of tumor necrosis factor-alpha (TNF-α) on the structure of the intestinal mucosa and the expression of tight junction  protein in a mouse model of ALF.We induced ALF using D-galactosamine/lipopolysaccharide  or GalN/TNF-α and assessed the results using transmission electron microscopy, immunohistochemistry, Western blotting, ELISA and real-time quantitative PCR. The effects of administration of anti-TNF-α IgG antibody or anti-TNF-α R1 antibody before administration of GalN/LPS or GalN/TNF-α, respectively, on TNF-α were also assessed.Morphological abnormalities in the intestinal mucosa of ALF mice were positively correlated with serum TNF-α level. Electron microscopic analysis revealed tight junction (TJ) disruptions, epithelial cell swelling, and atrophy of intestinal villi. Gut bacteria invaded the body at sites where TJ disruptions occurred. Expression of ZO-1 mRNA was significantly decreased in both ALF models, as was the level of ZO-1 protein. Prophylactic treatment with either anti-TNF-α IgG antibody or anti-tumor necrosis factor-a receptor1 (anti-TNF-α R1) antibody prevented changes in intestinal tissue ultrastructure and ZO-1 expression.TNF-α affects the structure of the intestinal mucosa, decreases expression of ZO-1, and affects the morphology of the colon in a mouse model of ALF. It also may participate in the pathophysiological mechanism of SBP complicated to ALF. Acute liver failure (ALF) is a devastating disease associated with high mortality. Spontaneous bacterial peritonitis (SBP), a common clinical disease, is one of the most severe complications of ALF and a major cause of death -4. HowevThe intestinal mucosa is a physical and metabolic barrier against toxins and pathogens in the lumen of the gut. Tight junctions (TJs) are the main structures responsible for restricting paracellular movement of compounds across the intestinal mucosa. Structurally, TJs are composed of cytoplasmic proteins, including the zona occludens proteins, ZO-1, ZO-2, and ZO-3 ,12 and tMale, six-to eight-week-old BALB/c mice  were obtained from the China Medical University . They were housed and cared for in rooms maintained at a constant temperature and humidity. Food and water were allowed ad libitum. Food was withdrawn the evening before the experiment. All animal experimental procedures were approved by the Ethics Committee of China Medical University before the commencement of the study.All mice were randomly divided into eight groups (n = 8 per group). One group of mice was given intraperitoneal injections of D-galactosamine  and LPS  to induce ALF. A second ALF-induction group was also given intraperitoneal injections of GalN (800 mg/kg body weight) and TNF-α . Two groups were given antibody treatments prior to ALF induction: one was given anti-TNF-α IgG  and the other was given anti-TNF-α R1 antibody . The anti-TNF-α IgG and anti-TNF-α R1 antibodies were injected via the vena caudalis 30 minutes and 15 minutes before GalN/LPS administration. There were four control groups, which were injected intraperitoneally with GalN, LPS, TNF-α, or NS.In summary, the eight groups were: 1) GalN/LPS; 2) GalN/TNF-α; 3) GalN control; 4) LPS control; 5) TNF-α control; 6) NS control; 7) anti-TNF-α IgG and GalN/LPS; and 8) anti-TNF-α R1 antibody and GalN/LPS. Mice in the first six aforementioned groups were euthanized 2, 6, 9, 12 and 24 h after treatment. Mice in the last two of the aforementioned groups were euthanized 9 h after administration of GalN/LPS. The study was approved by the Ethics Committee of China Medical University.Serum levels of alanine transaminase (ALT) were determined using an automatic analyzer . Serum levels of TNF-α were determined using an ELISA kit (R&D Systems) according to the manufacturer's protocol.Ultrathin (70 nm) intestinal sections were examined using a transmission electron microscope .2O2 for 20 minutes at room temperature and indirectly immuno-labeled using an ABC kit  according to the manufacturer's instructions. Slides were then blocked in goat serum for 30 min at 37°C and incubated with a rabbit anti-mouse polyclonal ZO-1 antibody  at 4°C overnight. For the negative controls, the primary antibody was replaced with PBS. This incubation was followed by incubation with biotinylated goat anti-rabbit IgG  diluted 1:300 in PBS for 2 h at room temperature. Sections were rinsed in PBS and then in distilled water. The slides were stained with 3, 3'-diaminobenzidine and counterstained with hematoxylin.Frozen intestinal tissue sections (5 μm thick) were fixed on glass slides by incubating them in acetone for 10 min at 4°C. The slides were incubated with 3% HIntestinal tissue samples were homogenized in lysis buffer . Proteins (50 μg) were electrophoresed using SDS-PAGE (8%) and transferred to a nitrocellulose membrane. Membranes were blocked with non-fat dried milk in TBS containing 0.05% Tween-20 (TTBS) for 1 h at room temperature and incubated with a rabbit anti-mouse polyclonal ZO-1 antibody  at 4°C overnight. After three washes in TTBS, the membranes were reacted with a 1:2000 dilution of alkaline phosphatase-labeled goat anti-rabbit IgG (Santa Cruz Biotechnology) for 2 h at room temperature. The immunoreaction was visualized using α-dianisidine and β-naphthyl acid phosphate .Total RNA was isolated from intestinal tissues using TRIzol Reagent . RNA was purified using DNase I and depurified using PI-PCI-EHCO. SYBR-green-based real-time PCR  was used to measure relative gene expression in each sample. First, we prepared an RNA standard  and determined standard curves for the ZO-1 gene and a house-keeping gene . PCR was performed using Taq DNA polymerase  and oligonucleotide primers for mouse ZO-1  and glyceraldehyde 3-phosphate dehydrogenase . PCR conditions were as follows: one cycle at 95°C for 30 minutes followed by 45 cycles of PCR amplification, each consisting of 95°C for 5 s and 60°C for 20 s. The concentration of mRNA was calculated according to the standard curve and then normalized to that of GAPDH.P values < 0.05 were considered significant. All data are presented as the mean ± SE.SPSS version 10.0 Software was used to perform the statistical analyses. All data were analyzed using analysis of variance (ANOVA) followed by a least-squares difference test. P < 0.01) (Table P < 0.01). Histopathological examination showed only spot or focal hepatonecrosis.Most ALF mice  died between 6 h and 12 h after the GalN/LPS injection. Serum ALT levels increased significantly 6 h after the GalN/LPS injection compared with the control groups   and mitochondrial matrices . The disruption of the intestinal mucosa and consequent bacterial invasion of the body in ALF may be caused by reduced levels of the TJ-associated protein, ZO-1, the production of which was not controlled by transcription. These changes were caused by an elevated serum level of TNF-α, as they were absent when TNF-α was blocked by anti-TNF-α IgG antibody or anti-TNF-α R1 antibody. This study confirmed that TNF-α damages TJs and affects the expression of ZO-1 protein ALF: Acute liver failure; GalN: D-galactosamine; LPS: lipopolysaccharide; TJ: tight junction; ZO-1: zona occludens 1; TEM: transmission electron microscopy; TNF-α: tumor necrosis factor-a; WB: Western blot; ALT: alanine transaminase; SBP: spontaneous bacterial peritonitis; TEM: transmission electron microscopy; TNF-α R: tumor necrosis factor-a receptor; NS: normal saline.The authors declare that they have no competing interests.HLS and PL participated in the conception and design of the study and drafted the manuscript. HLS and SL executed the study. HLS analyzed the data. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/9/70/prepub


TNF-alpha is a critical cytokine produced by Th1 cells while altered T helper 1 (Th1)-Th2 balance is found crucial for a successful pregnancy.A cohort of 132 Southern Chinese Han RSA patients and 152 controls constituted the subjects of this study. Two functional polymorphisms -308 and -238 of TNF-alpha were studied by association analysis.lack of association was found in TNF-alpha -308 SNP yet a significant difference was discovered in -238 polymorphism.This study suggested that TNF-alpha may be a risk factor in Chinese RSA patients. However the ethnic differences may also contribute to the results. Recurrent spontaneous abortion (RSA) is defined as three or more consecutive pregnancy losses before 22 gestational weeks or the spontaneous abortion of an embryo/fetus weighing less than 500 g and affecting an estimated 1 of every 100 couples preparing to have children . TTo our knowledge, this work firstly established an association between TNF-alpha gene polymorphisms and the development of URSA in Chinese population.TNF-alpha gene possibly not be a potential risk factor of URSA developing, but patients with URSA has a 3.95-fold higher frequency of -238 GG genotype adjusted by age, which demonstrated the association between -238 polymorphism and the URSA developing in Chinese population.In conclusion, the present study indicated that -308 polymorphism of The authors declare that they have no competing interests.CL collected all samples and performed the clinical tests. JW carried out the molecular genetic studies and performed the statistical analysis. SZ drafted the manuscript. BW participated in its design and coordination and helped to draft the manuscript. XM participated in the design of the study. All authors read and approved the final manuscript.


The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.Cells isolated from normal and degenerate nucleus pulposus regions of human intervertebral discs were cultured in alginate pellets and stimulated by the addition of 10 ng/mL TNF-α or IL-1β for 48 hours prior to RNA extraction. Quantitative real-time polymerase chain reaction was used to assess the effect of TNF-α or IL-β stimulation on the expression of matrix metalloproteinase (MMP)-3, -9 and -13, TNF-α, TNF receptor 1 (TNF-R1), TNF receptor 2 (TNF-R2), IL-1α, IL-1β, IL-1 receptor 1 (IL-1R1) and IL-1 receptor antagonist (IL-1Ra).MMP-3 and MMP-9 gene expressions were upregulated to a greater level by IL-1β than TNF-α. MMP-13 was upregulated by each cytokine to a similar extent. TNF-α and TNF-R2 expressions were upregulated by both TNF-α and IL-β, whereas TNF-R1 expression was not significantly affected by either cytokine. IL-1β and IL-1Ra expressions were significantly upregulated by TNF-α, whereas IL-1α and IL-1R1 were unchanged.MMP expression to the same degree as stimulation by IL-1β, but it does act to upregulate IL-1β expression as well as TNF-α and TNF-R2. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results support the hypothesis that, while TNF-α may be an important initiating factor in matrix degeneration, IL-1β plays a greater role in established pathological degradation.TNF-α does not induce  Disc degeneration is a major economic and social burden that affects large numbers of people. It is a major cause of back pain, which is one of the commonest causes of morbidity in the West. Within the UK, approximately 11 million people experience lower back pain for at least one week out of every month, and it is estimated to cost approximately £11 billion in lost production due to absence from work [The intervertebral disc (IVD) is a fibrocartilaginous tissue situated between the vertebrae of the spine. It provides stability and flexibility to the spinal column, allowing movement in all directions. The IVD is composed of a central gelatinous nucleus pulposus (NP), which provides the compressibility of the tissue, and a surrounding fibrous annulus fibrosus (AF). The NP is composed predominantly of the proteoglycan aggrecan and type II collagen and is highly hydrated, whereas the AF is made up of concentric lamellae of highly organised type I collagen fibres that provide the tensile strength and restrain the inner NP region. Molecular changes in degeneration include altered matrix synthesis, including a decrease in glycosaminoglycan production and an increase in collagen type I within the NP, and upregulation of matrix-degrading enzymes -5. This in situ zymography [Furthermore, we have shown that, while both IL-1 and TNF are expressed in IVD and upregulated with degeneration, degenerate IVDs show a much greater expression level of IL-1β than TNF-α and that, while the IL-1R1 was upregulated in degeneration, the TNF receptor 1 (TNF-R1) was not ,13,14. Hmography ,16. ThisAll human tissue was obtained in accordance with the Declaration of Helsinki, and ethical approval for these studies was obtained from the Trent Multi-centre Research Ethics Committee (reference 05/MRE04/3). Human IVD samples were obtained either from post-mortem samples or from surgery with the informed consent of patients or relatives. All samples were assessed histologically and graded for degeneration according to the method of Sive and colleagues . SamplesCells were isolated from the central NP region of the IVD by enzymatic digestion ; samples6 cells/mL  in 1.2te beads ,19. Cult2. All treatments were carried out in triplicate. This protocol has previously been shown to be sufficient for IL-1β to significantly upregulate gene expression of MMP-3 and -13 and ADAM-TS4 [ADAM-TS family .Standard culture media were replaced with 2 mL of media containing 10 ng/mL of either human recombinant IL-1β  or human recombinant TNF-α (AMS Biotechnology Ltd). Cells were stimulated for 48 hours and maintained at 37°C in a humidified atmosphere containing 5% CO® ND-1000 Spectrophotometer . cDNA was synthesised from 500 ng of RNA using Superscript™ II Reverse Transcriptase (Invitrogen Corporation) and random primers in accordance with the instructions of the manufacturer cDNA was stored at -20°C until required.Samples for RNA extraction were pelleted by centrifugation and the RNA was extracted using TRIzol™ and PureLink columns in accordance with the instructions of the manufacturer (Invitrogen Corporation). To prevent any DNA contamination of the RNA, the samples were treated with DNAse I solution , and RNA quality and quantity were determined using the NanodropGAPDH , TNF-α, TNF-R1, IL-1α, IL-1β, IL-1R1, IL-1Ra, MMP-3, -9, -13, aggrecan, collagen types I and II and SOX9 were designed for TaqMan polymerase chain reaction (PCR) using Primer Express software  based on the genomic sequences supplied in the GenBank database. Specificity for the primers and probes was confirmed by BLAST  analysis. Primers and probe for TNF receptor 2 (TNF-R2) were purchased from Applied Biosystems as a pre-designed assay (PDAR). Sequences for primers and probes are presented in Table Real-time primers and probes for All PCRs were set up in triplicate using a final volume of 25 μL (2.5 μL cDNA) and Universal Taq mastermix (Applied Biosystems). All primers were used at 900 nM, and probes were used at 250 nM. Reactions were carried out and analysed by an ABI Prism 7700 Detection System.GAPDH as the endogenous control gene and using the automatic settings to set baseline and threshold values. The data were then exported into Excel  for further analysis. Data were analysed using the 2-ΔΔCt method using the housekeeping gene GAPDH and untreated controls for normalisation [Initial analysis was performed using the 7000 System Sequence Detection Software package (Applied Biosystems) as a relative quantification study using U test.All samples were tested for normality using the Shapiro-Wilk test. As all data were non-parametric, they were then analysed by the Mann-Whitney MMP-3, -9 or -13 between untreated normal and degenerate NP cells (data not shown).There was no difference in the basal levels of gene expression for MMP-3 mRNA in both normal and degenerate NP cells (P = 0.05) and MMP-13 gene expression in normal cells (P = 0.05)  Figure . There ws Figure .MMP-3, -9 and -13 in both normal and degenerate NP cells (P = 0.05) , and the upregulation of MMP-13 was significantly greater in normal than degenerate cells (P = 0.05). There was no significant difference between the upregulation of MMP-3 in normal and degenerate NP samples.The addition of recombinant IL-1β for 48 hours resulted in a significant upregulation of mRNA of ) Figure . The uprMMP-3 mRNA was significantly greater by recombinant IL-1β than TNF-α in both normal and degenerate cells . IL-1β had a significantly greater effect than TNF-α on MMP-9 gene upregulation in degenerate NP cells , although the difference between IL-1β-associated MMP-9 upregulation and TNF-α-stimulated MMP-9 gene upregulation in normal NP cells was not significant. There was no significant difference between the effect of IL-β and TNF-α on MMP-13 gene upregulation in normal and degenerate NP cells.The upregulation of collagen type I gene was significantly higher in the degenerate cells than in the normal NP cells (P = 0.05). There was no significant difference in the basal gene expression levels between normal and degenerate cells for any of the other matrix genes examined (data not shown).The basal gene expression for the collagen type I gene expression in both normal and degenerate NP samples . Type II collagen mRNA was significantly downregulated in normal NP cells following the addition of TNF-α (P = 0.05), but expression was not significantly changed in degenerate cells .TNF-α and TNF-R2 genes in both normal and degenerate NP cells . TNF-R2 mRNA was upregulated to a greater extent in normal than in degenerate samples (P = 0.05).The addition of recombinant TNF-α for 48 hours resulted in a significant upregulation of s Figure . TNF-R1 s Figure . NeitherTNF-α and TNF-R2 genes in both normal and degenerate samples (P = 0.05) , whereas the TNF-R2 gene was upregulated to a greater extent in degenerate cells than normal NP samples (P = 0.05). TNF-R1 gene expression was not significantly affected by the addition of IL-1β in either normal or degenerate samples.The addition of exogenous IL-1β for 48 hours resulted in a statistically significant upregulation of ) Figure . The uprIL-1β and IL-1Ra genes in both normal and degenerate NP samples (P = 0.05)  that break down fibrillar collagens, gelatinases (MMP-2 and -9) that act on denatured collagens and collagen types IV and V, and stromelysins (including MMP-3) that degrade predominantly non-collagenous proteins such as proteoglycans and fibronectin and can activate the pro-collagenases. Having previously demonstrated that IL-1β downregulates explants , we inveexplants  showed tlleagues  demonstrolecules , so a grTNF-α and TNF-R2 in both normal and degenerate cells, although the levels of TNF-R1 remained unchanged. This finding is in keeping with previous studies from our group which have shown that TNF-α and TNF-R1 are expressed by the IVD, but the TNF-R1 expression is not increased in degeneration [Stimulation of NP cells by the addition of TNF-α resulted in the upregulation of neration . It is nneration , who fou2 (prostaglandin E2) IL-6 and MMP-1 regulations are mediated exclusively via TNF-R1, suggesting that, although the expression of TNF-R1 does not change with degeneration in the IVD, signalling through this receptor is critical for upregulation of degradative processes. TNF-α has been shown to upregulate MMP-9 gene expression in malignant carcinoma via TNF-R1 [TNF-R1 levels remain unchanged and TNF-R2 gene expression is increased, it is signalling through TNF-R1 that is important in upregulating metalloproteinases and inflammatory cytokines such as IL-1β and IL-6. However, studies on collagen-induced arthritis in TNF-R1 knockout mice have shown that the lack of TNF-R1 does not affect the incidence or severity of collagen-induced arthritis in the joints [TNF-R2 by TNF-α in NP cells remains to be elucidated.TNF-α can bind to and signal through either TNF-R1 or TNF-R2 receptors. Studies by Alsalameh and colleagues  on synova TNF-R1 , and TNFa TNF-R1 . This sue joints . Repeatee joints . The preIL-1β and IL-1Ra genes, but not IL-1α or IL-1R1, whereas IL-1β upregulates TNF-α and TNF-R2. We have also previously shown that IL-1β upregulates itself and IL-1α whilst downregulating IL-1R1 and having no effect on the expression of IL-1Ra in IVD [Our data demonstrate that TNF-α also upregulates a in IVD . The neta in IVD . This hya in IVD ,30. Treaa in IVD ,31,32. Hin situ zymography studies [One possibility is that TNF-α upregulates IL-1β, which then upregulates itself and IL-1α and further upregulates TNF-α. This would result in an increase in matrix-degrading activity and accelerating degeneration and would be consistent with the suggestion that TNF-α is involved in the early onset of degeneration, stimulating and driving the IL-1β signalling, as is thought to occur in articular cartilage and osteoarthritis. If TNF-α is involved only as an initiating factor in the early onset of degradation in the IVD and then becomes unimportant as the IL-1β degradative mechanisms take over (as has been suggested in cartilage), it would also explain why antagonists to IL-1 signalling, but not TNF, would inhibit matrix-degrading activity in established degradation, as shown in our  studies . HoweverMMP gene expression to the same degree as stimulation by IL-1β, but it does act to upregulate IL-1β gene expression as well as TNF-α and TNF-R2 mRNA. The net result of this would be an increased inflammatory environment and accelerated degradation of the matrix. These results are consistent with the hypothesis that, while TNF-α may be effective at an early stage of degeneration, IL-1β is a more potent stimulus as degeneration becomes prolonged and plays a greater role in established pathological degradation.In this study, we have shown that TNF-α downregulates matrix molecule genes at the same time that it upregulates matrix molecule-degrading genes within the normal and degenerate NP. TNF-α does not induce ADAM-TS: a disintegrin and metalloproteinase with thrombospondin motif; AF: annulus fibrosus; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL: interleukin; IL-1R: interleukin-1 receptor; IL-1Ra: interleukin-1 receptor antagonist; IVD: intervertebral disc; MMP: matrix metalloproteinase; NP: nucleus pulposus; PCR: polymerase chain reaction; TNF: tumour necrosis factor; TNF-R: tumour necrosis factor receptor.The authors declare that they have no competing interests.SJM-S carried out a proportion of the experimental work, supervised PWC, analysed the data and drafted the manuscript. PWC carried out the remainder of the experimental work. AJF helped to secure funding and contributed to the preparation of the final manuscript. JAH conceived and designed the study, secured funding and co-wrote the manuscript. All authors read and approved the final manuscript.


Although anti-LeY mAb-mediated TNF-α release paralleled ADCC activity, cytokine release required a higher concentration of sensitizing mAb than the induction of cytolysis. The determination of the FcγR classes involved in the induction of the distinct effector functions showed that anti-LeY mAb-induced cytolysis was triggered by interaction between anti-LeY mAbs and FcγRI. In contrast, mAb-induced TNF-α release mainly depended on the activation of monocyte FcγRII. Neutralization of TNF-α showed no influence on monocyte ADCC activity towards SKBR5 target cells. Our data indicate an independent regulation of anti-LeY mAb induced effector functions of ADCC and TNF-α release which seemed to be triggered by activation of different types of FcγR. © 2000 Cancer Research CampaignStimulation of monocytes by interaction of monoclonal antibodies (mAbs) with Fc gamma receptors (FcγRs) results in the activation of various monocyte effector functions. In the present investigation we show that the anti-Lewis Y (LeY) anti-tumour mAb ABL 364 and its mouse/human IgG1 chimaera induce both antibody-dependent cellular cytotoxicity (ADCC) and the release of tumour necrosis factor α (TNF-α) during mixed culture of monocytes with LeY


Mechanisms by which attainment of specific body sizes trigger developmental transitions to adulthood (e.g. puberty or metamorphosis) are incompletely understood. In Drosophila, metamorphosis is triggered by ecdysone synthesis from the prothoracic gland (PG), whereas growth rate is increased by insulin/insulin growth factor signalling (IIS). Transgene-induced activation of PI3K, the major effector of IIS, within the PG advances the onset of metamorphosis via precocious ecdysone synthesis, raising the possibility that IIS triggers metamorphosis via PI3K activation in the PG. Here we show that blocking the protein kinase A (PKA) pathway in the insulin producing cells (IPCs) increases IIS. This increased IIS increases larval growth rate and also advances the onset of metamorphosis, which is accompanied by precocious ecdysone synthesis and increased transcription of at least one ecdysone biosynthetic gene. Our observations suggest that IIS is regulated by PKA pathway activity in the IPCs. In addition, taken together with previous findings, our observations are consistent with the possibility that, in Drosophila, attainment of a specific body size triggers metamorphosis via the IIS–mediated activation of PI3K and hence ecdysone synthesis in the PG. In organisms such as Drosophila and mammals, growth occurs during an early juvenile phase, but at a certain age , organisms transition to non-growing, sexually mature adults. How do organisms sense when they are large enough to undertake the transition to adulthood? In humans of the western world, the age at puberty has decreased over the past 150 years, a phenomenon attributed to improved nutrition and hence increased growth rate, whereas Drosophila larvae reared on poor nutrients grow slowly and exhibit developmental delays. Thus, the timing of onset to adulthood is not fixed, but rather is affected by growth rate, suggesting that the hormones that trigger growth interact with the hormones that trigger developmental transitions. How these hormones might interact at the molecular level remains incompletely understood.InR, each decreased IIS and growth rate, and also delayed metamorphosis In Drosophila larvae, metamorphosis is triggered by the steroid hormone ecdysone To address these issues, we increased IIS by transgene manipulation within the IPCs and tested the effects of this increased IIS on growth rate and the timing of metamorphosis. We found that increased IIS significantly increased growth rate but had only a modest effect on final body size. In contrast, onset of metamorphosis was sharply advanced: larvae with increased IIS underwent metamorphosis about 36 hours earlier than wildtype, and this precocious development was accompanied by precocious synthesis of ecdysone and increased transcription of at least one ecdysone biosynthetic gene. We conclude that IIS is rate limiting for metamorphosis, and advances the onset of metamorphosis even in the absence of changes to other signalling systems. Such an effect of increased IIS on the timing of metamorphosis has not been previously reported. In addition, combined with previous work demonstrating that PI3K activity in the PG is also rate limiting for metamorphosis Creb and PKA mutants To evaluate the effects of altered IIS on growth rate and the timing of metamorphosis, we began by altering activity of genes of the protein kinase A (PKA) pathway within the insulin producing cells (IPCs). We had two reasons for hypothesizing that altered PKA pathway activity within the IPCs might alter IIS. First, PKA and its downstream transcription factor Creb activate transcription of the mammalian insulin gene Gal4/UAS system dilp2-Gal4 driver, which expresses specifically in the IPCs PKAR*DNCreb2 transgene To test this possibility, we inhibited PKA signalling specifically in the IPCs by use of the dilp2>PKAR* and DNdilp2>Creb2 larvae grew faster than wildtype controls  on developmentally-staged larvae to measure transcript levels of the Thor gene, which encodes initiation factor 4E binding protein and is repressed transcriptionally by IIS Thor transcript levels were decreased in dilp2>PKAR* and DNdilp2>Creb2 compared to dilp2>YFP 108 and 120 hours AEL . We found that controls . For exaours AEL . Third, dilp2>PKAR* and DNdilp2> Creb2 larvae, final pupal and adult size was altered only modestly. The dilp2>PKAR* and DNdilp2>Creb2 genotype did not significantly affect either female or male adult weight  caused a precocious metamorphosis disembodied (dib) and phantom (phm)Why did the increased growth rate in dib and hence precocious synthesis of ecdysone. We allowed developmentally staged larvae to proceed to pupariation and found that dilp2>PKAR* and DNdilp2>Creb2 larvae pupariated and ultimately eclosed as adults about 36 hours before dilp2>YFP  weighed 28% and 30% greater, respectively than arm>YFP adults (not shown). Thus, it appears that ubiquitous dilp overexpression and IPC-specific dilp overexpression affect body size differently.It was previously reported that increased IIS, caused by transgene-induced overexpression of dilps, increased body size dilp2>dilp2 adults were about 25% longer than wildtype dilp2>dilp2 pupae and controls . Furthermore, the advancement of metamorphosis reported in It was previously reported that controls . The disOur results lead to three conclusions. First, IIS is a rate-limiting step for metamorphosis. It was previously shown that decreased IIS delays metamorphosis Manduca sexta that show that PTTH application increases levels of phospho-Erk, a target of Ras/Raf, within the PG The proper timing of metamorphosis requires a second hormone-mediated signalling pathway in addition to insulin. Genetic ablation of the neurons expressing prothoracicotropic hormone (PTTH) delay pupariation and metamorphosis as a consequence of attenuation of ecdysone biosynthetic gene transcription αs and activates adenylate cyclase and PKA Our work suggests that PKA/Creb2 activity in the IPCs inhibits IIS. Although the extracellular ligands regulating IPC PKA activity are unknown, possible regulators include adipokinetic hormone (AKH), which has functional similarity to glucagon PKAR* or DNCreb2, then absence of IRS-2 could increase insulin signalling by blocking insulin responsiveness and thus the recently-described The mechanism by which PKA/Creb2 inhibition activates IIS remains unknown. In mammals, Creb carries out both transcriptional activation and feedback inhibition of transcription at the insulin promoter Altered growth rate, achieved by altered nutrition or endocrine disruptions, can also affect the timing of puberty in mammals. For example, genetic disruption of mouse growth hormone signalling, which decreases insulin growth factor (IGF) synthesis, delays puberty dilp2-Gal4 line was provided by Eric Rulifson , UAS-PKAR* was provided by Dan Kalderon , DNUAS-Creb2 lines was provided by John Kiger  and UAS-dilp2 was provided by Gyunghee Lee . All other stocks were provided by the Drosophila Stock Center at Bloomington, Indiana.All fly stocks were maintained on standard cornmeal/agar media at 22°C. The Third instar larvae were dissected in 1× PBS, fixed in 4% formaldehyde in 1XPBS-T (0.1% TritonX) for 15 minutes at room temperature and washed 3 times, 10 minutes each time, in 1XPBS-T. Primary rabbit anti- phospho-CREB  was incubated at 1∶50 overnight at 4°C. After three 10 minute 1XPBS-T washes secondary goat anti-rabbit antibody  was incubated at 1∶500 for 2 hrs at room temperature. The samples were washed as above and mounted in UltraCruz mounting media containing DAPI . Images were collected using a Zeiss confocal laser-scanning microscope (40× objective).Larvae for Western blots, weight and Q-PCR were pre-laid for three days, and then were staged by serial transfer of parent flies  at 12 hour intervals.4O7P2, 20 mM NaF, 1.5 µM pepstatin A, 2.3 µM leupeptin and 100 µM PMSF). Samples were centrifuged at 18000 g for 30 minutes at 4°C. Equal amounts of protein lysates were aliquoted, 2× sample buffer was added, and samples were boiled for 5 min. Samples were then resolved on 8% SDS-PAGE gel and transferred to nitrocellulose. The blots were blocked in 5% non-fat milk and incubated with rabbit anti-Drosophila pAkt antibody at 1∶1000  and rabbit anti-Akt antibody at 4°C overnight. Anti-vinculin 1∶500 was used as a loading control (Santa Cruz). HRP-conjugated secondary antibodies were used and chemiluminescence was detected on film, and digitized.Staged larvae were collected, frozen and incubated on ice for 15 minutes in lysis buffer . To measure dib transcript levels, we used the forward primer sequence 5′GCCCAAGCTCACCAGATTGA3′, the reverse primer sequence 5′TGCAGACGAGCTCCAAAGGT3′ and the probe sequence 5′TTTGGAATTAACCTGTTTGCT3′. To measure Thor transcript levels we used the RPII140 expression assay (Applied Biosystems) and RPII 140 was used as the house keeping gene.RNA extractions from staged larvae, cDNA preparations and transcript analysis by Q-PCR were performed with the TaqMan gene expression assay (Applied Biosystems) as described in Caldwell et al. (2005). The


This was due to overproduction of the antioxidant glutathione, which decreased the nuclear factor (NF)-κB activity known to control the expression of survival genes. These findings were confirmed in vivo, in nude mice bearing U937 tumours coinjected with recombinant hGH and the NF-κB -inducing anticancer drug daunorubicin, to avoid the in vivo toxicity of TNF-α. This study therefore highlights one of the various properties of hGH that may have potential clinical implications.In addition to its primary role as growth factor, human growth hormone (hGH) can also participate in cell survival, as already documented by its protective effect on human monocytes or human promyelocytic leukaemia U937 cells exposed to a Fas-mediated cell death signal. However, despite similarities in the molecular events following Fas and TNF- Hypoploid cell assessment was measured on ethanol-permeabilised U937 cells exposed or not to 10 ng ml−1 of TNF-α cultured overnight with or without 400 ng ml−1 of cycloheximide (CHX), by staining with PI, as previously described . Specificity was assessed by incubating nuclear extracts obtained from TNF-α-stimulated cells with nonradiolabelled NF-κB probe (5′-ACAAGGGACTTTCCGCTGGGGACTTTCCAG-3′) or mutated NF-B (5′-ACAACTCACTTTCCGCTGCTCACTTTCCAG-3′) oligonucleotide probe, as previously reported ±rhGH. In other separate experiments, we analysed nuclear extracts from U937 cell lines cultured in the absence or in the presence of: TNF-α ±CHX , or anti-Fas mAb  . In both cases, specificity was assessed by using the probes described above. Alternatively, prior to nuclear extractions, U937 cells were incubated with 10 ng ml−1 of TNF-α alone or in addition to various doses of glutathione ethyl-ester .Cell lines were cultured for 90 min in the presence of 10 ng ml5 cells with or without exposure to 5 ng ml−1 of TNF-α were separated on 10% SDS–polyacrylamide gel and electroblotted onto nitrocellulose membranes. In separate experiments, U937 cells were incubated with 5 ng ml−1 of TNF-α alone or in addition to 20 mM of GSH and U937-hGH cells were incubated with 5 ng ml−1 TNF-α alone or in addition to 50 mM of diethyl maleate (DEM) (Sigma), in conditions already used in our models (κBα (Santa-Cruz Biotechnology) or anti-IκB Abs  after striping, and then with anti-rabbit peroxidase-labelled second Abs (Amersham). They were developed using an enhanced chemiluminescence detection system .Cell lysates obtained from 5 × 10−1 of total protein measured in cell extracts by the Bradford method.The total glutathione level was measured by the glutathione reductase recycling method, as previously described (7) were first injected subcutaneously into irradiated 6- to 8-week-old male nu/nu Swiss mice in order to obtain solid tumours. Another group of mice was then engrafted with fragments from these tumours (4 mm3), pooled and randomly assigned to four groups of 10 mice: animals injected with saline solution; animals injected with 1.5 mg kg−1 of daunorubicin (dosage determined as previously reported (−1 of recombinant exogenous hGH (rhGH) based on the study of the in vivo effects of rhGH in a murine model of sepsis induced by Escherichia coli =d2 (mm2) × D (mm)/2, where d and D are the smallest and perpendicular diameters, respectively. For ethical reasons, the maximum tumour volume accepted was 2000 mm3. In a separate experiment, mice were treated as described above and were killed at various times. Fixed tumours were analysed by epifluorescence for in situ apoptosis with terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL) (Roche) or for in situ NF-κB activation with rabbit anti-P65 Abs revealed with fluorescein-labelled goat anti-rabbit Abs, and nuclei were stained with DAPI. Animals were killed under CO2 anaesthesia. All experiments were carried out under the conditions established by the European Union (Directive 86/609/EEC).U937 cells . In contrast, hGH had no additional effect on apoptosis of cells exposed to TNF-α in the presence of the protein synthesis inhibitor CHX, known to potentiate TNF-α-induced U937 cell death .Based on our previous studies demonstrating that stable expression of hGH in cell lines could mimic the effect of exogenous GH , as compared to nonexposed cells (lane 1), was no longer observed in U937-hGH cells treated under similar conditions . Specificity was assessed by incubating nuclear extracts obtained from TNF-α-treated control cells with nonradiolabelled consensus NF-κB (lane 5) or mutated NFκB (lane 6) probe. Interactions of these nuclear extracts were also observed with anti-P65 (lane 7) and, to a lesser extent, anti-P50 (lane 8) Abs, confirming the detection of NF-κB.As inhibition of NF-imulator . As illuκB activity, in separate experiment, parental U937 cells were coincubated with TNF-α and various concentrations of rhGH. As shown in α-mediated NF-κB-DNA binding activity (lane 4) was decreased by addition of 50 ng ml−1 (lane 6) or 500 ng ml−1 (lane 7) of rhGH.To confirm the effect of hGH on NF-κB can interact with Fas-mediated apoptosis in various situations (κB translocation using nuclear extracts from cells exposed to anti-Fas mAb (α (lane 2), and also to TNF-α±CHX (lane 3), compared to unstimulated cells (lane 1). These protein–DNA complexes were specifically competed by a consensus NF-κB probe but not by the mutated probe . The present experiment also confirmed that NF-κB translocation was strongly inhibited by hGH in response to a TNF-α stimulus (lane 6), and was markedly diminished in response to a TNF-α+CHX stimulus (lane 7).NF--Fas mAb . No protκB can be closely monitored by its interaction with members of the IκB family. In response to various stimuli, IκB undergoes proteolytic degradation, which allows nuclear translocation of NF-κB leading to gene activation  , a GSH analogue which can enter cells due to its hydrophobic ester groups, increased cell death in TNF-α-treated U937 cells   with the37 cells . These rof TNF-α . The prein vivo, we decided to use the anticancer drug daunorubicin, as TNF-α, one of the main cytokines involved in sepsis , while no fluorescent nuclei were observed in the two other groups. In addition, the limited nuclear staining revealed with an anti-P65 NF-κB subunit Ab, evidenced a marked decrease in NF-κB activation in tumour cells of mice treated with both daunorubicin and rhGH, compared to the daunorubicin-treated group (P<0.0001)  . Both ef−1, as defined in Materials and Methods) combined with injections of 5 mg kg−1 of rhGH, while tumours remained unaffected by treatment with daunorubicin only. Tumour volumes were significantly different from the 6th day after initiation of treatment until the 12th day, when comparing animals treated with rhGH and daunorubicin to animals treated with daunorubicin only . No further comparisons were possible due to death of animals in the daunorubicin-treated group at day 12. No protective effects were observed in mice receiving saline (Ctrl) or rhGH only. Given the high growth rate of U937 cells in vivo and the design of the present experiments in which only a single cure was given to the mice, the complete eradication of tumours could not be achieved. However, the survival of mice with a tumour below 300 mm3 was determined. As shown in P<0.008).In parallel, the growth rate of U937 tumours was measured in these mice. As shown in It should be noted that coadministration of rhGH and daunorubicin did not induce any toxicity or body weight loss (data not shown).α receptors  . Although unusual, this type of situation has already been described by other authors in cells from IκB α-deficient mice in response to TNF-α  . One of B family , or IκBβα induces the release of ROIs   . These prylation , or alterylation . It is t


The low-density lipoprotein receptor-related protein 1 (LRP1) plays critical roles in lipid metabolism, cell survival, and the clearance of amyloid-β (Aβ) peptide. Functional soluble LRP1 (sLRP1) has been detected in circulating human placenta; however, whether sLRP1 is also present in the central nervous system is unclear.Here we show that abundant sLRP1 capable of binding its ligands is present in human brain tissue and cerebral spinal fluid (CSF). Interestingly, the levels of sLRP1 in CSF are significantly increased in older individuals, suggesting that either LRP1 shedding is increased or sLRP1 clearance is decreased during aging. To examine potential effects of pathological ligands on LRP1 shedding, we treated MEF cells with Aβ peptide and found that LRP1 shedding was increased. ADAM10 and ADAM17 are key members of the ADAM family that process membrane-associated proteins including amyloid precursor protein and Notch. We found that LRP1 shedding was significantly decreased in MEF cells lacking ADAM10 and/or ADAM17. Furthermore, forced expression of ADAM10 increased LRP1 shedding, which was inhibited by ADAM-specific inhibitor TIMP-3.Our results demonstrate that LRP1 is shed by ADAM10 and ADAM17 and functional sLRP1 is abundantly present in human brain and CSF. Dysregulated LRP1 shedding during aging could alter its function and may contribute to the pathogenesis of AD. Alzheimer's disease (AD) is the most common cause of dementia in elderly. The central hypothesis of AD is the amyloid hypothesis which proposes that accumulation of amyloid-β (Aβ) peptide, a small peptide with a high propensity to form aggregates in the brain, is the primary factor driving AD pathogenesis -3. The lLRP1 is synthesized as a precursor that migrates on SDS-gel with an apparent molecular size of 600 kD . En routsLRP1 levels were measured in the CSF of normal controls and AD patients by Western blot Figure  and RAP The sLRP1 levels were also analyzed in human brain tissue  by Western blot. Cortex and hippocampus of human brain tissue were separated into membrane and soluble fractions, and were blotted with LRP1 antibody. As shown in Figure To examine potential effect of pathological ligands on LRP1 shedding, mouse embryonic fibroblast (MEF) cells were treated with Aβ42 or vehicle control for 48 h. Media and cell lysates were collected and blotted with two LRP1 antibodies ,14,15 toADAM10 and ADAM17 are members of the ADAM metalloprotease family and play critical roles in ectodomain shedding of several transmembrane proteins including APP . To undeBecause TIMP-3 is a natural inhibitor of matrix metalloproteinases , it was 2+-dependent ligand binding domains and is bound to the membrane-spanning LRP1-85 subunit. Functional soluble LRP1 (sLRP1) has been detected in circulating human placenta; however, whether sLRP1 is also present in the central nervous system is unclear. Here we show that sLRP1 is abundantly present in human CSF in the forms of LRP1-515 and LRP1 fragments, which are recognized by LRP1 antibody and bind to the LRP1 ligand RAP. In human brain tissues, sLRP1 is also detected in the soluble fractions of both cortex and hippocampus. The precise nature of the LRP1 fragments needs to be further characterized by mass spectrometry using highly purified sLRP1.LRP1 belongs to the low-density lipoprotein receptor family, the members of which share many structural and functional characteristics. The LRP1-515 subunit of LRP1 contains multiple Cain vitro. It is possible that increased LRP1 shedding in AD leads to its deposition in amyloid plaques [Kang et al. found that the brains of AD patients had significantly lower levels of LRP1 than that of age-matched controls. Moreover, they also showed that in the brains of normal individuals, LRP1 levels are decreased substantially with age. The changes in LRP1 levels could be due to LRP1 shedding in the brain. We tested this hypothesis by comparing sLRP1 levles in normal controls and AD patients and found that the levels of sLRP1 in CSF are significantly increased in older individuals. This suggests that either LRP1 shedding is increased or sLRP1 clearance is decreased during aging. However, we did not detect significant differences in sLRP1 levels between normal controls and AD patients. This was somewhat surprising as we found that Aβ42 significantly increases LRP1 shedding  plaques  rather tThe detection of sLRP1 in conditioned medium of MEF cells provides a model system for elucidating the mechanism of LRP1 shedding. ADAM family members are involved in ectodomain shedding of various cell surface proteins such as growth factors, receptors and their ligands, cytokines, and cell adhesion molecules . Among tThe identification of the soluble forms of LRP1 in human CSF and brain tissue introduces a new dimension into the biology of this unique molecule, especially in neuropathology and aging. Further studies are required to understand the structures of the LRP1 fragments and establish their physiological role in the brain and CSF. Dysregulated LRP1 shedding during aging could alter its function and may contribute to the pathogenesis of AD.Functional sLRP1 is abundantly present in human brain and CSF, and is likely increased during aging. LRP1 shedding is mediated by ADAM10 and ADAM17 and is up-regulated by Aβ42. Increased LRP1 shedding during aging could contribute to the pathogenesis of AD.S-transferase expression vector and isolated as described previously [Human recombinant RAP was expressed in a glutathione eviously . All tiseviously ,14,15. P2 at 37°C. CHO LRP1-null cells were cultured in Ham's F12 medium supplemented with 10% fetal calf serum in humidified air with 5% CO2 at 37°C.MEF cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum in humidified air with 5% COCHO LRP1-null cells were transfected with the appropriate cDNAs: LPR1 minireceptor (mLRP4), RAP, ADAM10, ADAM17 and/or TIMP3. Twenty-four hours after transfection, media was collected and concentrated, and cells were rinsed, gently scraped into PBS (pH 7.4), and pelleted. Cells were then lysed in lysis buffer, and the lysate and concentrated media were used for Western blot.2, with EDTA-free Complete proteinase inhibitors) using a Polytron homogenizer. After two-low speed spins , the homogenate was centrifuged 30 min at 100,000 × g at 4°C to separate the membrane fraction (pellet) from the soluble fraction. The pellet was resuspended in 50 mM Tris (pH8.0), 80 mM NaCl, 2 mM CaCl2, with EDTA-free Complete proteinase inhibitors. Triton X-100 was then added to both the membrane and soluble fraciton to a final concentration of 1%. The membrane fraction was passed through a 28-gauge needle and cleared by a second centrifugation at 100,000 × g to remove insoluble material.Preparation of soluble and membrane fractions has been described before . BrieflyCell and tissue samples were lysed on ice in lysis buffer . CSF samples were directly mixed with sample buffer. Protein concentration was determined in each sample using a Protein Assay kit (Bio-Rad). Equal amount of protein was used for SDS-PAGE. Immunoreactive bands were visualized by enhanced chemiluminescence and exposed to film. In RAP ligand blotting, blots were probed with 20 nM RAP for 1 h at room temperature, and then with RAP-specific antibody. For densiometric analyses, immunoreactive bands were scanned using a Kodak Digital Science DC290 Zoom camera and quantified using Kodak Digital Science image analysis software.All quantified data represent an average of at least triplicate samples. Error bars represent standard deviations. Statistical significance was determined by Student's t-test and P < 0.05 was considered significant.The authors declare that they have no competing interests.QL and GB designed all the experiments. QL carried out all the studies, participated in acquisition of data, performed the statistical analysis, and drafted the manuscript. Under QL's supervision, JZ and HT carried out the transfection and Western blot study and participated in data acquisition. GB supervised QL, JZ and HT in experiments and participated in preparation of the manuscript. MMV and SE provided human CSF and brain tissues. KR provided ADAM10-KO and ADAM17-KO MEF cell lysates and media. All authors read and approved the final manuscript.


We investigated the influence of recombinant human tumour necrosisfactor alpha (TNF-α) and its derivatives termed muteins III, V,VI—in which the first 3 to 7 amino acids of native TNF-α have beenreplaced—on the survival time of mice inoculated with leukaemiaL1210 or leukaemia P338. TNF-α prolonged the survival of mice withleukaemia L1210 but did not have any therapeutic activity inleukaemia P388-bearing mice. Muteins-treated mice with leukaemiaP388 lived longer than animals receiving TNF-α, while thoseinoculated with leukaemia L1210 did not show any significantprolongation of life compared with the TNF-α treated group. Theresults presented in this report indicate that the antileukaemicactivity of TNF-α is governed at least in part by the nature of theN-terminal amino acids.