Genotype-expression association analysis using linear regression may produce different test results depending on whether founders only or all pedigreed members are used. This difference is not due to the correlation of samples within a pedigree, because linear mixed models have been applied to account for that correlation. We investigated the possibility that the difference is due to a dependence of expression levels on, among other things, the generation number in the pedigree. Indeed, of the 30 or so studied expression quantitative traits, several of them show significant dependence on the generation number. We propose to use all pedigree members in genotype-expression association analyses whenever the complete genotyping information is available. The genotype-expression association analyses were performed by Morley et al.  and Cheuy ~ ax+b), the x variable in different samples is allowed to be correlated as long as the y variable is independent conditional on x. Using pedigree members might be a potential problem in genotype-expression association by regression analysis if expression levels in some pedigrees are systematically higher or lower than other pedigrees, thus the parameter b, for example, can vary from one pedigree to another in a random fashion. Note that this is a different issue from the correlated samples for allele frequency estimation in case-control analysis [In linear regression models  association signal [cis action makes much biological sense, but also because these genes are under linkage peaks. We used these genes in our study, but relaxed the cis-acting requirement: the SNP that shows the strongest association signal will be checked for our comparative study between using founders only and using all pedigree members. This procedure usually selects trans-acting SNPs, and thus the chance for false-positive SNP-eQT association is higher.Morley et al. found 27 genes that exhibit strong A comparison of using founders and using all pedigree members might detect something else: consistency between gene expression in founders and in non-founders. If pedigree founders are not a representative subset of samples of a pedigree, using founders only will lead to biased selection. Recent studies have shown that many eQTs may be age-dependent , which mcis linkage signal and strong association signals, and 2 eQTs that are of great interests for our investigation of rheumatoid arthritis, HLA-DRB1 [tran-acting); and SNP-eQT pairs used here are mostly different from those in Cheung et al. [We selected 28 eQTs: 26 of the 27 gene expressions (there is no probe set in GAW data that matches ICAP-1A) listed in Table HLA-DRB1  and PTPNHLA-DRB1 . After uyij = a + εi + bxij + εij, where i is the pedigree index and j is the person index; and random effects on both a and the slope b (MM2 for mixed model 2): yij + a + εi + (b + δi)xij + εij. As for the age effect, we simplified the issue by examining the effect of generation number  on expression level, disregarding pedigrees (labeled as "GC model" for generation as a covariate): yij = a + bxij + c2gij 2,+ c3gij 3,+ εij. Note that the generation covariate is coded as a factor with two dummy variables, g2  and g3 .When all pedigree members are used (194 samples), we considered two models to account for possible pedigree-specific effects on eQT: a random effect on the intercept a (MM1 for mixed model 1):t-tests for the expression levels by either combining genotypes 0 and 1 as one group (and genotype 2 as the second group), or combining genotype 1 and 2 as a group (and genotype 0 as another group). The best p-value of the two tests was selected.To check the robustness of linear regression in which an additive model is assumed, we also carried out two To examine whether founders tend to have higher or lower expression levels with respect to the non-founders, we determined the percentile value for each founder in a pedigree, for a specific eQT. For that eQT, this percentile value was averaged over all pedigrees. If the averaged founder percentile was close to 50%, there was no founder selection bias; and if it was close to 100% or 0%, founders were considered as a biased subset.Both SAS and R statistical packages were used.p-value, instead of a larger, insignificant one. For the 31 SNP-eQT pairs listed in Additional file p-values are increased in the all-pedigree-member data set , despite a tripling of the sample size.As seen in Additional file t-tests by grouping samples with the heterozygous genotype to those with one of the homozygous genotypes. For founders, the only SNP-eQT pair with p-value smaller than 4.4 × 10-6(0.01/2263) is CSTB-rs157334 . When all pedigree members are used, CSTB-rs157334 is still the only pair that is significant at this level .Because the linear regression used here implies an additive model, we also carry out two -6 level: CSTB-rs157334 and HSD17B12-rs1334334. When MM2 mixed model is applied, only CSTB-rs157334 exhibits a p-value close to that level (5.3 × 10-6). Interestingly, this is the only cis-acting pair among those listed in Additional file When the MM1 mixed model is applied to the 194-sample data, only two SNP-eQT pairs remain significant at the 4.4 × 10p-values are listed in Additional file b), generation 2 (c2), and generation 3 (c3). Note that in this regression, pedigree member information is discarded.To investigate why all-pedigree-member data set may exhibit a different regression result from the founders-only data set, we examined whether eQT changes from generation to generation. This is a simplified version of examining the age effect, as generation 1 (founders) are older than the second and the third generations. Under the GC regression model described in the Methods section, three -6 level after accounting for the generation effect: the two pairs mentioned above as well as RPS26-rs720428, CTSH-rs1021639, and GSTM1-rs1039337. On the other hand, significant association with the generation variable has been observed for these eQTs: DDX17 (4.8 × 10-15) on generation 2; PTPN22 (2.8 × 10-20), CGI-96 (8.4 × 10-15), HLA-DRB1 (1.7 × 10-8), ZNF85 (3.5 × 10-8), IL16 (2.2 × 10-6), and CSTB (6.6 × 10-6) on generation 3. Interestingly, even though CSTB has a significant association with generation 3, it has an even stronger association with the genotype of rs157334.Five SNP-eQT pairs in Additional file A very simple check on whether founders tend to have different expression levels from non-founders is to calculate their percentile value with respect to other samples in the pedigree. For example, for a 14-member pedigree, each member has a percentile value ranging from 1/14 to 1, in an increment of 1/14, and is determined by their expression level of a particular eQT. For 28 eQTs , their average, standard deviation, and median percentile values are listed in Table To view more directly the relative location of founders' expression with respect to other pedigree members, Figure p-value = 0.001 level. These nine eQTs are CSTB, RPS26, DDX17, CGI96, TM7SF3, IL16, ZNF85, PTPN22, and HLA-DRB1, consistent with the result from the percentile value calculation; 3) for the founder-pedigree interaction term, four eQTs are significant at the p-value = 0.001 level.Yet another simple check for the founder effect on expression is the two-way ANOVA with pedigree as one factor and founder/non-founder as the second factor. Our result shows that 1) pedigree-dependence of the expression is significant for most eQTs (with the exception of four to five eQTs); 2) founder-dependence of expression is significant for nine eQTs at the Although it does not apply to the GAW data, in a practical setting, genome-wide genotyping information may only be available for the first stage of a study. In this situation, one may select SNPs that show promising association signals, and only type these SNPs for the rest of the pedigree (second stage) in order to save cost. It is also suggested by Van Steen et al. that two-stage design also helps to ease the multiple testing problem . If the p-values. From these results, for each eQT, the minimum p-values of all 2263 SNPs can be recorded. There are 47 eQTs that have a minimum p-value lower than 4.4 × 10-6. Now we assume in the second-stage that these selective SNPs are typed for non-founders. For the corresponding eQT-SNP pair, another linear regression analysis on all 194 members can be carried out, as well as a linear regression analysis with the generation variable as covariates. If we do that, among these 47 eQT-SNP pairs, only two remain significant at the 4.4 × 10-6 level for the 194-sample data set: CSTB-rs157334 and HSD17B12-rs1334334. Interestingly, these two pairs are the only overlap between the 47-pair set and the 31-pair set listed in Additional file To test where a two-stage design would lead us, we imagine a hypothetical situation in which we do not have the genotyping information for non-founders. Then we would first exhaustively perform all possible genotype-expression linear regression analyses for the 56 founders. There are more than 8 millions possible SNP-eQT pairs, leading to an equal number of Because the eQTs listed in Table We have shown that besides pedigree founders, pedigree members can also be used in a SNP-eQT association analysis. The issue of relatedness among pedigree members can be handled by mixed models, either with one or two random effects to account for pedigree-specific expression variations. Using all pedigree members has other advantages, such as increasing the sample size, checking the consistency among samples, and detecting possible age dependence in expression levels.CEPH: Centre d'Etude du Polymorphisme HumanGAW: Genetic Analysis Workshop 15GC: generation as a covariate (linear regression model)eQT: expression quantitative traitMM: mixed model (with 1 or 2 random effects)SNP: single-nucleotide polymorphismThe author(s) declare that they have no competing interests.p-values in regression analyses for 31 selected SNP-eQT pairsComparison of Click here for file


Isolated limb perfusion (ILP) with tumour necrosis factor alpha  and melphalan has shown impressive results in patients with irresectable soft tissue sarcomas and stage III melanoma of the extremities. The mechanisms of the reported in vivo synergistic anti-tumour effects of TNF-alpha and melphalan are not precisely understood. We have developed an ILP model in the rat using a non-immunogenic sarcoma in which similar in vivo synergy is observed. The aim of this present study was to analyse the morphological substrate for this synergistic response of TNF-alpha in combination with melphalan to shed more light on the pathomechanisms involved. Histology of the tumours from saline- (n = 14) and melphalan-treated (n = 11) rats revealed apparently vital tumour cells in over 80% of the cross-sections. Interstitial oedema and coagulation necrosis were observed in the remaining part of the tumour. Haemorrhage was virtually absent. TNF-alpha (n = 22) induced marked oedema, hyperaemia, vascular congestion, extravasation of erythrocytes and haemorrhagic necrosis (20-60% of the cross-sections). Oedema and haemorrhage suggested drastic alterations of permeability and integrity of the microvasculature. Using light and electron-microscopy, we observed that haemorrhage preceded generalised platelet aggregation. Therefore, we suggest that the observed platelet aggregation was the result of the microvascular damage rather than its initiator. Remarkably, these events hardly influenced tumour growth. However, perfusion with the combination of TNF-alpha and melphalan (n = 24) showed more extensive haemorrhagic necrosis (80-90% of the cross-sections) and revealed a prolonged remission (mean 11 days) in comparison with the other groups of rats. Electron microscopical analysis revealed similar findings as described after TNF-alpha alone, although the effects were more prominent at all time points after perfusion. In conclusion, our findings suggest that the enhanced anti-tumour effect after the combination of TNF-alpha with melphalan results from potentiation of the TNF-alpha-induced vascular changes accompanied by increased vascular permeability and platelet aggregation. This may result in additive cytotoxicity or inhibition of growth of residual tumour cells.


Phosphatidylcholine (PC) is a major lipid of the gastrointestinal mucus layer. We recently showed that mucus from patients suffering from ulcerative colitis has low levels of PC. Clinical studies reveal that the therapeutic addition of PC to the colonic mucus using slow release preparations is beneficial. The positive role of PC in this disease is still unclear; however, we have recently shown that PC has an intrinsic anti-inflammatory property. It could be demonstrated that the exogenous application of PC inhibits membrane-dependent actin assembly and TNF-α-induced nuclear NF-κB activation. We investigate here in more detail the hypothesis that the exogenous application of PC has anti-inflammatory properties.PC species with different fatty acid side chains were applied to differentiated and non-differentiated Caco-2 cells treated with TNF-α to induce a pro-inflammatory response. We analysed TNF-α-induced NF-κB-activation via the transient expression of a NF-κB-luciferase reporter system. Pro-inflammatory gene transcription was detected with the help of a quantitative real time (RT)-PCR analysis. We assessed the binding of TNF-α to its receptor by FACS and analysed lipid rafts by isolating detergent resistant membranes (DRMs).The exogenous addition of all PC species tested significantly inhibited TNF-α-induced pro-inflammatory signalling. The expression levels of IL-8, ICAM-1, IP-10, MCP-1, TNF-α and MMP-1 were significantly reduced after PC pre-treatment for at least two hours. The effect was comparable to the inhibition of NF-kB by the NF-kB inhibitor SN 50 and was not due to a reduced binding of TNF-α to its receptor or a decreased surface expression of TNF-α receptors. PC was also effective when applied to the apical side of polarised Caco-2 cultures if cells were stimulated from the basolateral side. PC treatment changed the compartmentation of the TNF-α-receptors 1 and 2 to DRMs.PC induces a prolonged inhibition of TNF-α-induced pro-inflammatory signalling. This inhibition may be caused by a shift of the TNF-α receptors at the surface to lipid rafts. Our results may offer a potential molecular explanation for the positive role of PC seen in clinical studies for the treatment of ulcerative colitis. Inflammatory bowel disease (IBD) is the result of a chronic intestinal inflammatory response. While the exact pathogenesis of IBD remains incompletely understood, it is likely that the initiation of the immune response is triggered by luminal factors . The natin vitro phagosome system -PCIn previous publications, we have already shown that PC has an effect on pro-inflammatory gene transcription downstream of NF-κB . In ordeInterestingly, some genes were up-regulated faster than others. This led us to classify the genes into two groups: 1) early up-regulated genes , where a significant increase in transcript levels could be detected within 30 min; and 2) late up-regulated genes  which responded later. PC pre-treatment reduced the transcriptional levels of both groups of genes significantly. The effect lasted for up to 120 min. Analysing later time points (240 min) did not show a significant difference anymore. The only exception was MMP-1, where a significant difference could also be detected after 240 min  or PC 18:2/18:2 . None of the tested PC species affected the interaction of TNF-α to its receptors or changed the density of TNF-α receptors at the cell surface  and TNF-α-R2 (CD120b). After binding TNF-α, TNF-α-R1 recruits a death domain (DD)-containing adaptor molecule, namely the TNF-α-R1-associated death domain protein (TRADD). TRADD serves as a platform for recruiting additional mediators . It bind1. It could be speculated that PC or LPC inhibits the association of TNF-α to its receptor. To exclude such an effect, we performed experiments in which the cells were treated with PC before TNF-α stimulation and quantified the binding of TNF-α to its receptor by FACS. FACS analysis could detect no quantitative differences between PC or LPC treated and non-treated cells. This makes a decreased receptor binding unlikely.2. It has been previously suggested that TNF-α receptors, upon substrate binding, might be endocytosed and that the signalling response occurs in endosomes, as demonstrated in other receptors such as the β2-adrenergic receptor, the endothelin receptor and many other G-coupled protein receptors -56. Thus3. A third scenario seems more likely. Mammalian membranes are lipid bilayers consisting of 300–400 different lipid species that are ordered in vertical and lateral dimensions. This order is maintained with the help of a highly regulated concert of enzymes. Adding exogenous lipids would lead to a change in the lipid assembly of the membrane and therefore would influence membrane-dependent processes. We have previously speculated that PC might be present in a freely mobile form and a pool that is attached to proteins . Protein4. Not only proteins but also membrane lipid compartments are necessary for TNF-α signalling to occur. It has been previously shown that TNF-α binding recruits TNF-α-R1 to lipid rafts . Rafts aTo get closer to the scenario in the mucosa, polarised Caco-2 cells were analysed as well. Although polarised cells could only be stimulated by basolateral exposure to TNF-α, an inhibitory effect of PC could be observed from both the apical and basolateral sides. The cause of this phenomenon requires further investigation; however, a possible explanation may be that TNF-α signalling occurs after internalisation and that apical and basolateral vesicles mix with each other in the endosomal compartments . AnotherIn summary, our results further support the idea that exogenous PC and LPC have anti-inflammatory properties. Our findings suggest that PC (LPC) inhibits the signalling responses via NF-κB possibly due to changes in the plasma membrane. This is a potential mechanism explaining how PC can influence the course of ulcerative colitis and provides the molecular foundation for the benefit seen in our clinical studies.DD: death domain; DRMs: detergent resistant membranes; IBD: inflammatory bowel disease; ICAM-1: intercellular adhesion molecule 1; IL-8, -1β: interleukin 8, 1β; IP-10: interferon inducible protein 10; LPC: lysophosphatidylcholine; MCP-1, -2, and -3: monocyte chemoattractant proteins 1, 2, and 3; NF-κB: nuclear factor-κB; ns: not significant; PC: phosphatidylcholine; PE: phosphatidylethanolamine; RIP: receptor-interacting protein; SD: standard deviation; SEM: standard error of the mean; SM: sphingomyelin; TNF-α: tumour necrosis factor α; TNF-α-R21, -R2: tumour necrosis factor α receptor 1, receptor 2; TRADD: TNF-α-R1-associated death domain protein; TRAF2: TNF-receptor-associated factor 2; UC: ulcerative colitis.The authors declare that they have no competing interests.RE precipitated in the design of the study and in the data analysis. He drafted the manuscript. AB, IT and PJ carried out the experiments. TG performed the RT-PCR analysis. JF, WS and GG were involved in the design of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/9/53/prepubCo-treatment of Caco-2 cells with TNF-α and phospholipids. Cells were grown in 3.5 cm dishes and the effects of different PC species on the inhibition of TNF-α-induced NF-κB-activation were analysed via the transient expression of a NF-κB-luciferase reporter system. (A) Cells were co-treated with 10 ng TNF-α and 200 μmol of the respective lipid. Luciferase activity was analysed 4 h after stimulation. Interestingly, PC 18:2/18:2 , a PC species with two unsaturated side chains, was the most effective one compared to both PC 16:0/16:0  and PC 16:0/18:2  (p < 0.05). (B) Cells were co-treated with 10 ng/ml TNF-α and the indicated amounts of PC 16:0/16:0. PC inhibited NF-κB activation in a dose-dependent manner. The strongest effect was seen with 200 μmol PC which was significantly different from all other PC concentrations tested. The value was arbitrarily set to 100% in cells treated with TNF-α but not with phospholipids. Results presented here are representative for three others carried out independently. Data are shown as mean and SD (n = 3). Asterisks assign statistically different values from Tukey's post hoc test compared to control ; crosses indicate significant differences from +TNF-α after treatment with different phospholipids or phospholipid concentrations .Click here for file3H]-PC 16:0/16:0 into Caco-2 cellsUptake of -PC 16:0/16:0. Methods: Caco-2 cells were grown to 80% confluence in 5 cm2 dishes and incubated for various times with different concentrations of 1-, 2-dipalmitoyl-3-phosphatidyl-[N-methyl-[3H]-choline  and 1-, 2-dipalmitoyl-glycero-3-phosphocholine (PC 16:0/16:0) at a ratio of 1:250. After washing, cells were incubated with NaOH (1 M) for 10 min. Both the cell lysates and the supernatants were analysed with counting solution in a scintillation counter (Beckman Coulter LS 6500) as done previously [3H]-methyl-choline]-L-dipalmitoyl-phosphatidylcholine ([3H]-PC) (50 Ci/mmol) was purchased from New England Nulcear, . Results: (A) The uptake reaches a plateau with increasing time. (B) Within the first 5 min of incubation with PC, the uptake was almost linear. An additional PC uptake was not detectable after 1 h. This uptake kinetic probably indicates that the inhibitory effect of exogenous PC correlates to the amount of PC incorporated into the cells. The plateau after 1 h likely explains why no additional effect on NF-κB activation was seen with longer pre-treatment times  Co-treatment with 200 μmol of PC 16:0/16:0  resulted in a significant inhibition of the TNF-α-induced up-regulation. Up-regulation of selected genes after TNF-α stimulation might be classified into two groups: 1) early up-regulated genes (such as IL-8) and 2) late genes . The experiment depicted is representative of three others with the same results. Data are expressed as mean and SEM (n = 3). Asterisks assign statistically different values from Tukey's post hoc test  comparing +TNF-α and +TNF-α +PC at each time point; crosses indicate significant differences of control compared to both +TNF-α and +TNF-α +PC at each time point .Click here for fileEffect of SN 50 and PC on TNF-α-induced gene activation. Sub-confluent Caco-2 cells were stimulated with TNF-α (10 ng/mL) to induce an up-regulation of several selected genes. Pre-treatment with the NF-κB inhibitor SN 50 (50 mg/mL) for 30 min resulted in a significant reduction of the TNF-α-induced up-regulation. This reduction was similar to that found in cells treated with PC, possibly indicating that both treatments influence the same pathway. Data are expressed as mean and SEM (n = 3). Asterisks assign statistically different values from Tukey's post hoc test of +TNF-α to all other values at each time point ; At no time point could a significant difference of +TNF-α +PC and +TNF-α +SN 50 be detected with the exception ICAM-1 at 120 min. Crosses indicate significant differences of control compared to +TNF-α, TNF-α +SN 50 and +TNF-α +PC at each time point .Click here for fileNeither PC 16:0/16:0 nor PC 18:2/18:2 showed any effect on the binding of TNF-α to its cell surface receptors. Vero cells were taken for FACS analyses because of their regular cell shape. They were either left untreated or were pre-treated for 1 h prior to the addition of TNF-α-biotin with a 200 μM preparation of PC 16:0/16:0  or PC 18:2/18:2 . The PC-treated (TNF-α-biotin + PC 16:0/16:0 or TNF-α-biotin + PC 18:2/18:2) and untreated (TNF-α-biotin) cells were then collected for staining with biotinylated TNF-α and avidin-FITC. Soybean trypsin inhibitor that had been biotinylated in the same degree was used as a negative control. Neutralized TNF-α biotin with anti-TNF-α blocking antibody was used as a specificity control. Receptor binding activity was determined by flow cytometric analysis with a 488 nm wavelength laser excitation. Results shown here were representative for three other experiments carried out independently.Click here for file


The following information was omitted from the Funding section: YT was supported by the Mitsubishi Foundation.


Iron is both essential and toxic for cells and impaired iron homeostasis has been shown to cause or potentiate various forms of liver injury. Research in our laboratory suggests that iron also plays a pivotal role in intracellular signaling for NF-kappa B activation in hepatic macrophages (HM). Our results showed: 1) HM from alcohol-fed rats had a increase in the nonheme iron content accompanied by NF-kappa B activation; 2) iron chelation normalized nonheme iron concentration and blocked enhanced NF-kappa B activation and TNF-alpha expression in these cells; 3) LPS-induced NF-kappa B activation was also blocked by iron chelator; 4) iron directly induced TNF-alpha expression via IKK and NF-kappa B activation in normal HM. We propose that iron acts as an independent proinflammatory molecule via induction of the intracellular signaling for NF-kappa B activation in HM and primes the liver for chronic inflammation and injury. Our earlier study showed that hepatic macrophages (HM) from rats fed ethanol and high fat diet had a significant 70% increase in the nonheme iron content as compared to controls . This st1+) but not cupric (Cu2+) copper also stimulated TNF-alpha release at 50 micromolar to less extent. Thus, these results demonstrate direct stimulation of HM TNF-alpha release by iron and copper in a redox status dependent manner. We then tested whether Fe2+ stimulates TNF-alpha promoter in cultured HM. The promoter activity was indeed increased 2~3 fold with 10~50 micromolar Fe2+. Cu1+ (50 micromolar) also slightly increased TNF-alpha promoter activity but not Cu2+ or Fe3+  in a serum-free condition. These results establish that Fe2+ activates TNF-alpha promoter in a NF-kappa B dependent manner.Direct addition of ferrous but not ferric iron in cultured HM increased TNF-alpha release 8 fold at 10 and 50 micromolar during a 4 hr treatment period without cell toxicity. Cuprous (CuAs shown in the top panel of Figure Our studies to date strongly suggest the causal link between iron and activation of NF-kappa B in HM in both normal and alcohol-fed rats. These findings raise a question as to how iron signals to activate NF-kappa B. Since NF-kappa B is a redox sensitive transcription factor and ROS are implicated in its activation ,4, it is


Drawbacks to this production system include contamination of VLP preparations with baculovirus and the necessity for routine maintenance of infectious baculovirus stock. We used gag transgene integration and expression up to 100 cell passages, and although the level of VLPs produced was low compared to baculovirus-produced VLPs, they appeared similar in size and morphology to baculovirus-expressed VLPs. In a murine immunogenicity study, whereas baculovirus-produced VLPs elicited good CD4 immune responses in mice when used to boost a prime with a DNA vaccine, no boost response was elicited by transgenically produced VLPs.Transgenic cell lines maintained stable piggyBac integron.Transgenic insect cells are stable and can produce HIV Pr55 Gag VLPs for over 100 passages: this novel result may simplify strategies aimed at making protein subunit vaccines for HIV. Immunogenicity of the Gag VLPs in mice was less than that of baculovirus-produced VLPs, which may be due to lack of baculovirus glycoprotein incorporation in the transgenic cell VLPs. Improved yield and immunogenicity of transgenic cell-produced VLPs may be achieved with the addition of further genetic elements into the  Human immunodeficiency virus type 1 (HIV-1) is responsible for the current infection of over 20 million people and the death of over 2 million living in sub-Saharan Africa [gag DNA vaccine prime [The HIV-1 precursor structural protein Pr55 Gag has been targeted as a potential candidate in vaccine studies as it is able to self-assemble and bud from a variety of cell systems to form non-replicating and non-infectious virus-like particles (VLPs) with good humoral and cell-mediated immune responses in animals. To date, Gag VLPs have been generated using various eukaryotic expression systems, but most often via the baculovirus-based transient protein expression system in insect cell cultures ,3. We hane prime ,5.However, there are significant potential drawbacks to use of the baculovirus expression system: these include the necessity for constant maintenance of baculovirus stocks, the need for fresh batch infections to be made each time the product is required, and co-purification of recombinant baculovirus or baculovirus proteins with VLPs. The creation of a transgenic cell line for continuous culture and protein production may provide a way to bypass production issues arising with the use of baculovirus and to overcome potential safety issues with baculovirus particle contamination of VLP preparations.Spodoptera frugiperda insect cells in culture include the random integration of an entire expression plasmid into the insect genome through recombination under antibiotic selection [piggyBac transposable element has been widely studied and is favoured as a useful tool in insect transgenesis due to its simplicity of movement and often high frequency of transformation [Trichoplusia ni and is a member of the TTAA-target site-specific class of transposable elements [piggyBac vector with the transgene, while supplying the transposase in trans [The only reported attempts to transform election -11. Tranelection ,13. The ormation . This clelements . It exclin trans .piggyBac vector system. This wide range of utility for this element makes it an attractive genetic tool. piggyBac transposons are favored over other elements as they are able to transpose large DNA fragments (9.6-14 kb) [A number of whole insects from species spanning three orders ,16 as we6-14 kb)  making t6-14 kb) ,21-23.piggyBac transposon-mediated mutagenesis of cultured Spodoptera insect cell lines have been reported to date. Here we report the creation of transgenic Spodoptera frugiperda cell lines using the piggyBac system to express HIV-1 Gag protein, with the aim of developing a system for continuous production of HIV-1 Gag VLPs for vaccine studies. The immunogenicity of these transgenically expressed VLPs is compared to that of baculovirus produced VLPs in BALB/c mice.No studies involving Spodoptera frugiperda-derived Sf21 cell line (Invitrogen) was maintained as a monolayer at 27°C in TC-100 insect medium (Sigma) supplemented with 10% (v/v) foetal bovine serum (FBS), 50 μg/ml neomycin, 69.2 μg/ml penicillin G and 100 μg/ml streptomycin. The Spodoptera frugiperda-derived Sf9 cell line (Invitrogen) was maintained as a shaking culture at 27°C, 140 rpm in SF900 II SFM media (Gibco) supplemented with 10 μg/ml gentamicin. The High 5™ cell line is derived from Trichoplusia ni (Invitrogen) and was maintained as a shaking culture in Express Five® SFM media (Gibco) supplemented with 10 ug/ml gentamicin and 18 mM glutamine.The gag DNA sequence used in this study was derived from the DNA vaccine plasmid pTHgagC [gag sequence used encodes a myristoylation signal responsible for directing the myristoylated Pr55 Gag protein to the host cell membrane where it embeds, aggregates and buds off as VLPs which are "coated" in host cell outer membrane [® Baculovirus Expression Vector System [6 Sf9 cells/ml were infected with baculovirus encoding human codon optimised HIV-1 subtype C gag under control of the polh promoter at a multiplicity of infection (MOI) of 2-10, and VLPs were harvested from the culture supernatant 72 h post infection.The human codon-optimised HIV-1 subtype C  pTHgagC ,25. The membrane ,27. Bacur System ,27,28. BpiggyBac minimal construct, pXLBacII has been previously described [piggyBac transposase which is necessary for transposition to occur. Instead, the piggyBac transposase open reading frame is encoded on a separate helper plasmid. We used as helper plasmids; pCasper-hs-orf (pCasper), in which the transposase gene is controlled by the hsp70 promoter, and pBSII-IE1-orf (pBSII-IE1), in which the transposase gene is controlled by the baculovirus immediate early (ie1) promoter. These plasmids have been described previously [gag gene , were included for comparison of Gag expression levels with and without the inclusion of an intron [The escribed ,15. pXLBeviously ,30. Conseviously ,32. Consgag gene was coupled to different regulatory elements and cloned into the piggyBac minimal construct pXLBacII, to be used in transfections  to enable antibiotic selection of transgenic cells. This construct was created by cloning the ie1-gag-polyA fragment from the pXLBSII-IE1Gag construct, downstream of the neo gene in pXLNeo  for 5 hours. DNA amounts of 1 to 5 μg were used to transfect 1.5 × 106 Sf21 cells per 2 ml, and the ratios of the transgene to helper plasmids were varied from 1:1, 7:1 and 1:2 [Each of the pXLBacII constructs containing the  and 1:2 ,34. Two  and 1:2 ,30. A co6 transgenic insect cells using the Dneasy Tissue Extraction kit (Qiagen) and subjected to PCR screening. Primers were designed to amplify the transgenes (gag or neomycin) to assess their presence and stability over several passages  to 0.007 ng (± 7 × 105 copies) of the pXLBacIINeoGag vector and amplified according to the manufacturer's protocol . Amplified products were subsequently digested with Mlu1 (which cuts twice within the NeoGag region) and analysed on a 0.8% agarose gel with ethiduim bromide staining.To confirm that the pXLBacII vector is absent from the transgenic insect cell culture, and hence not contributing to Gag expression, rolling circle amplification  was carried out on DNA isolated from harvested cells. RCA is based on a bacteriophage ϕ29 DNA polymerase that exponentially amplifies single- or double-stranded circular DNA templates by rolling circle amplification. To confirm the absence of inhibitory elements, the isolated DNA sample was spiked with 0.7 ng  according to the manufacturer's instructions.NeoGag construct, together with the pCasper helper, was transfected into Sf21 cells. Selection using 1 mg/ml geneticin  [The , Sigma) ,6 began piggyBac (PB)-produced VLPs were harvested from 1.5 × 109 transgenic NeoGag insect cells and baculovirus produced (BV) VLPs were harvested from 24 × 107 baculovirus infected cells. Cell culture supernatants were clarified by low speed centrifugation at 1000 g for 10 minutes. VLPs in these supernatants were pelleted at 4°C by ultracentrifugation in a Beckman SW32ti rotor at 26 000 rpm for 90 minutes. The pellets were then resuspended in phosphate buffered saline  and fractionated by centrifugation using a 10-50% Optiprep® (Sigma) step gradient at 26 000 rpm in a Beckman SW32ti rotor for 4 hrs at 4°C. Three light-scattering bands observed at the 10/20%, 20/30% and 30/40% Optiprep® interphases  were collected and pooled. VLPs were pelleted, after dilution of the Optiprep® with PBS, at 26 000 rpm for 90 minutes in a SW32ti rotor then resuspended in l × PBS. Both PB and BV VLP preparations were confirmed negative for the presence of endotoxins .Gradient purified samples were adsorbed onto carbon coated copper grids, stained with 2% uranyl acetate and visualized with a LEO 912 transmission electron microscope.PB and BV VLP samples were analysed on Coomassie stained gels and anti-p24 western blots. Aliquots of purified PB and BV VLPs were fractionated by electrophoresis through a 10% denaturing SDS polyacrylamide gel and either stained with 0.1% Coomassie Blue stain to assess the relative purity of the preparation, or blotted to a nitrocellulose membrane  using a semi-dry electroblotter (Hoefer) for 1.5 h at 15 V. Pre-stained molecular weight standards  and a serial dilution of a HIV-1 Pr41 Gag positive control  was included on the gel. Membranes were probed with 1:10 000 dilution of anti-p24 rabbit antiserum  followed by a 1:5000 dilution of anti-rabbit alkaline phosphatase--conjugated secondary antibody (Sigma). Membranes were developed with Nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate .The banding patterns of purified Gag concentrations were determined by comparing calculated densities of the Pr55 bands in experimental and control samples using gel imaging software (Syngene). VLP concentrations were determined by calculating densities of the Pr55 band on western blots rather than using p24 ELISA quantification, as the Pr55 content is a more reliable indicator of the actual VLP concentration in the sample than the p24 content. VLP preparations were formulated with 15% trehalose to 100 ng Gag/100 μl PBS then stored at -70°C.PB and BV VLPs was assessed by western blot analysis as described above using a 1:10 000 dilution of anti-baculovirus envelope gp64 antibody , followed by a 1:5000 dilution of anti-mouse alkaline phosphatase--conjugated secondary antibody (Sigma). Membranes were developed with NBT, as above.Baculovirus gp64 envelope glycoprotein content of PB VLPs and BV VLPs when given alone, and their ability to boost a response to a prime with the matched HIV-1 subtype C DNA vaccine, pTHgagC [PB Gag VLPs or BV Gag VLPs on day 0, and spleens from VLP-inoculated mice were harvested on day 12. To assess VLP boosting of a DNA prime, mice were given a single dose of pTHGagC on day 0 and boosted with a single dose of either PB Gag VLPs or BV Gag VLPs given on day 28 and spleens were harvested on day 40. Mice vaccinated with pTHGagC on day 0 without any boosting on day 28 were used as a DNA primed, unboosted control group and their spleens were likewise harvested on day 40. All procedures were carried out according to guidelines and with approval of the UCT Animal Research Ethics Committee. Cell mediated immune responses were determined using splenocytes in Interferon gamma (IFN-γ) and interleukin 2 (IL-2) ELISPOT assays (BD Pharmingen) with Gag CD8 peptide (AMQMLKDTI), and Gag CD4 (13) and Gag CD4 (17) peptides  as previously described [We compared the immunogenicity in BALB/c mice of  pTHgagC ,25,35. Vescribed .piggyBac transformation and subsequent Gag VLP expression. For reasons of practical handling, all further experiments were carried out using only Sf21 cell lines, for small scale amplification, and Sf9 cell lines, for large scale amplification (Sf9 cells being more suitable to this because they were maintained as a shaking culture versus Sf21 cells that were maintained as a monolayer culture). Sf21 cells and Sf9 cells expressed the Gag protein at similar levels for each construct tested. The highest p24 expression level obtained was from cell lines transgenic for the gag gene coupled to the ie1 promoter and the pBSII-IE1 polyA tail (derived from pXLBacSII-IE1Gag). Expression from these cell lines ranged between 100-1000 pg per 1.5 × 106 cells. Previous work suggested that inclusion of an intron adjacent to a gene can stimulate its transcription and possibly enhance protein expression levels [gag gene was placed under the hsp70 or actin 5C promoters. Further investigation into transgenic hsp70-gag cell lines confirmed the integration of the construct  for at least 100 cell passages. Table piggyBac vector and the transgenes was carried out on several transgenic insect cell lines. Table gag gene and the 3' piggyBac border (3' ITR), product 2 is an amplification of the 5' piggyBac border (5' ITR). Product 1 and 2 would be absent if canonical piggyBac transposon mutagenesis had occurred. Product 3 is the 3' end of the neomycin gene and the 5' end of the gag gene, providing evidence of transgene integrity. The majority of results indicate that the plasmid was linearised at the 3' ITR prior to integration of the entire intact piggyBac plasmid, possibly through a recombination mechanism unrelated to tranposition. A study in Aedes aegypti using piggyBac transformation revealed similar non-canonical integration of sequences from both donor and helper plasmids [hsp70 promoter in transgenic cell lines transfected with the pCasper helper plasmid (data not shown). However, these results are inconclusive in predicting whether recombination or transposon insertion occurred, as single insect cell clones were not isolated post transfection and mixed populations may exist.To examine the nature of the integration event that had occurred, PCR screening of the plasmids ,36. No eNeoGag cells to eliminate the possibility of transient Gag expression from a persistent non-integrated circular piggyBac vector. As seen in Figure NeoGag plasmid (lane 2) was detected in the transgenic DNA sample. The minimum amount of pUC19 DNA that can be amplified using RCA is 1 pg (± 3 × 105 copies) . To discount the possibility that non-amplification could be attributed to a low copy of the circularised DNA or the presence of inhibitors in the isolated DNA sample, transgenic insect DNA extract was spiked with 10-fold dilutions of the pXLBacIINeoGag plasmid prior to amplification. A minimum of 70 pg (± 7 × 106 copies) of spiked construct was detectable. No amplification of 7 pg of spiked plasmid (± 7 × 105 copies) occurred. This copy number approaches the limits of detectability for the RCA assay. Together these results suggest that absence of detectable RCA amplification in transgenic Sf21 NeoGag cells is not due to inhibitor presence but is rather due to absence of non-integrated plasmid in the samples.Rolling circle amplification (RCA) was carried out on DNA extracted from transgenic Sf21 PB and BV gradients revealed particles of size 120-150 nm in the 10/20% and 20/30% Optiprep® interphases. VLP size range and morphology resembled that of VLPs previously reported [PB VLPs and BV VLPs were similar in size and shape although BV VLPs appeared more defined and compact than the PB VLPs .As seen in Figure PB VLPs elicited a similar response in the absence of baculovirus elements, mice were inoculated with equal quantities of either BV or PB VLPs. Because the study reported here was designed in proof of concept, we conducted only a standard heterologous prime-boost immunogenicity experiment, in which we compared the boost potential of PB VLPs versus BV VLPs, using the same dosage which our group had previously found optimal when using BV VLPs to elicit a boosted immunological response in DNA inoculated mice: that is, 100 ng of HIV-1C VLPs administered as 2 × 50 ng inoculated into each quadriceps muscle alone or as a boost to DNA primed mouse at day 28 . IFN-γ and IL-2 ELISPOT analysis -specific IFNγ and IL-2 producing cells but no GagCD8-specific cells in response to BV VLP vaccination. These BV VLPs boosted pTHGagC responses. A 1.8 fold increase in the response to the GagCD8 peptide was detected in the IFN-γ ELISPOT assay. The low cumulative response to the GagCD4(13) and GagCD4(17) peptide of 50 IFN-γ sfu/106 splenocytes induced by pTHGagC increased 12 fold to 602 sfu/106 splenocytes when pTHgagC primed mice were boosted with these BV VLPs. No boost of the DNA vaccine induced GagCD8 response was detected in IL-2 assays. Although pTHgagC did not induce Gag-specific CD4+ T cells that produced IL-2, a high cumulative magnitude of 390 sfu/106 splenocytes Gag-specific CD4(13) and GagCD4(17) T cells producing IL-2 was detected in response to a DNA prime BV VLP boost.It has previously been shown that BV Gag VLPs enhance a cellular immune response in mice when used as a boost to a pTHgagC DNA prime ,27. To as Figure  showed tpiggyBac transposon mutagenesis as a novel method of generating gag-transgenic insect cell lines for continuous production of HIV-1 Gag VLPs. Expression of Gag VLPs from these transgenic cell lines proved stable for at least 100 cell passages: this is a novel result, which may have valuable implications in future HIV and other vaccine work.Gag epitopes elicit robust CTL responses that effectively control HIV-1 viral load in the early phases of HIV-1 infection, and Gag is therefore thought to be highly suitable as a vaccine candidate to elicit CTL . HIV-1 Ggag transgene. No improvement in Gag protein expression was noted when we cloned hr5 and hr3 baculovirus-derived enhancer elements [piggyBac construct, nor when we included the transactivator during transfection [piggyBac system does not demonstrate overproduction inhibition [hsp70 or actin 5C promoters were not active in the Spodoptera transgenic cell lines, and so no further work was done with these constructs. The Bombyx mori actin 3C promoter and Drosophila ubiquitin promoter have proven active in a variety of insect species [Spodoptera insect cell culture as an alternative means to possibly improve transgene expression.Using immunodetection and EM we verified that Gag VLPs were secreted from transgenic insect cell lines. However, the yields were low (at least 1000 times lower than from the baculovirus production system) and therefore we tested the ability of various regulatory elements to improve protein expression of the elements  or introelements  into thesfection ,39. Diffhibition . In prel species  and shoupol (un-translated region of the nucleopolyhedrovirus polyhedron gene) is able to improve transgene expression when placed upstream to the promoter [Gal4/UAS or FLP/FRT system [Although Gag VLP yields obtained in this preliminary study were low, we are confident that there are several approaches that can be employed to improve protein expression yields. Inclusion of a Gal4 DNA binding domain as an N-terminal fusion to the transposon has been shown to increase the number of transposition events  which inpromoter . Poor trT system ,42,43, oT system . TranspoT system , leadingHowever, it is possible that constitutive production of Gag protein is toxic to the cell, so only low expressers survive. In this case, inducible expression systems could be explored.piggyBac transgenically expressed VLPs. While BV VLPs were able to induce a good CD4 immune response in mice when administered as a boost to a DNA prime, PB VLPs, on the other hand, elicited no immune response, showing that Gag VLPs are not intrinsically as highly immunogenic as previously thought, and that baculovirus-derived elements probably enhance Gag VLP immunogenicity. Baculovirus-expressed Gag VLPs include trace amounts of insect cell and baculoviral contaminants  that are not efficiently removed during purification, as well as incorporated baculovirus envelope proteins. Deml et al proposed that these contaminating components act as "danger signals" that can activate an innate immune response [PB VLPs and BV VLPs to stimulate dendritic cells, as this could provide insight into the observed differences in immunogenicity between PB and BV VLPs in the study reported here.We conducted a comparative study in mice to assess the relative immunogenicity of baculovirus-produced VLPs versus response ,26. It hresponse  as well response  contain PB VLPs are "coated" in host cell outer membrane, but gp64 would be available for incorporation only into the BV VLPs (Figure PB VLPs were similar in size and morphology to BV VLPs, as seen by EM, they did not appear to be as compact or sharply defined in shape as BV VLPs (Figure PB VLPs lacked baculovirus gp64 incorporation into the VLPs outer membrane coating, the more defined shape of the BV VLPs compared to that of the PB VLPs may be accounted for by the incorporation of gp64 into the BV VLP outer membrane. It is likely that incorporation of gp64 onto the surface of baculovirus expressed VLPs facilitates BV VLP uptake into APC by promoting membrane fusion between BV VLPs and host cells, thereby enhancing the resultant immune response to VLP Gag antigens conditioned by APCs. Transgenically produced VLPs lack gp64 and this may result in less efficient cellular uptake of VLPs, with resultant lower immunogenicity.Baculovirus has been shown to transduce mammalian cells, which could lead to adjuvanting of immune responses ,48. A res Figure . Since Pet al. [piggyBac transposon vectors to transiently express two gene products. In this system, the two genes were placed under the control of bidirectional promoters which in turn were enhanced by a single enhancer element. Such dual expression systems could be used to co-express immune enhancer elements [The use of molecular adjuvants or incorporation of gp64 onto VLP surfaces could be utilised to improve the immunogenicity of these particles expressed from transgenic insect cell lines. Shi et al.  demonstrelements  or immunpiggyBac-transformed insect cell lines would be carried out by transgene integration site elimination. It has been demonstrated that elimination of the piggyBac transposon integration sites adjacent to the integrated transgene renders the element immobile to further transposase exposure [Once a cell line has been established that transgenically expresses Gag VLPs at a high level and with enhanced immunogenicity features, permanent stabilisation of the transgene in exposure ,53.This study serves as a basis to indicate the potential of a transgenic insect cell expression system as an alternative to the baculovirus-insect cell production system. Stably transformed cells produced VLPs reliably over 100 passages; purification of VLPs was also easier than in the baculovirus system due to lack of heterologous virus particles. However, further work is needed to improve VLP expression levels and their immunogenicity.piggyBac transposons and protocols and helped with the ms. A-LW was overall study director and obtained the funding, and helped in drafting the ms. EPR participated in study design and supervision and helped to revise the manuscript. All authors read and approved the final version.AL participated in the design of the study, carried out most of the experimental work and drafted the manuscript. FT conceived the study, participated in the design of the study, and helped in drafting and revising the manuscript. ES performed the immunological experiments described in the study and participated in experimental design. MF provided the


Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.The dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid .We isolated second site suppressors of the temperature sensitive phenotypes caused by one The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function. In addition to DnaA, the proteins needed to load the replicative helicase at the origin are also needed for replication initiation. In Bacillus subtilis, these include DnaD, DnaB (not to be confused with the E. coli replicative helicase DnaB), and DnaI. These proteins are conserved in low GC content Gram-positive bacteria and in some cases are known to be required for replication initiation {e.g., B. subtilis, all are needed to load the helicase (DnaC in B. subtilis) at oriCpriA in replication restart have been described in both E. coliB. subtilisE. coli, these mutations are typically in the gene for the helicase loader (E. coli dnaC) B. subtilis, these mutations are in dnaB, part of the helicase loading machinery Initiation of DNA replication is an important event in the cell cycle. In bacteria, several proteins are required for initiation, but not elongation, of replication. DnaA is the most highly conserved replication initiation protein and is found in virtually all bacteria dnaD and dnaB gene products are both essential proteins that interact with each other in vitro B. subtilis and dnaB is needed for enrichment of the oriC region in membrane fractions dnaD and dnaB cause a block in replication initiation at the non-permissive temperatures. Four temperature sensitive mutations in B. subtilis dnaB have been described: dnaB134ts , dnaB1ts, dnaB27ts, and dnaB19tsB. subtilis dnaD, dnaD23ts, has been described The dnaD23ts and dnaB134tsdnaB, dnaBS371P, that causes a serine to proline change at amino acid 371. That is, dnaBS371P is an extragenic suppressor of the temperature sensitive phenotype of dnaD23ts cells, and an intragenic suppressor of the temperature sensitive phenotype of dnaB134ts cells dnaBS371P, also called dnaB75, had been isolated previously based in its ability to bypass the need for PriA in replication restart dnaD23tsB. subtilis cells, indicating an increased interaction between these proteins in vivo Previously, we described the isolation and characterization of suppressors (temperature resistant revertants) of the temperature sensitive phenotypes caused by dnaD23ts mutation, three different intragneic suppressors have been described dnaD23ts mutation. We found that one of the intragenic suppressors caused increased interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction between wild type DnaD and the mutant DnaBS371P In addition to the extragenic suppressor of the dnaBts mutations, dnaB19ts and dnaB134ts. In both cases, intragenic and extragenic suppressors were isolated. None of the extragenic suppressors were in replication genes and most appeared to be informational suppressors.We also isolated suppressors of two different E. coli and B. subtilis. Transformations were done using standard procedures mls marker.Media and growth conditions were as previously described E. coli strains used for cloning and procedures used for strain constructions were as previously described B. subtilis strains are listed in trpC and pheA mutations. Suppressor strains are listed in dnaB and dnaD alleles discussed here are summarized in dnaD23ts causes an alanine to threonine change at amino acid 166 (A166T) dnaB19ts causes an alanine to threonine change at amino acid 379 (A379T) and dnaB134ts  causes a lysine to glutamic acid change at amino acid 85 (K85E) DnaD is 232 amino acids and 7 or 108dnaD23ts (KPL73), dnaB19ts (MER271), or dnaB134ts (KPL69) cells (grown in LB medium at 30°C) onto LB plates and incubating overnight at either 45°C or 48°C, essentially as described Spontaneous suppressors were isolated by plating approximately 10dnaB19ts following mutagenesis with ethyl methanesulfonate (EMS). For the mutagenesis, dnaB19ts (MER271) cells were grown in LB medium at 30°C to an OD600 = 0.4. Cells were washed twice with LB and resuspended to an OD600 = 1.0. Seven independent pools of cells were mutagenized in LB with 1.2% EMS (Sigma) for 30 minutes at 30°C and then washed twice with LB. Cells were grown for four generations at 30°C in LB without mutagen for recovery, and plated on LB at 45°C or 48°C for selection of temperature resistant suppressors.We also isolated suppressors of dnaA, dnaB, dnaC (helicase), and dnaD using antibiotic-resistant markers linked to the wild-type allele of each gene. For dnaA, dnaB, and dnaD, a Tn917 insertion containing the chloramphenicol resistance gene (cat) from the plasmid pTV21Δ2 linked to each gene was used as a selectable marker 917ΩHU163 is linked to dnaA, zhb-83::Tn917 is linked to dnaB, and Tn917ΩHU151 is linked to dnaDdnaC (helicase) was done with the spectinomycin resistance gene associated with a dnaC-myc fusion from strain KPL314 dnaD and dnaB were amplified by PCR and the products either sequenced directly or cloned and then sequenced. For suppressors thought to be in alanine tRNA genes, the relevant tRNA genes were amplified by PCR and either sequenced directly or cloned and sequenced. The rrnO locus was cloned into the integrative vector pGEMcatSuppressor mutations were classified and mapped essentially as described dnaD alleles dnaD23Δ154-155, dnaD23W188L, dnaD23T148M, and dnaD23A138G were amplified by PCR from the appropriate chromosomal DNA and inserted into the vector pGBDU-C3 to create in frame fusions of DnaD23Δ154-155, DnaD23W188L, DnaD23T148M, and DnaD23A138G to the DNA binding domain (DBD) of the Gal4 transcription factor dnaB that cause altered interaction between DnaB and DnaD indicate that these proteins are likely targets for regulatory factors. One approach to study DnaB and DnaD, and to possibly identify regulators of their functions, is to identify second site mutations that suppress phenotypes caused by dnaB and dnaD mutations. In the case of intragenic second site suppressors, this approach also has the potential to provide structure-function insights. To these ends, we isolated and characterized second site suppressors of a temperature sensitive mutation in dnaD and two different temperature sensitive mutations in dnaB. Temperature resistant revertants were isolated and characterized phenotypically to determine which were most likely to be second site suppressors and not simple back mutations. Isolates that were more temperature resistant than the original mutant, but also more temperature sensitive than the true wild type were chosen for further analyses. Most of the second site suppressors were intragenic and the dnaD and dnaB alleles discussed in this work are listed in DnaD and DnaB are involved in multiple protein and DNA interactions. The existence of mutations in dnaD23ts mutation  causes an alanine to threonine change at amino acid 166 of DnaD dnaBS371P mutation (We isolated 25 independent spontaneous suppressors (temperature resistant revertants) of the temperature sensitive phenotype caused by the mutation , line 1 dnaD+ cells. They grew as well as wild-type through the full range of temperatures at which wild-type grows. We chose not to characterize these because it was not clear that they represented second site suppressors.The remaining 20 apparently intragenic suppressor mutants fell into six classes based on differences in growth and colony phenotypes on LB plates at a series of temperatures. The phenotypes of one group of 9 independent revertants were indistinguishable from those of wild type dnaD  leads to deletion of two amino acids in DnaD, amino acids 154 (asp) and 155 (gln). We refer to the allele of dnaD that contains both this deletion and the original mutation that causes the temperature sensitivity as dnaD23Δ154-155 , dnaD325 causing a leucine to isoleucine change also at amino acid 193 (dnaDL193I), and dnaD326 causing a deletion of glutamine and aspartate at amino acids 155 and 156 (dnaDΔ155-156). All three of these suppressors are different from the alleles described here, although the 2 amino acid deletions are remarkably similar. That we isolated different alleles in our experiments indicates that none of the selections have been saturated.Three intragenic suppressors of dnaDts selection, dnaBS371P, allowed for detection of an interaction between DnaB and DnaD in a yeast two-hybrid assay dnaBS371P is located at the region of DnaB that is similar to a family of phage replication proteins dnaDts were also located in the phage homology region of DnaD. We used yeast two-hybrid analysis dnaDts intragenic suppressors allowed for a detectable interaction between mutant DnaD and wild-type DnaB.We previously found that the only extragenic suppressor isolated in the B. subtilis, the dnaD23Δ154-155 mutation allows for increased or unregulated interactions to occur between DnaB and DnaD, just as dnaBS371P does We found that DnaD23Δ154-155 and DnaB interact in the yeast two-hybrid assay. We fused DnaB to the activation domain (AD) and DnaD23Δ154-155 to the DNA binding domain (DBD) of the yeast transcription factor Gal4. In this system, a physical interaction between the Gal4 AD and DBD domains will drive expression of ADE2, thereby allowing for growth of the yeast on medium lacking adenine dnaD23A138G did not yield reproducible results, and the intragenic suppressor dnaDA166S was not tested.In contrast to the yeast two-hybrid results indicating increased interaction between DnaD23Δ154-155 and DnaB, we did not detect physical interactions between the other mutant forms of DnaD and DnaB. Wild-type DnaB did not detectably interact in the two-hybrid assay with either DnaD23T148M or DnaD23W188L . The intThere are multiple mechanisms by which intragenic suppressors could partly restore function to the DnaDA166T (DnaD23ts) mutant protein. Wild type DnaD is an oligomer The DnaDA166T (DnaD23ts) mutant protein has decreased binding to ssDNA in vitro, and decreased cooperativity of binding dnaD23ts that is in dnaB. This extragenic suppressor, dnaBS371P, causes increased interaction with DnaD dnaD23ts restore the normally cooperative DNA binding and/or cause increased interaction with DnaB, although any potential increase in interaction with DnaB was not detected in a yeast two-hybrid assay.One of the intragenic suppressors, DnaD23Δ154-155, caused increased interaction with DnaB, possibly contributing to the mechanism of suppression. The loss of these two residues likely uncovers an otherwise hidden DnaB binding site. This phenotype is similar to that caused by the extragenic suppressor of dnaDA166T (dnaD23ts) by altering some other aspect of DnaD function. For example, the suppressors could alter the oligomeric state of DnaD, affect interaction with DnaA, or possibly make DnaD less sensitive to uncharacterized negative regulatory factors affecting replication initiation. The suppressors are not in the N-terminal region of DnaD that contains the main oligomerization activity (aa 1–128) It is also possible, although probably less likely, that the suppressors compensate for the defect caused by dnaB134ts (dnaBK85E). In addition, we isolated a total of 31 independent suppressors of dnaB19ts (dnaBA379T), some spontaneous and some following EMS mutagenesis  As reported previously, dnaB134ts fell into four groups based on growth and colony phenotypes at a series of temperatures. All groups contained members that were linked to dnaB and the sequence of at least one representative from each group was determined   or DnaBA379T (DnaB19ts). Little is known about the mechanistic defects caused by these dnaB mutations, so it is difficult to know if the suppressors restore normal function or compensate for decreased function by increasing some other aspect of DnaB function. Wild type DnaB is found in membrane fractions of cells and is involved in the enrichment of oriC in membrane fractions dnaB134ts, it is plausible that some of the other intragenic suppressors could work similarly.As with dnaB19ts were extragenic, as they were not linked to dnaB. All of these mutations caused allele-specific suppression and the mutants formed very small colonies on LB plates. Cells appeared normal in phase contrast microscopy (data not shown). Suppressors from one of these groups were linked to Tn917ΩHU163 (near dnaA), but did not appear to be in dnaA. A ribosomal RNA and tRNA gene cluster (rrnO) is also linked to this transposon, and based on the growth phenotype and linkage, we suspected that the suppressors might be informational. We PCR-amplified and sequenced trnO-ala from the rrnO operon of one of the suppressor mutants and found a mutation in the tRNA-ala gene that changes the alanine anti-codon to one that will recognize a threonine codon . We found that, in the three additional mutants that were characterized, each had a mutation that changes the anti-codon from recognizing an alanine codon to recognizing a threonine codon. In total, two sequenced isolates were in trnA-ala, and one each was in trnB-ala and trnO-ala (Based on the finding that a mutation in dnaB134ts (dnaBK85E) suppressors were not linked to dnaB and thus were extragenic. These caused phenotypes similar to those of the suppressors of dnaB19ts that were in tRNA genes. That is, all of the extragenic suppressors of dnaB134ts were allele-specific and caused slow growth. One group of suppressors (917ΩHU163 (near the rrnO-trnO operon) and suppressors in the other group were not linked to the rrnO operon and were not characterized further.Two groups of the pressors , line 6 rrnO operon from one suppressor mutant into the integration vector pGEMcat. When integrated into the chromosome of a dnaB134ts (dnaBK85E) mutant, this clone was able to suppress the temperature sensitive phenotype, indicating that it contained the suppressor mutation. We sequenced the two tRNA genes in the rrnO operon, trnO-ala and trnO-ile, from all four suppressors in this class, i.e., the cloned rrnO operon and the other three that were not cloned. In all four suppressors, trnO-ala and trnO-ile were wild type. This was not completely surprising since the mutation in dnaB134ts is in a lysine codon (changing it to glutamate) and not in an alanine or isoleucine codon.We cloned the dnaB134ts were not likely to be in any regulatory factor affecting replication initiation. Rather, we suspect that, while not in trnO-ala or trnO-ile, they might still be informational suppressors, and at least the one we cloned is likely to be in one of the rRNA genes of the rrnO operon (dnaB134ts that appears to be in the rrnO cluster, but that is not in either of the tRNA genes in the rrnO operon, is in the 16S rRNA gene and might be causing translational misreading, thereby allowing for synthesis of some wild type DnaB protein.These extragenic suppressors of O operon , line 6.dnaDts and dnaBts mutants did not result in the identification of new regulators of DNA replication. Most of the revertants were intragenic and some were allele-specific informational suppressors. The nature of these mutations have functional implications for DnaB and DnaD.The isolation of temperature resistant revertants of dnaB19ts mutation has some interesting implications regarding DnaB function. The efficiency of suppressor tRNAs that suppress missense mutations is generally very low dnaB19ts cells implies that the amount of wild-type DnaB necessary for the cell is much lower than the amount of DnaB found in wild-type cells. It may be that there is excess DnaB in wild-type cells, and thus decreasing the concentration of functional DnaB is not harmful to cells. In addition, since DnaB is a tetramer, it is possible that mixed tetramers are functional and perhaps only a single wild type protein in a tetramer is sufficient for function and restoration of temperature resistant growth to dnaB19ts cells. We have not tested these possibilities.The isolation of mutations in tRNA-ala genes that are informational suppressors of the temperature sensitive phenotype caused by the dnaD23ts that increase interaction with DnaB, and mutations in dnaB that increase interaction with DnaD and suppress dnaD23ts, together indicate that increased interaction between these proteins might be an important part of suppression. Perhaps DnaB and DnaD naturally interact in vivo, as indicated by weak interactions in vitro. The mutations that increase this interaction could either strengthen the interaction or increase the frequency of interaction, perhaps by bypassing a normal regulatory step. The increased interaction could restore a function defective in the mutant protein or enhance a different function, thereby compensating for the defect in the mutant protein. The mechanisms by which these mutations affect DnaD and DnaB should become clearer with more structural analyses of these essential replication initiation proteins.Intragenic suppressors of


The wound healing process and production oftumour necrosis factor alpha (TNF-α) by peritonealcells of 7-day and 14-day obstructive jaundice (OJ)and sham-operated rats were investigated. In thestudy the skin wound breaking strength was measured,In addition such histological and biochemicalparameters as fibroblast and endothelial cell proliferation,inflammatory cell infiltration and hydroxyprolinecontent were evaluated in polyurethanesponge discs implanted subcutaneously into rats.TNF-α production by peritoneal exudate cells (PEC),both spontaneous and lipopolysaccharide (LPS)-induced was determined by a bioassay. In OJ rats theprocess of both early as well as late phase of healingwas impaired. The breaking strength of skin woundwas decreased, the fibroblast and endothelial cellproliferation and collagen deposition, as well as hydroxyprolinecontent were diminished. In 7 day OJthe numbers of inflammatory cells in the implantswere lowered with a subsequent slight increase onday 14 of OJ. The spontaneous and LPS induced TNF-α production by PEC were significantly higher in 7day OJ as compared with sham-operated controls. Onday 14 of OJ the LPS-induced TNF-α level was, incontrast, much lower and did not differ much fromthe spontaneous TNF-α production. We concludethat the impairment of wound healing in OJ resultsfrom disturbances in functioning of the immunesystem caused by systemic endotoxaemia.


To design hybrid tumour necrosis factor-alpha  applicable to systemic anti-tumour therapeutic use, we assessed the relationships among the molecular size of hybrid TNF-alpha, in vitro bioactivity and in vivo anti-tumour potency. Natural human TNF-alpha was covalently modified with polyethylene glycol (PEG) of various number-average molecular weights . The in vitro bioactivity of PEG-modified TNF-alpha s decreased with an increase in the degree of PEG modification, irrespective of the molecular weight of PEG. This decrease in the specific bioactivity markedly increased with an increase in the molecular weight of the attached PEG. The in vivo anti-tumour effects of the hybrid TNF-alpha s with a molecular size from 100 to 110 kDa, which had more than 50% of specific bioactivity of native TNF-alpha, were significantly superior to other PEG-TNF-alpha s. These hybrid TNF-alpha s showed over ten times greater anti-tumour effects than native TNF-alpha. Thus, the molecular size, which was determined by the degree of PEG modification and PEG molecular weight, influences the specific activity and anti-tumour effects of hybrid TNF-alpha.


Transplantation of bone marrow cells from wild-type mice restored the synthesis of TNF, corrected the splenic microarchitecture, repopulated the lamina propria with IgA-producing plasma cells, and normalized the serum immunoglobulin levels of TNF-LTα deficient mice. Furthermore, the formation of germinal centers in the spleen and the defective Ig class switch in response to a T-cell-dependent antigen is corrected. These data demonstrate that most TNFproducing cells are bone-marrow-derived, and that the immunodeficiency due to TNF-LTα deletion can be corrected to a large extent by normal bone marrow, cell transplantation.Mice with inactivated TNF-LT


In serum, the levels of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis factor (TNF), serum amiloid-A (SAA) and Fe++ were never different from controls, indicating that no systemic inflammatory changes were induced. Locally the exudate volume and the number of leukocytes recruited into the pouch increased progressively until 7 days after carrageenan. The same was true for PGE2 production. We could not measure IL-1 but the production of IL-6 and TNF reached a maximum after 5-24 h then quickly decreased. Anti-TNF antibodies inhibited cell migration by 50% 24 h after treatment. Pretreatment with interleukin 10 (IL-10) inhibited TNF production almost completely and cell migration by 60%. Carrageenan-induced inflammation was modulated by anti-inflammatory drugs. Pretreatment with dexamethasone (DEX) or indomethacin (INDO) inhibited cell migration and reduced the concentration of TNF in the exudate. Production of PGE2 or vascular permeability did not correlate with the number of cells in the pouch. Local TNF seems to play an important role in this model, particularly for leukocyte migration in the first phase of the inflammatory process. In conclusion, the air pouch seems to be a good model for studying the regulation of the early events of local inflammation, particularly the role of cytokines and cell migration.We used the mouse air pouch model of inflammation to study the interaction between cytokines, prostaglandin E


Changes of the tumor necrosis factor-alpha (TNF-α) system have been shown to be involved in the development of psychiatric disorders and are additionally associated with changes in body weight as well as endocrine and metabolic changes in psychiatric patients. TNF-α might, for example, contribute to the pathogenesis of depression by an activation of the hypothalamo-pituitary-adrenocortical (HPA) axis, an activation of neuronal serotonin transporters and the stimulation of the indoleamine 2,3-dioxygenase which leads to tryptophan depletion. On the other hand, during an acute depressive episode, an elevated HPA axis activity may suppress TNF-α system activity, while after remission, when HPA axis activity has normalized the suppression of the TNF-α system has been shown not to be apparent any more.In narcoleptic patients, soluble TNF receptor (sTNF-R) p75 plasma levels have been shown to be elevated, suggesting a functional role of the TNF-α system in the development of this disorder.Additionally, psychotropic drugs influence the TNF-α system as well as the secretion and the effect of hormones which counteract or interact with the TNF-α system such as the intestinal hormone ghrelin. However, only preliminary studies with restricted sample sizes exist on these issues, and many open questions remain. The brain and the immune system are the two major adaptive systems of the body. Both influence and regulate each other. Factors stemming from the immune system, such as cytokines and chemokines, and factors derived from the central nervous system, for example hormones, neurotransmitters and neuropeptides, have an influence on both, the brain and the immune system. And there is growing evidence that infectious and immune factors may contribute to the pathogenesis of neurodevelopmental disorders including autism, mental retardation, and schizophrenia . More spvia the pituitary, and through direct neuronal influences via the sympathetic, parasympathetic, peptidergic and sensory innervation of peripheral tissues. Alternatively, certain cytokines such as interleukin (IL)-1, IL-6 and TNF-α released during an immune response and other processes the immune system is involved in modulate brain activity. These cytokines act on the brain via a fast transmission pathway involving primary afferent nerves innervating the bodily site of inflammation and a slow transmission pathway involving cytokines originating from the choroid plexus and circumventricular organs and diffusing into the brain parenchyma by volume transmission. They alter neurotransmitter networks activity and, thus induce sleepiness, fatigue, loss of appetite and decreased libido. These symptoms have been described as “sickness behaviour” and are related to the behavioral changes of depression [The central nervous system affects the immune system mainly through the neuroendocrine outflow pression .Among the different ways of bidirectional influence between the immune system and the central nervous system, one cytokine has recently attracted a lot of scientific interest: TNF-α. This review focuses on the TNF-α system, although other cytokines do also contribute to the interaction between the brain and the immune system and, in part, amplify the effects of TNF-α.et al. as a soluble factor released by host cells that caused necrosis of a transplanted tumor [TNF-α is a 185 amino acid glycoprotein hormone, which was isolated in 1975 by Carswell ed tumor .TNF-α is a multifunctional signaling molecule with important functions in inflammation and apoptosis . It playVia these receptors, TNF-α is mitogenic in normal cells; however, TNF-α initiates apoptosis in transformed cells causing DNA fragmentation and cytolysis. Whether TNF-α induces cell differentiation or apoptosis via the TNF-Rs depends on the signaling pathway activated within the cell. The TNF-α-induced survival pathway seems to be mediated by the transcription factor nuclear factor-kappaB (NF-KB), as recent studies have demonstrated that cells in which the NF-KB signaling pathway is blocked are more likely to undergo apoptosis in response to TNF-α [The action of TNF-α at the cellular level is mediated by two cell surface receptors, TNF-R p55 and TNF-R p75. TNF-R p55 is expressed in most tissues, whereas expression of TNF-R p75 is highly regulated and typically found in cells of the immune system. Accordingly, in the vast majority of cells, TNF-R p55 appears to be the key mediator of TNF-α signaling, whereas in the lymphoid system, TNF-R p75 seems to play a major role . When boto TNF-α .In cases in which TNF-α induces apoptosis, it has been shown that one of the crucial intracellular signaling event is the sequential activation of caspases, a family of cysteine proteases .in vivo by inhibiting the ability of cytokines to bind to their membrane receptors and thus inhibiting a biological response, elevated plasma levels of sTNF-R p75 indicate an inflammatory process in several diseases, for example rheumatoid arthritis [1) shows a simplified scheme of the TNF-α system and the components described above.The membrane-bound TNF-Rs can be proteolytically cleaved from the cell membrane by the proteolytic action of a disintegrin metalloproteinase called TNF-α converting enzyme (TACE) . Thereforthritis . Fig. , have been localized to neutrophils, lymphocytes, and macrophages, which suggests that ghrelin may be involved in the process of immune modulation. And indeed, ghrelin has potent anti-inflammatory properties through modulation of secretion of both pro-inflammatory and anti-inflammatory cytokines from macrophages . OverallIn obesity, the white adipose tissue is characterized by an increased production and secretion of a wide range of inflammatory molecules including TNF-α, which may have local effects on white adipose tissue physiology but also systemic effects on other organs. Interestingly, weight loss is associated with a reduction in the macrophage infiltration of white adipose tissue and an improvement of the inflammatory profile of gene expression. Several factors derived not only from adipocytes but also from infiltrated macrophages probably contribute to the pathogenesis of insulin resistance. Most of them are overproduced during obesity, including leptin, TNF-α, IL-6 and resistin . et al. [Macrophages exist in separate types of differentiation, but the nature of adipose tissue macrophages is largely unknown. However, in a study of Zeyda et al.  basal anTherefore, it could be shown by several investigations that body fat mass influences TNF-α plasma levels , 66. Howet al. concluded that the positive associations of obesity and visceral adiposity with elevated cytokine levels suggest the importance of reducing obesity and visceral adiposity to prevent elevations in cytokine levels and associated diseases [As increased pro-inflammatory cytokine levels are known to be associated with cardiovascular disease , 41, 81,diseases .2) is ment to show the interactions between the TNF-α system and the HPA axis and its negative feedback mechanism.Cytokines such as TNF-α link the non-specific immune system to the hypothalamo-pituitary-adrenocortical (HPA) axis: inflammatory cytokines — such as TNF-α and its soluble receptors p55 and p75 — released during infection and inflammation activate the HPA system at the hypothalamic, pituitary, and adrenal level resulting in the release of cortisol as the most important negative feedback signal to prevent an overshoot of the ongoing host defense , 89, 96;Additionally, cytokines such as TNF-α exert other effects on the hypothalamic level. The pro-inflammatory cytokines IL-1, IL-6 and TNF-α have been most investigated for their pyrogenic action. The experimental evidence demonstrates the role of these secreted proteins in modulating the fever response. An association between cytokine levels in serum and cerebrospinal fluid and fever, the finding of the presence of cytokine receptors on various cell types in the brain and demonstration of the effects of pharmacological application of cytokines and of their neutralizing antibodies on the fever response and fever studies on cytokine- and cytokine receptor-transgenic models underline the strong influence of cytokines on hypothalamic thermoregulation .Cytokine-induced loss of appetite is consistently observed during cytokine immunotherapy in humans. Investigators have also shown that cytokines induce anorexia when administered peripherally or into the brain. Anorexigenic cytokines by central action include IL-1, IL-6, leptin, IL-8, TNF-α, and interferon (IFN)- γ. Cellular approaches indicate that modulation of hypothalamic activity is involved in cytokine-induced anorexia. Cytokine action involves the modulation of specific neurons that are proposed to participate in the control of feeding, i.e. glucose-sensitive neurons in the lateral hypothalamic area and hypothalamic ventromedial nucleus . AppetitOne condition already mentioned in which TNF-α is produced and secreted into the blood is obesity. Nowadays the concurrence of rich food and physical idleness is an increasing problem and established risk factor for various diseases in affluent countries. Different combinations of obesity, diabetes, hypertriglyceridemia and insulin resistance cause hepatic steatosis, which can trigger necroinflammation and fibrosis. Patients with steatohepatitis exhibit ultrastructural mitochondrial lesions. Mitochondria play a major role in fat oxidation and energy production but also leak reactive oxygen species (ROS) and are the main cellular source of ROS. In patients with steatosis, mitochondrial ROS may oxidize hepatic fat deposits. Lipid peroxidation products impair the flow of electrons along the respiratory chain, which may cause overreduction of respiratory chain components, further increasing mitochondrial ROS formation and lipid peroxidation. Lipid peroxidation, in turn, causes further cytokine induction .Several psychiatric disorders are reported to be associated with alterations of the cytokine system. In this article, hypotheses regarding associations of major depression, an affective disorder, and narcolepsy, a sleep disorder, with the TNF-α system are demonstrated. Additionally, one should keep in mind that the TNF-α system is as well involved in the development of other brain disorders such as multiple sclerosis, Parkinson’s disease and Alzheimer’s disease .Modern neurobiological methods have revealed pathophysiological mechanism associated with depression. The monoamine hypothesis, which was advocated in the 1950s, emphasizes that the deficiency of monoamine neurotransmitters, for example serotonin, brings about depressive symptoms. This theory played an important role in promoting the development of new antidepressants and it is underlined by genetic findings of polymorphisms of serotonin transporter gene associated with depression. Neuroendocrine studies have revealed the HPA axis dysfunctions in depressive patients and increased activity of HPA axis are considered as state marker of depression . SeveralAlterations in plasma cytokine levels have repeatedly been found in patients suffering from affective disorders , 52, andet al. reported data suggesting that chronic HPA system overactivity in depressed patients suppresses the production of inflammatory cytokines [In contrast, Schuld ytokines . In anotytokines . Therefoet al. hypothesized that cytokines might also activate neuronal serotonin transporters. This idea would underline the theory of a serotonin deficiency during depression and the pharmacodynamic mechanism of selective serotonin reuptake inhibitors (SSRI) in the treatment of depression, because SSRIs lead to recovery from depression via deactivation of serotonin transporters. Indeed, Zhu et al. found TNF-α stimulated serotonin uptake in both a rat embryonic raphe cell line and in mouse midbrain and striatal synaptosomes. These results provided evidence that pro-inflammatory cytokines can acutely regulate neuronal serotonin transporter activity. A mitogen-activated protein kinase may be in involved in this mechanism [As pro-inflammatory cytokines and serotonergic homeostasis have both been implicated in the pathophysiology of major psychiatric disorders, Zhu echanism .Pro-inflammatory cytokines such as IL-1 and TNF-α affect the tryptophan metabolism directly or indirectly by stimulating the enzyme indoleamine 2,3-dioxygenase which leads to a peripheral depletion of tryptophan . And theTherefore, we can hypothesize three mechanisms how cytokines may lead to depression or depressive symptoms: the activation of the HPA axis, the activation of neuronal serotonin transporters and the stimulation of the indoleamine 2,3-dioxygenase which leads to tryptophan depletion.Narcolepsy is a disabling sleep disorder characterized by excessive daytime sleepiness, cataplexy, hypnagogic hallucination, and sleep paralysis . Since tet al. [Furthermore, a small number of studies suggest that certain cytokine-producing genes may predispose to narcolepsy. Hohjoh et al.  conducteIn a sample of 30 narcoleptic patients in comparison to 120 gender- and age-matched and 101 gender-, BMI- and age-matched randomly selected normal controls, sTNF-R p75 levels were consistently elevated in the narcoleptic patients, even if compared to gender-, BMI- and age-matched counterparts. It was concluded that narcoleptic patients show increased plasma levels of sTNF-R p75 suggesting a functional alteration of the TNF-α cytokine system and further corroborating a possible pathogenic role of the immune system in this sleep disorder . One posvia an activation of a neuroendocrine system such as the HPA axis, the activation of neurotransmitter transporters such as the serotonin transporter, the influence on the metabolism of neurotransmitters and the autoimmune destruction of neurons.In conclusion, TNF-α may lead to psychiatric disorders In addition to its immunological and metabolic properties, TNF-α promotes nerve cell growth and differentiation, and is cytotoxic to oligodendrocytes . The genThus, TNF-α might be involved in the process of neurodevelopment and neurodegeneration, which could link to the pathogenesis of Schizophrenia by subtle alterations in neuronal number and density . A functvia glutamate release from hemichannels of activated microglia in an autocrine manner. This mechanism may contribute to neuronal damage in neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis and multiple sclerosis [The TNF-α system has also been shown to be involved in the development of other brain diseases such as multiple sclerosis (MS). In MS patients, the inflammatory demyelinisation seems to be mediated by the presence of TNF-α leading to myelin and oligodendrocyte destruction . One souclerosis .Weight gain in patients on psychotropic medication is a frequent side effect of prominent clinical relevance, occurring under antipsychotics and antidepressants.In recent years, studies have been conducted on neuroendocrine and neuroimmunological mechanisms involved in appetite and weight regulation. Regarding these mechanisms, the TNF-α system might play a particular role. All drugs investigated so far that induce weight gain - clozapine, olanzapine, amitriptyline and mirtazapine – also clearly activate the TNF-α system , 75, 85.in vitro production of TNF-α in healthy volunteers [Recently, the influence of the mood stabilizer carbamazepine on leptin and the TNF-α system has been investigated, too. Lithium, for example, demonstrated significant immunoregulatory effects by increasing the lunteers , and thelunteers .A longitudinal study on the influence of carbamazepine and lithium on TNF-α plasma levels has been conducted. An increase in the body mass index and in TNF-α and its soluble receptor levels over the 4 weeks of treatment was found in both treatment groups .Is not clear, however, whether the activation of the TNF-α system by psychotropic drugs is the cause or a consequence of weight gain during psychopharmacological treatment. As mentioned above, recent data indicate that white adipose tissue is infiltrated by macrophages, which may be a major source of locally produced pro-inflammatory cytokines , in case1.Activation of the TNF-α system seems to be specific for psychotropic drugs that induce weight gain, since drugs which did not affect or even decreased weight  did not influence the TNF-α system at all , 75, 85.Abnormal liver function tests are frequently encountered in patients receiving psychopharmacological treatment . All kinThis finding suggests that the effects of psychotropic drugs on the TNF-α cytokine system – independent of weight gain – might contribute to early changes in liver enzyme levels. This idea is in line with present evidence already mentioned earlier that cytokines might be involved in liver damage , 40, 97.In a small study with 24 depressed patients we sought to determine whether changes in TNF-α and its receptor plasma levels early during antidepressant therapy are predictors of psychopharmacologically induced weight change. Changes of weight during the first week of treatment, but no parameter of the TNF-α system strongly predicted weight change until endpoint of the study. Therefore, changes regarding the TNF-α system do not seem to be a clinically useful predictor of future weight development during psychopharmacological therapy , but to As mentioned above, the effects of the TNF-α system may be modulated or even counteracted by the intestinal hormone ghrelin. Ghrelin recently became a target of research regarding investigations on weight gain during psychopharmacological treatment. Regarding antipsychotic treatment for example, it was reported that olanzapine would increase plasma ghrelin levels in patients with schizophrenia  and thatRegarding antidepressant treatment, the role of ghrelin seems to be much more complex and the various study results are not sufficient to lead to a common hypothesis for the role of ghrelin in antidepressant treatment. For example, tryptophan – the precursor molecule of serotonin – enhances ghrelin expression and secretion , antidepet al. assessed the effect of etanercept on fatigue and symptoms of depression associated with the condition. 618 patients with moderate to severe psoriasis received double-blind treatment with placebo or 50 mg twice-weekly etanercept. The primary efficacy endpoint was a 75% or greater improvement from baseline in psoriasis area and severity index score at week 12. Secondary endpoints included the functional assessment of chronic illness therapy fatigue scale, the Hamilton Depression Rating Scale (HAMD), the Beck depression inventory (BDI), and adverse events. In this study, greater proportions of patients receiving etanercept had at least a 50% improvement in the HAMD or BDI at the endpoint of the study; patients treated with etanercept also had significant and clinically meaningful improvements in fatigue. The improvements in symptoms of depression were less correlated with objective measures of skin clearance or joint pain. The authors concluded that etanercept treatment might relieve fatigue and symptoms of depression associated with this chronic disease [It is pharmacologically possible to give a TNF-α blocker to a patient. One of these new drugs is etanercept. It can, for example, be used for the treatment against the autoimmune disease psoriasis. Psoriasis has substantial psychological and emotional effects. Therefore, Tyring  disease . This obOne should also consider, whether cytokines could be involved in the development of morphological changes of hippocampus and down regulation of neurotrophin observed in depressive patients. But no investigations are available regarding these associations. 1) [Recent genetic research points at the importance of caspase genes for the predisposition to major depression , and cas1) . These aThis review focuses on plasma levels of TNF-α and its soluble receptors. Other inflammatory and anti-inflammatory cytokines act together with TNF-α and should therefore at least be mentioned. We chose the TNF-α system as an issue of this review, because significant findings regarding its involvement in brain disorders - such as depression, narcolepsy, multiple sclerosis, Alzheimer’s and Parkinson’s disease - and psychopharmacology have been reported in several study reports . In the As the majority of the mentioned studies are cross-sectional, caution is recommended regarding conclusions about the causality. The conclusion that elevated cytokine levels may contribute to the pathogenesis of depression is derived from the literature combining the conclusions of several studies, but can not be substantiated from longitudinal studies. Therefore, it would be necessary to strengthen experimental and longitudinal clinical research in animals and humans investigating the effect of cytokines on mood over a certain period of time.et al. [One could argue that the cited studies reported mainly plasma levels of TNF-α and its soluble receptors. This is an important demur. On the other hand, Sorge  reportedet al.  reportedTo conclude, only preliminary studies with restricted sample sizes exist on most of the mentioned issues, and many open questions remain.


A wide variety of evidence has pointed to a critical role of TNF-α in tumour proliferation, migration, invasion and angiogenesis. The function of TNF-α as a key regulator of the tumour microenvironment is well recognised. We will emphasise the contribution of TNF-α and the nuclear factor-κB pathway on tumour cell invasion and metastasis. Understanding the mechanisms underlying inflammation-mediated metastasis will reveal new therapeutic targets for cancer prevention and treatment.Tumour necrosis factor-alpha (TNF- It was peatedly . By contα is synthesised as a transmembrane protein with a molecular mass of 26 kDa and the pro-peptide is secreted as a soluble 17-kDa molecule on cleavage by TNF-α-converting enzyme (TACE). Although activated macrophages are the major source of TNF-α, it can also be produced by a variety of other cells, such as fibroblasts, astrocytes, Kupffer cells, smooth muscle cells, keratinocytes and a wide variety of tumour cells, including B-cell lymphoma, breast and colon carcinomas. Increasing evidences indicate that TNF-α acts as tumour-promoting factor and is linked to all steps of tumourigenesis including transformation, proliferation, angiogenesis, invasion and metastasis in many cancers. Tumour necrosis factor-α has a particularly important role in tumour microenvironment and promotes tumour cell migration and invasion, however, the mechanism by which TNF-α facilitates these events remains elusive. In this study, we discuss the molecular mechanisms of TNF-α-induced tumour migration and invasion, particularly focusing on the contribution of TNF-α–NF-κB–Snail pathway.Tumour necrosis factor-α secretion can be induced by conserved structural elements common to microbial pathogens, such as lipopolysaccharide (LPS), that are bound by Toll-like receptors (TLRs) -κB and NF-AT signalling pathways, and thereby enhance translational efficiency by a mechanism targeting consensus 3′-untranslated AU-rich elements (ARE) in mRNA  and TNF-α receptor II  (κB activation versus that of TNF-R2. The TNF-R1 ligation induces receptor trimerisation and the recruitment of the adaptor protein TNF-R1-associated death domain protein (TRADD) that binds to a specific death domain (DD) in the cytoplasmic domain of TNF-R1. TNF-R1-associated death domain protein also recruits TNF receptor-associated factor (TRAF2) and activates IκB kinase (IKK) through receptor-interacting protein (RIP). The RIP1 is ubiquitinated in a TRAF2-dependent manner during TNF-R1 activation and is essential for TNF-α-induced IKK and NF-κB activation. RIP1 knock-out cells fail to activate IKK in response to TNF-α. The TRAF2 recruits the IKK complex to the activated TNF-R1 by interacting with the LZ motifs of IKKα and IKKβ. The IKK complex consists of IKKα, IKKβ and NEMO . In the classical activation pathway, activated IKKβ phosphorylates specific serine residues of IκB in a NEMO-dependent manner, leading to IκB phosphorylation, ubiquitination and proteosome-mediated degradation. The degradation of IκB releases the transcription factors NF-κB, which translocates to the nucleus, binds the κB site and activates gene transcription. Nuclear factor-κB is composed of five distinct but structurally related subunits, p50, p52, c-Rel, RelA and RelB. These subunits can form various homodimeric and heterdimeric complexes; each combination of subunits has a specific signalling function  . The TLR or p80) . The TNFo TNF-R1 . Althoug complex  and not α is a key mediator of inflammation and cancer  to support lung metastasis  in macrophages and through enhanced MMPs production in tumour cells , IL-8 and intercellular adhesion molecule-1 . The TNF-α enhances the invasive property of cancer cells by inducing EMT through Snail- or ZEB1/ZEB2-dependent mechanisms  in the control of embryogenesis (β) and NF-κB pathways  in which the level of TNF-α is elevated. These cells lose the expression E-cadherin and they dissociate from the tumour mass and infiltrate into the surrounding stroma. It will be interesting to know whether the synergistic interaction of Snail and β-catenin is required for EMT induction and tumour cell invasion at the invasive front , infliximab , pegylated recombinant humanised sTNF-R1, pegylated humanised anti-TNF-α fragment (CDP870) and TNF-α synthesis inhibitors (p38 kinase inhibitors) have now been used to treat various tumours (Tumour necrosis factor- tumours . However